CN101090980A - Modified human growth hormone - Google Patents

Abstract

The present invention provides modified growth hormone polypeptides and uses thereof.

Description

modified human growth hormone

Cross References to Related Applications

This application claims priority to U.S. Provisional Patent Application No. 60/638,616, filed December 22, 2004, and U.S. Provisional Patent Application No. 60/727,996, filed October 17, 2005, which applications The full text of the specification is incorporated herein.

technical field

The present invention relates to growth hormone polypeptides modified with at least one non-naturally encoded amino acid.

Background technique

Growth hormone (GH) supergene family (Bazan, F. Immunology Today 11: 350-354 (1990); Mott, H.R. and Campbell, I.D. Current Opinion in Structural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J.N. (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINERECEPTORS) represent a group of proteins with similar structural features. Each member of this protein family contains a four-helix bundle, the general structure of which is shown in Figure 1 . Although many members of this family remain to be identified, some of them include the following proteins: growth hormone, prolactin, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin Interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit) , IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, epsilon interferon, granulocyte Colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage-colony-stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) (“GH supergene family "). Although members of the GH supergene family generally have limited amino acid or DNA sequence identity, they share similar secondary and tertiary structures. Shared structural features allow easy identification of new members of gene families. The general structures of the family members hGH, EPO, IFNα-2 and G-CSF are shown in Figure 2, Figure 3, Figure 4 and Figure 5, respectively.

Human growth hormone (hGH) is one of the members of the GH supergene family. Human growth hormone is involved in many regulation of normal human growth and development. This naturally occurring single-chain pituitary hormone consists of 191 amino acid residues and has a molecular weight of approximately 22 kDa. hGH exhibits numerous biological effects including, inter alia, linear growth (somatogenesis), lactation, activation of macrophages, and insulin-like and diabetogenic effects (Chawla, R. et al., Ann. Rev. Med. 34:519- 547 (1983); Isaksson, O. et al., Ann. Rev. Physiol, 47: 483-499 (1985); Hughes, J. and Friesen, H., Ann. Rev. Physiol, 47: 469-482 (1985 )).

The structure of hGH is well known (Goeddel, D. et al., Nature 281:544-548 (1979)), and the three-dimensional structure of hGH has been solved by X-ray crystallography (de Vos, A. et al., Science 255:306-312 (1992)). The protein has a compact globular structure comprising four amphipathic alpha-helical bundles called A-D connected by loops starting from the N-terminus. hGH also contains four cysteine residues that participate in the formation of two intramolecular disulfide bonds: C53 pairs with C165 and C182 pairs with C189. This hormone is not glycosylated and has been expressed in secreted form in E. coli (Chang, C et al., Gene 55:189-196 (1987)).

A number of naturally occurring hGH mutants have been identified. These mutants include hGH-V (Seeburg, DNA 1:239 (1982); U.S. Patent Nos. 4,446,235, 4,670,393, and 4,665,180, which are incorporated herein by reference) and hGH-containing residues 32-46. (Kostyo et al., Biochem. Biophys. Acta 925:314 (1987); Lewis, U. et al., J. Biol. Chem., 253:2679-2687 (1978)). In addition, numerous hGH variants have been reported resulting from post-transcriptional, post-translational, secretion, metabolic processing, and other physiological processes (Baumann, G., Endocrine Reviews 12:424 (1991)).

The biological effects of hGH result from its interaction with specific cellular receptors. This hormone is a member of a family of homologous proteins that includes placental lactogen and prolactin. However, hGH is unusual among family members in that it exhibits broad species specificity and interacts with cloned somatogenic receptors (Leung, D. et al., Nature 330:537-543( 1987)) or the prolactin receptor (Boutin, J. et al., Cell 53:69-77 (1988)). Based on structural and biochemical studies, functional maps of the prolactin- and somatic-binding domains have been proposed (Cunningham, B. and Wells, J., Proc. Natl. Acad. Sci. 88:3407 (1991)). The hGH receptor is a member of the hematopoietic/cytokine/growth factor receptor family that includes several other growth factor receptors such as interleukin (IL)-3, interleukin-4 and interleukin G-6 receptor, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, erythropoietin (EPO) receptor, and G-CSF receptor. See, Bazan, Proc. Natl. Acad. Sci USA 87:6934-6938 (1990). Members of the cytokine receptor family contain four conserved cysteine residues and a tryptophan-serine-X-tryptophan-serine motif located just outside the transmembrane region. Conserved sequences are thought to be involved in interactions between proteins, see, eg, Chiba et al., Biochim. Biophys. Res. Comm. 184:485-490 (1992). The interaction between hGH and its receptor (hGHbp) extracellular domain is one of the best understood hormone-receptor interactions. High-resolution X-ray crystallographic data (Cunningham, B. et al., Science, 254:821-825 (1991)) have shown that hGH has two receptor binding sites and uses unique sites on the molecule to sequentially bind to the two Receptor molecules bind. The two receptor binding sites are referred to as site I and site II. Site I includes the carboxy-terminus of helix D and part of helix A and the A-B loop, while site II encompasses the amino-terminal region of helix A and a portion of helix C. Binding of GH to its receptor occurs sequentially with site I binding first. Site II then engages a second GH receptor, leading to receptor dimerization and activation of intracellular signaling pathways that lead to the response of hormone-producing cells. The hGH mutein that had introduced the G120R substitution into site II was able to bind a single hGH receptor but was unable to dimerize both receptors. The mutein is estimated to act as an hGH antagonist in vitro, possibly by occupying the receptor site but not activating intracellular signaling pathways (Fuh, G. et al., Science 256:1677-1680 (1992)).

Recombinant hGH is used as a therapeutic agent and has been approved for the treatment of many indications. Deficiency of hGH leads, for example, to dwarfism, a condition that has been successfully treated for over a decade by exogenous administration of the hormone. In addition to hGH deficiency, hGH has also been approved for the treatment of renal failure (in children), Turner's Syndrome, and cachexia in AIDS patients. Recently, the Food and Drug Administration (FDA) has approved hGH for the treatment of GH-independent short stature. hGH is also currently being investigated for the treatment of aging, frailty, short bowel syndrome and congestive heart failure in the elderly. Target groups for hGH therapy include children with idiopathic short stature (ISS) and adults with GHD-like symptoms.

Currently, recombinant hGH is sold as a daily injectable product, of which there are currently five main products on the market: Humatrope ^TM (Eli Lilly & Co.), Nutropin ^TM (Genentech), Norditropin ^TM (Novo-Nordisk), Genotropin ^TM (Pfizer) and Saizen/Serostim ^(TM ) (Serono). However, a major challenge with the use of growth hormone as a therapeutic agent is that the protein has a short half-life in vivo, and therefore it must be administered by daily subcutaneous injections for maximum efficacy (MacGillivray et al., J. Clin. Endocrinol. Metab. 81:1806-1809 (1996)). Much effort has been focused on methods to improve the administration of hGH agonists and antagonists by reducing production costs, making administration of drugs to patients easier, improving efficacy and safety profiles, and creating other properties that would provide a competitive advantage. For example, Genentech and Alkermes previously marketed Nutropin Depot ^™ , a depot formulation of hGH for pediatric growth hormone deficiency. Although the depot formulation allowed for less frequent dosing (every 2-3 weeks rather than daily), it was also associated with adverse side effects such as decreased bioavailability and pain at the injection site, and was withdrawn in 2004. out of the market. Recently, another product, Pegvisomant ^TM (Pfizer), has also been approved by the FDA. Pegvisomant ^™ is a genetically engineered analog of hGH that acts as a highly selective growth hormone receptor antagonist indicated for the treatment of acromegaly (van der Lely et al., The Lancet 358:1754-1759( 2001)). Although some of the amino acid side chain residues in Pegvisomant ^™ are derivatized with polyethylene glycol (PEG) polymers, the product is still administered once daily, indicating suboptimal pharmaceutical properties. In addition to pegylated and depot formulations, other routes of administration including hGH inhaled and oral formulations are in early stages of preclinical and clinical development, and none have yet been approved by the FDA. Therefore, there is a need for polypeptides that exhibit growth hormone activity but also provide longer serum half-life and (thus) better optimal therapeutic levels of hGH and increased therapeutic half-life.

The covalent linkage of hydrophilic polymer poly(ethylene glycol) (abbreviated as PEG) is a way to increase the water solubility and bioavailability of many biologically active molecules including proteins and peptides, and especially hydrophobic molecules, increasing their Serum half-life, methods of increasing its therapeutic half-life, modulating its immunogenicity, modulating its biological activity or prolonging its circulation time. PEG has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are important. To optimize the desired properties of PEG, the overall molecular weight and hydration state of the PEG polymer attached to the bioactive molecule must be high enough to impart the favorable properties normally associated with PEG polymer attachment, such as increased water solubility and circulation half-life) without adversely affecting the biological activity of the parent molecule.

PEG derivatives are often linked to biologically active molecules through reactive chemical functional groups (such as lysine, cysteine, and histidine residues), the N-terminus, and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the site most suitable for modification by polymer attachment plays an important role in receptor binding and is required to maintain the biological activity of the molecule. Thus, indiscriminate attachment of polymer chains to these reactive sites on bioactive molecules often results in a significant reduction or even complete loss of the bioactivity of the polymer modified molecule. R. Clark et al., (1996), J. Biol. Chem, 271:21969-21977. To form conjugates with sufficient polymer molecular weight to confer the desired advantages on the target molecule, prior art methods typically involve the random attachment of numerous polymer arms to the molecule, thereby increasing the risk of reduced or even complete loss of biological activity of the parent molecule.

The reactive site that forms the locus for attachment of the PEG derivative to the protein is dictated by the protein structure. Proteins, including enzymes, are composed of various α-amino acid sequences having the general structure H ₂ N—CHR—COOH. The α-amino moiety ( _H2N-- ) of one amino acid is linked to the carboxyl moiety (--COOH) of an adjacent amino acid to form an amide bond, which can be expressed as --(NH--CHR--CO) _n --, where the subscript "n" may be in the hundreds or thousands. The fragment represented by R may contain reactive sites for maintaining the biological activity of the protein and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there is an _--NH2 group at the ε position as well as at the α position. ε--NH ₂ can react freely under alkaline pH conditions. Much of the art in the field of derivatizing proteins with PEG has been devoted to the development of PEG derivatives for attachment to lysine residues ε-- _NH2 moieties present in proteins. "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 2003, pp. 1-17. However, these PEG derivatives all share the limitation that they cannot be selectively localized among the often infinite number of lysine residues present on the surface of proteins. Where, for example, lysine residues present in the active site of an enzyme are important for protein activity, or where lysine residues play a role in regulating the interaction of a protein with other biomolecules, as in In the case of receptor binding sites, this limitation can be a significant limitation.

A second and equally important complication of existing protein PEGylation methods is that PEG derivatives can undergo undesired side reactions with residues other than those desired. Histidine contains a reactive imino moiety structurally denoted --N(H)--, but many chemically reactive species that react with ε-- _NH2 can also react with --N(H)--. Similarly, the side chain of the amino acid cysteine has a free sulfhydryl group represented structurally as -SH. In some cases, PEG derivatives of ε-- _NH2 specific to lysine will also react with cysteine, histidine or other residues. This can create complex heterogeneous mixtures of PEG-derivatized bioactive molecules and risks disrupting the activity of the targeted bioactive molecule. It would be desirable to develop PEG derivatives that allow the introduction of chemical functional groups at individual sites within proteins that would then enable one or more PEG polymers to bind to biologically active molecules in a defined and predictable manner on the protein surface. Selective coupling at specific sites.

In addition to lysine residues, much effort in the art has been directed towards the development of activated PEG reagents targeting other amino acid side chains including cysteine, histidine and the N-terminus. See, e.g., U.S. Patent No. 6,610,281, which is incorporated herein by reference; and "Polyethylene Glycol and Derivatives for Advanced PEGylation," Nektar Molecular Engineering Catalog, 2003, pp. 1-17. Cysteine residues can be introduced into the structure of proteins in a site-selective manner using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moieties can be reacted with PEG derivatives having thiol-reactive functional groups . However, this approach is complicated because the introduction of free sulfhydryl groups complicates the expression, folding and stability of the resulting protein. Thus, it would be desirable to have a method of introducing chemical functional groups into biologically active molecules that would enable the selective coupling of one or more PEG polymers to proteins while simultaneously binding to sulfhydryl groups and other chemical compounds commonly present in proteins. The functional groups are compatible (ie, do not engage in undesired side reactions with sulfhydryl groups and other chemical functional groups commonly found in proteins).

As can be seen from a sampling of the art, these have been developed for linking side chains of proteins (in particular linking the _--NH2 moiety on the lysine amino acid side chain and the -SH moiety on the cysteine side chain) Many of the derivatives have proven problematic in their synthesis and use. Some derivatives form labile bonds with proteins that are susceptible to hydrolysis and thus break down, degrade or are otherwise unstable in an aqueous environment such as in the bloodstream. Some derivatives form more stable bonds but are prone to hydrolysis before bond formation, which means that reactive groups on PEG derivatives may be inactivated before proteins can be attached. Some derivatives are somewhat toxic and thus less suitable for in vivo use. Some derivatives react too slowly to be practically applicable. Some derivatives result in the loss of protein activity by linking to sites that give rise to protein activity. Some derivatives are not specific for where they will be attached, which also leads to loss of desired activity and to lack of reproducibility of results. To overcome the challenges associated with modifying proteins with poly(ethylene glycol) moieties, more stable (e.g., U.S. Patent No. 6,602,498, which is incorporated herein by reference) or poly(ethylene glycol) moieties that can be attached to molecules and surfaces have been developed. PEG derivatives that selectively react with the thiol moiety of (eg, US Patent No. 6,610,281, which is incorporated herein by reference). Clearly, there is a need in the art for PEG derivatives that are chemically inert in physiological environments until selective reactions are required to form stable chemical bonds.

Recently, a novel technique in the field of protein science has been reported that promises to overcome many of the limitations associated with site-specific modification of proteins. Specifically, new components have been added to the prokaryote Escherichia coli (Escherichia coli, E.coif) (for example, L. Wang et al., (2001), Science 292:498-500) and the eukaryotic In the protein biosynthetic machinery of the organism Sacchromyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), the machinery has enabled non-genetically encoded amino acids to be used in living Incorporated into protein in vivo. Using the methods described, many novel amino acids with novel chemical, physical or biological properties (including photoaffinity-labeled and photoisomerized amino acids, photocrosslinked amino acids (see, e.g., Chin, JW et al., ( 2002) Proc.Natl.Acad.Sci.USA 99:11020-11024; and Chin, JW et al., (2002) J.Am.Chem.Soc. 124:9026-9027), ketoamino acids, heavy atom-containing amino acids and glycosylated amino acids) were efficiently and with high fidelity incorporated into proteins in E. coli and yeast in response to the amber codon TAG. See, e.g., JW Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Chin and PG Schultz (2002), ChemBioChem 3(11):1135-1137; JW Chin et al., (2002), PNAS United States of America 99:11020-11024; and L. Wang and PGSchultz, (2002), Chem.Comm. , 1:1-11. All documents are incorporated by reference in their entirety. The studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups not present in proteins such as keto, alkynyl and azide moieties for the 20 common genetically encoded amino acids are chemically inert with respect to all functional groups present in and may be used to react efficiently and selectively to form stable covalent bonds.

The ability to incorporate non-genetically encoded amino acids into proteins allows the introduction of amino acids that provide naturally occurring functional groups (such as ε- _NH2 for lysine, sulfhydryl-SH for cysteine, imino group for histidine, etc.). Chemical functional groups for valuable surrogates. Some chemical functional groups are known to be inert to those present in the 20 common genetically encoded amino acids but can react completely and efficiently to form stable bonds. For example, it is known in the art that azido and ethynyl groups undergo Huisgen [3+2] cycloaddition reactions under aqueous conditions in the presence of catalytic amounts of copper. See, eg, Tornoe et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev et al. (2002) Angew. Chem.Int.Ed. 41:2596-2599. By introducing an azide moiety into the protein structure, for example, we are able to incorporate moieties that are chemically inert to the amine, sulfhydryl, carboxylic acid and hydroxyl groups present in proteins but also smoothly and efficiently combine with acetylene moieties A functional group that reacts to form a cycloaddition product. Importantly, in the absence of the acetylene moiety, the azide moiety remains chemically inert and unreactive in the presence of other protein side chains and under physiological blood conditions.

In particular, the present invention addresses problems associated with GH polypeptide activity and manufacture, and also addresses the manufacture of hGH polypeptides with improved biological or pharmacological properties, such as improved therapeutic half-life.

Contents of the invention

The invention provides members of the GH supergene family, including GH (eg, hGH) polypeptides, comprising one or more non-naturally encoded amino acids.

In some embodiments, a GH (eg, hGH) polypeptide comprises one or more post-translational modifications. In some embodiments, a GH (eg, hGH) polypeptide is linked to a linker, polymer, or biologically active molecule. In some embodiments, a GH (eg, hGH) polypeptide is linked to a bifunctional polymer, a bifunctional linker, or at least one additional GH (eg, hGH) polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises poly(ethylene glycol) moieties. In some embodiments, the non-naturally encoded amino acid is linked to the water-soluble polymer by a linker or linked to the water-soluble polymer by a bond. In some embodiments, the poly(ethylene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is a GH (eg, hGH) polypeptide.

In some embodiments, a GH (eg, hGH) polypeptide comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety. In some embodiments, at least one amino acid is a non-naturally encoded amino acid.

Regions of GH (eg, hGH) can be illustrated as follows, where the middle row indicates the amino acid position in hGH.

Helix A Helix B Helix C Helix D

[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184 -191]

N end A-B ring B-C ring C-D ring C end

In some embodiments, at any position in one or more of the following regions corresponding to hGH secondary structure as follows: 1-5 (N-terminal), 6-33 (helix A), 34-74 (helix A and Region between helix B, A-B loop), 75-96 (helix B), 97-105 (region between helix B and helix C, B-C loop), 106-129 (helix C), 130-153 (helix The region between C and helix D, C-D loop), 154-183 (helix D), 184-191 (C-terminus) (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3), will One or more non-naturally encoded amino acids are incorporated. In other embodiments, a non-naturally encoded amino acid is substituted at a position selected from the group consisting of each of the following residue positions: residues 1-5, 32-46, 97-105, 132-149, and 184-191 (from hGH SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, one or more non-naturally encoded amino acids are incorporated into GH (e.g., hGH) at one or more of the following positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 2, or of SEQ ID NO: 1 or 3 corresponding amino acids).

In some embodiments, at one or more of the following positions: positions 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186, and 187 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3), substituted with one or more non-naturally encoded amino acids .

In some embodiments, at one or more of the following positions: positions 29, 33, 35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 187 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3), substituted with one or more non-naturally encoded amino acids.

In some embodiments, at one or more of the following positions: positions 35, 88, 91, 92, 94, 95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145 and 155 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3), substituted with one or more non-naturally encoded amino acids.

In some embodiments, one or more non-naturally encoded Amino acid substitutions. In some embodiments, one or more unnatural Coded amino acid substitutions.

In some embodiments, the non-naturally occurring amino acid is attached to the water soluble polymer at one or more of these positions including, but not limited to: Before position 1 (i.e., at the N-terminus) , position 1, 2, 3, 4, 5, 8, 9, 11, 12, 1 5, 1 6, 1 9, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95 ,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129 ,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154 , 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurring amino acid at one or more of said positions is linked to a water soluble polymer: positions 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO : 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurring amino acid at one or more of said positions is linked to a water soluble polymer: positions 35, 92, 143, 145 (SEQ ID NO: 2, or SEQ ID NO: the corresponding amino acid of 1 or 3).

Human GH antagonists include, but are not limited to, those at positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, Substitution at 119, 120, 123 and 127 or addition at position 1 (i.e. at the N-terminus) or any combination thereof (SEQ ID NO: 2, or SEQ ID NO: 1, 3 or any other GH sequence those antagonists of the corresponding amino acids).

In some embodiments, the GH (e.g., hGH) polypeptide comprises a modulating GH (e.g., hGH) polypeptide for GH (e.g., hGH) when compared to the affinity of a corresponding unsubstituted, added or deleted GH (e.g., hGH) Substitution, addition or deletion of affinity for polypeptide receptors. In some embodiments, the GH (e.g., hGH) polypeptide comprises a substitution that increases the stability of the GH (e.g., hGH) polypeptide when compared to the stability of a corresponding unsubstituted, added, or deleted GH (e.g., hGH), Added or missing. In some embodiments, the GH (e.g., hGH) polypeptide comprises an amino acid substitution selected from the group consisting of: F10A, F10H, F10I; M14W, M14Q, M14G; H18D; H21N in hGH SEQ ID NO: 2 ; G120A; R167N; D171S; E174S; F176Y, 1179T or any combination thereof. In some embodiments, the GH (e.g., hGH) polypeptide comprises a protein that modulates the immunogenicity of the GH (e.g., hGH) polypeptide when compared to the immunogenicity of a corresponding unsubstituted, added, or deleted GH (e.g., hGH). Replacement, addition or deletion. In some embodiments, the GH (e.g., hGH) polypeptide comprises a GH (e.g., hGH) polypeptide that modulates the serum half-life of the GH (e.g., hGH) polypeptide when compared to the serum half-life or circulation time of a corresponding unsubstituted, added, or deleted GH (e.g., hGH) or substitutions, additions or omissions of cycle times.

In some embodiments, the GH (e.g., hGH) polypeptide comprises a substitution that increases the water solubility of the GH (e.g., hGH) polypeptide when compared to the water solubility of a corresponding unsubstituted, added, or deleted GH (e.g., hGH), Added or missing. In some embodiments, the GH (e.g., hGH) polypeptide comprises a GH (e.g., hGH) polypeptide that causes production of the GH (e.g., hGH) polypeptide in the host cell when compared to the solubility of a corresponding unsubstituted, added, or deleted GH (e.g., hGH). Solubility-increasing substitutions, additions or deletions. In some embodiments, the GH (e.g., hGH) polypeptide comprises expression of the GH (e.g., hGH) polypeptide in the host cell when compared to the expression or synthesis of a corresponding unsubstituted, added or deleted GH (e.g., hGH) A substitution, addition or deletion that enhances or enhances in vitro synthesis. In some embodiments, the hGH polypeptide comprises the amino acid substitution G120A. hGH polypeptides comprising such substitutions retain agonist activity and maintain or increase expression levels in the host cell. In some embodiments, the GH (e.g., hGH) polypeptide comprises an increased protease resistance of the GH (e.g., hGH) polypeptide when compared to the protease resistance of a corresponding unsubstituted, added, or deleted GH (e.g., hGH) substitution, addition or deletion.

In some embodiments, amino acid substitutions in a GH (eg, hGH) polypeptide can be made with naturally occurring or non-naturally occurring amino acids, with the proviso that at least one substitution is with a non-naturally encoded amino acid.

In some embodiments, the non-naturally encoded amino acid comprises a carbonyl, acetyl, aminooxy, hydrazino, hydrazide, semicarbazide, azido, or alkynyl group.

In some embodiments, the non-naturally encoded amino acid comprises a carbonyl group. In some embodiments, the non-naturally encoded amino acid has the structure,

wherein n is 0-10, R _is alkyl, aryl, substituted alkyl, or substituted aryl; R is _H , alkyl, aryl, substituted alkyl, and substituted aryl and R ₃ is H, an amino acid, a polypeptide or an amino-terminal modification group, and R ₄ is H, an amino acid, a polypeptide or a carboxyl-terminal modification group.

In some embodiments, the non-naturally encoded amino acid comprises an aminooxy group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazide group. In some embodiments, the non-naturally encoded amino acid comprises a hydrazine group. In some embodiments, the non-naturally encoded amino acid residue comprises a semicarbazide group.

In some embodiments, the non-naturally encoded amino acid residue comprises an azide group. In some embodiments, the non-naturally encoded amino acid has the structure,

wherein n is 0-10; R is _alkyl , aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is _0-10 ; R is H, an amino acid, a polypeptide, or an amino-terminal modification group, and _R is H, an amino acid, a polypeptide, or a carboxyl-terminal modification group.

In some embodiments, the non-naturally encoded amino acid comprises an alkynyl group. In some embodiments, the non-naturally encoded amino acid has the structure,

wherein n is 0-10; R is _alkyl , aryl, substituted alkyl or substituted aryl; X is O, N, S or absent; m is _0-10 , R is H, Amino acid, polypeptide or amino terminal modification group, and R ₃ is H, amino acid, polypeptide or carboxy terminal modification group.

In some embodiments, the polypeptide is a GH (eg, hGH) polypeptide agonist, partial agonist, antagonist, partial antagonist, or inverse agonist. In some embodiments, a GH (eg, hGH) polypeptide agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises poly(ethylene glycol) moieties. In some embodiments, the GH (e.g., hGH) polypeptide agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-naturally encoded amino acid and one or more post-translational modifications , linkers, polymers or bioactive molecules. In some embodiments, the non-naturally encoded amino acid linked to the water soluble polymer is present in the site II region (comprising the surface of the AC helix bundle, the amino terminal region of helix A, and a portion of helix C) of the GH (e.g., hGH) polypeptide. protein region). In some embodiments, a GH (e.g., hGH) polypeptide comprising a non-naturally encoded amino acid linked to a water soluble polymer acts by preventing a GH (e.g., hGH) polypeptide antagonist from interacting with a second GH (e.g., hGH) polypeptide. The receptor molecules bind to prevent dimerization of the GH (eg, hGH) polypeptide receptor. In some embodiments, G120 in SEQ ID NO: 2 (hGH) is substituted with an amino acid other than glycine. In some embodiments, G120 in SEQ ID NO: 2 is replaced with arginine. In some embodiments, G120 in SEQ ID NO: 2 is substituted with a non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid linked to the water soluble polymer is present within the receptor binding region of the GH (eg, hGH) polypeptide or interferes with receptor binding of the GH (eg, hGH) polypeptide.

The invention also provides an isolated nucleic acid comprising a polynucleotide that hybridizes under stringent conditions to SEQ ID NO: 21 or 22, wherein the polynucleotide comprises at least one selector codon. In some embodiments, the selector codon is selected from the group consisting of amber codons, ochre codons, opal codons, single codons, rare codons, pentabase codons and four base codons.

The invention also provides methods of making GH (eg, hGH) polypeptides linked to water-soluble polymers. In some embodiments, the methods comprise contacting an isolated GH (eg, hGH) polypeptide comprising a non-naturally encoded amino acid with a water soluble polymer comprising a moiety reactive with the non-naturally encoded amino acid. In some embodiments, a non-naturally encoded amino acid incorporated into a GH (e.g., hGH) polypeptide is reactive to a water soluble polymer that is otherwise reactive to any of the 20 common amino acids. Reactive. In some embodiments, a non-naturally encoded amino acid incorporated into a GH (e.g., hGH) polypeptide is reactive to a linker, polymer, or biologically active molecule that is additionally reactive to 20 any of the common amino acids without reactivity.

In some embodiments, the water-soluble GH (e.g., hGH) polypeptide is prepared by reacting a GH (e.g., hGH) polypeptide comprising a carbonyl-containing amino acid with a poly(ethylene glycol) molecule comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group. A GH (eg, hGH) polypeptide linked to a sexual polymer. In some embodiments, the aminooxy, hydrazino, hydrazide, or semicarbazide group is attached to the poly(ethylene glycol) molecule through an amide bond.

In some embodiments, the water soluble polymeric compound is prepared by reacting a poly(ethylene glycol) molecule comprising a carbonyl group with a polypeptide comprising a non-naturally encoded amino acid comprising an aminooxy, hydrazine, hydrazide, or semicarbazide group. A GH (eg, hGH) polypeptide linked to a substance.

In some embodiments, the GH (e.g., hGH) polypeptide linked to a water soluble polymer is prepared by reacting a GH (e.g., hGH) polypeptide comprising an alkynyl-containing amino acid with a poly(ethylene glycol) molecule comprising an azide moiety. hGH) polypeptide. In some embodiments, the azido or alkynyl group is attached to the poly(ethylene glycol) molecule through an amide bond.

In some embodiments, the GH (e.g., hGH) polypeptide linked to a water-soluble polymer is prepared by reacting a GH (e.g., hGH) polypeptide comprising an azide-containing amino acid with a poly(ethylene glycol) molecule comprising an alkyne moiety. hGH) polypeptide. In some embodiments, the azido or alkynyl group is attached to the poly(ethylene glycol) molecule through an amide bond.

In some embodiments, the poly(ethylene glycol) molecule has a molecular weight between about 0.1 kDa and about 100 kDa. In some embodiments, the poly(ethylene glycol) molecule has a molecular weight between 0.1 kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecules are branched polymers. In some embodiments, each arm of the poly(ethylene glycol) branched polymer has a molecular weight of between 1 kDa and 100 kDa or between 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the GH (eg, hGH) polypeptide comprises a polyalkylene glycol moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into a GH (eg, hGH) polypeptide comprises a carbonyl, aminooxy, hydrazide, hydrazine, semicarbazide, azido, or alkynyl group. In some embodiments, the non-naturally encoded amino acid residue incorporated into a GH (eg, hGH) polypeptide comprises a carbonyl moiety and the water soluble polymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into a GH (eg, hGH) polypeptide comprises an alkyne moiety and the water soluble polymer comprises an azide moiety. In some embodiments, the non-naturally encoded amino acid residue incorporated into a GH (eg, hGH) polypeptide comprises an azide moiety and the water soluble polymer comprises an alkyne moiety.

The invention also provides compositions comprising a GH (eg, hGH) polypeptide comprising a non-naturally encoded amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer.

The invention also provides cells comprising a polynucleotide encoding a GH (eg, hGH) polypeptide, the polynucleotide comprising a selector codon. In some embodiments, the cells comprise an orthogonal RNA synthetase and/or an orthogonal tRNA for substituting a non-naturally encoded amino acid into a GH (eg, hGH) polypeptide.

The invention also provides methods of making GH (eg, hGH) polypeptides comprising non-naturally encoded amino acids. In some embodiments, the method comprises culturing a cell comprising a polynucleotide encoding a GH (e.g., hGH) polypeptide, an orthogonal RNA synthetase, and/or an orthogonal tRNA under conditions to allow GH (e.g., hGH) hGH) polypeptide expression; and purifying the GH (eg, hGH) polypeptide from the cells and/or culture medium.

The invention also provides methods of increasing the therapeutic half-life, serum half-life, or circulation time of a GH (eg, hGH) polypeptide. The invention also provides methods of modulating the immunogenicity of GH (eg, hGH) polypeptides. In some embodiments, the methods comprise substituting a non-naturally encoded amino acid for any one or more amino acids in a naturally occurring GH (e.g., hGH) polypeptide and/or linking the GH (e.g., hGH) polypeptide to molecules, polymers, water-soluble polymers or bioactive molecules.

The invention also provides methods of treating a patient in need of such treatment with an effective amount of a GH (eg, hGH) molecule of the invention. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a GH (e.g., hGH) polypeptide and a pharmaceutically acceptable carrier, the GH (e.g., hGH) Polypeptides comprise non-naturally encoded amino acids. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer.

The invention also provides GH (e.g., hGH) polypeptides comprising the sequence set forth in SEQ ID NO: 1, 2, 3, or any other GH polypeptide sequence except that at least one amino acid is substituted with a non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises poly(ethylene glycol) moieties. In some embodiments, the non-naturally encoded amino acid comprises a carbonyl, aminooxy, hydrazide, hydrazine, semicarbazide, azido, or alkynyl group. In some embodiments, a non-naturally encoded amino acid is substituted at a position selected from the group consisting of each of the following residue positions: residues 1-5, 82-90, 117 from SEQ ID NO: 3 (hGH) -134 and 169-176.

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a GH (e.g., hGH) polypeptide comprising the sequence shown in SEQ ID NO: 1, 2, 3 Or any other GH polypeptide sequence wherein at least one amino acid is substituted by a non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid comprises a carbohydrate moiety. In some embodiments, the water soluble polymer is linked to the polypeptide through a carbohydrate moiety. In some embodiments, the linker, polymer or biologically active molecule is linked to the GH (eg, hGH) polypeptide through a carbohydrate moiety.

The invention also provides GH (eg, hGH) polypeptides comprising a water-soluble polymer covalently linked to the GH (eg, hGH) polypeptide at a single amino acid. In some embodiments, the water soluble polymer comprises poly(ethylene glycol) moieties. In some embodiments, the amino acid covalently linked to the water soluble polymer is a non-naturally encoded amino acid present in the polypeptide. In some embodiments, a non-naturally encoded amino acid is substituted at position 35, 92, 143, or 145 of SEQ ID NO 2.

The invention provides GH (e.g., hGH) polypeptides comprising at least one linker, polymer, or biologically active molecule, wherein the linker, A polymer or biologically active molecule is attached to the polypeptide. In some embodiments, the polypeptide is mono-PEGylated. The invention also provides GH (e.g., hGH) polypeptides comprising a linker, polymer, or biologically active molecule linked to one or more non-naturally encoded amino acids, wherein ribosomal action renders the A non-naturally encoded amino acid is incorporated into the polypeptide.

In another embodiment, conjugation of a hGH polypeptide comprising one or more non-naturally occurring amino acids to another molecule including, but not limited to, PEG provides a unique chemical reaction utilized for conjugation to the non-natural amino acid. Substantially pure hGH. Conjugation of hGH comprising one or more non-naturally encoded amino acids to another molecule such as PEG can be performed with other purification techniques performed before or after the conjugation step to provide substantially pure hGH.

The present invention further provides a hormone composition comprising growth hormone (GH) linked by a covalent bond to at least one water-soluble polymer, wherein said covalent bond is an oxime bond. In some embodiments, the GH is human growth hormone (hGH), such as a sequence at least about 80% identical to SEQ ID NO:2; in some embodiments, the sequence is the sequence of SEQ ID NO:2. The GH may comprise one or more non-naturally encoded amino acids (NEAAs), such as NEAAs comprising a carbonyl group (eg, a keto group), such as NEAA which is p-acetylphenylalanine. In some embodiments, the oxime linkage is between NEAA and the water soluble polymer. The GH may be substituted with p-acetylphenylalanine at a position corresponding to position 35 of SEQ ID NO:2. In some embodiments, the water soluble polymer includes one or more polyethylene glycol (PEG) molecules. The PEG can be linear, eg, a linear PEG having a molecular weight between about 0.1 kDa and about 100 kDa, or between about 1 kDa and about 60 kDa, or between about 20 kDa and about 40 kDa, or about 30 kDa. In some embodiments, the PEG is a branched PEG, eg, a branched PEG having a molecular weight between about 1 kDa and about 100 kDa, or between about 30 kDa and about 50 kDa, or about 40 kDa. In some embodiments, the GH is linked to the water-soluble polymers through a plurality of covalent bonds, at least one of which is an oxime bond. In some of these embodiments, the GH is human growth hormone (GH, e.g., hGH), e.g., a GH having a sequence at least about 80% identical to SEQ ID NO: 2 (e.g., hGH); in some The sequence described in the embodiments is the sequence of SEQ ID NO:2. In some embodiments where the GH (eg, hGH) is linked to a plurality of water soluble polymers, the GH comprises a plurality of NEAAs.

In some embodiments, the invention provides a GH composition comprising a GH (e.g., hGH) comprising the sequence of SEQ ID NO: 2, wherein the GH (e.g., hGH) is linked to a 30 kDa linear PEG via an oxime bond, and Wherein the oxime bond is formed with p-acetylphenylalanine substituted at a position corresponding to position 35 of SEQ ID NO:2.

In some embodiments, the present invention provides a hormonal composition comprising GH (e.g., hGH) linked by an oxime bond to at least one linear PEG, wherein the GH (e.g., hGH) comprises SEQ ID NO: 2 amino acid sequence and contain at least one NEAA substituted at one or more positions selected from the group consisting of the following respective residue positions: residues 1-5, 6-33, 34-74, 75-96, 97-105 , 106-129, 130-153, 154-183, and 184-191. In some embodiments, NEAA is substituted at one or more positions selected from the group consisting of each of the following residue positions: before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5 , 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103 ,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136 ,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172 , 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192 (ie, at the carboxyl terminus of the protein). In some embodiments, NEAA is substituted at one or more positions selected from the group consisting of residue positions 35, 92, 131, 134, 143, and 145. In some embodiments, NEAA is substituted at one or more positions selected from the group consisting of residue positions 30, 35, 74, 92, 103, 143, and 145. In some embodiments, NEAA is substituted at one or more positions selected from the group consisting of residue positions 35, 92, 143, and 145. In some embodiments, position 35 is substituted with NEAA. In some embodiments, at least one NEAA is p-acetylphenylalanine. In some embodiments, the PEG has a molecular weight of between about 0.1 kDa and about 100 kDa, or between about 1 kDa and about 60 kDa, or between about 20 kDa and about 40 kDa, or about 30 kDa.

In other embodiments, the present invention provides a method of preparing a GH (e.g., hGH) linked to a water-soluble polymer via an oxime linkage, comprising making a GH (e.g., hGH) comprising a carbonyl-containing NEAA in a suitable Contact with PEG hydroxylamine under conditions that form an oxime bond. The NEAA may contain a keto group, eg, a carbonyl group. NEAA may be p-acetylphenylalanine. In some embodiments that contain acetylphenylalanine, the acetylphenylalanine is substituted at a position in GH (e.g., hGH) corresponding to amino acid 35 in SEQ ID NO: 2. In some embodiments, the PEG hydroxylamine is monomethoxy PEG (MPEG) hydroxylamine. In some embodiments, the MPEG hydroxylamine is linear, eg, about 20-40 kDa or about 30 kDa linear MPEG. In some embodiments, the MPEG hydroxylamine is a linear 30 kDa monomethoxy-PEG-2-aminooxyethylamine carbamate hydrochloride. In some embodiments, produced by introducing (i) a nucleic acid encoding GH (e.g., hGH), wherein the nucleic acid has been modified to provide a selector codon for NEAA incorporation, and (ii) NEAA into an organism GH (eg, hGH) comprising NEAA, the cellular machinery of the organism is capable of incorporating NEAA into proteins in response to (i) a selector codon of the nucleic acid. In some embodiments, the reaction conditions to form an oxime bond comprise mixing MPEG with GH (e.g., hGH) to produce an MPEG-GH (e.g., hGH) mixture, wherein the ratio of MPEG:GH (e.g., hGH) is from about 5 to 10, a pH of about 4 to 6; and gently stirring the MPEG-GH (eg, MPEG-hGH) mixture at room temperature for about 10 to 50 hours. In some embodiments, the method further comprises purifying the GH (eg, hGH), eg, to a purity of at least about 99%.

Cellular machinery includes, but is not limited to, orthogonal tRNAs and/or aminoacyl tRNA synthetases.

In other embodiments, the present invention provides a pharmaceutical composition comprising a hormonal composition comprising at least one water-soluble polymer linked by a covalent bond and a pharmaceutically acceptable excipient. Growth hormone, wherein the covalent bond is an oxime bond. In some embodiments, the GH is a GH such as hGH. In some embodiments, the GH comprises NEAA. In some embodiments, the water soluble polymer comprises PEG, such as linear PEG. In some embodiments, PEG is a linear PEG of about 30 kDa, and GH is GH substituted with p-acetylphenylalanine at a position corresponding to amino acid 35 of SEQ ID NO: 2 (e.g., hGH), and An oxime bond is formed between acetylphenylalanine and PEG.

In some embodiments, the present invention provides a method of treatment by administering to an individual in need of treatment an effective amount of a hormonal composition comprising a Growth hormone (GH), wherein the covalent bond is an oxime bond. In some embodiments, GH is hGH. In some embodiments, the GH comprises NEAA. In some embodiments, the water soluble polymer comprises PEG, such as linear PEG. In some embodiments, PEG is a linear PEG of about 30 kDa, and GH is hGH substituted with p-acetylphenylalanine at a position corresponding to amino acid 35 of SEQ ID NO: 2, and at the position of p-acetylphenylalanine Formation of oxime bond with PEG. In some embodiments, the individual treated is a human. In some embodiments, the individual treated has pediatric growth hormone deficiency, idiopathic short stature, childhood-onset adult growth hormone deficiency, adult-onset adult growth hormone deficiency, or secondary growth hormone deficiency Hormone deficiency. In some embodiments, GH is hGH. In some embodiments, the GH comprises NEAA. In some embodiments, the water soluble polymer comprises PEG, such as linear PEG. In some embodiments, the PEG is a linear PEG of about 30 kDa, and the GH (e.g., hGH) is substituted with p-acetylphenylalanine at a position corresponding to amino acid 35 of SEQ ID NO: 2, and the An oxime bond is formed between the amino acid and PEG.

In some embodiments, the present invention provides a method of treatment by administering to an individual in need of treatment an effective amount of a hormonal composition comprising a compound covalently linked to at least one water-soluble polymer. Growth hormone (GH), wherein the water soluble polymer is a linear polymer, and wherein the hormonal composition is administered at a frequency of only once a week, once every two weeks, or once a month. In some embodiments, the polymer is PEG. In some embodiments, the GH comprises NEAA. In some embodiments, the polymer is linked to the GH through an oxime bond.

In some embodiments, the invention provides a hormonal composition comprising GH (eg, hGH), wherein the GH (eg, hGH) has a mean serum half-life of at least about 12 hours when administered subcutaneously to a mammal. In some embodiments, the GH (eg, hGH) comprises NEAA. In some embodiments, the hormonal composition further contains a water-soluble polymer, such as PEG, eg, linear PEG.

In some embodiments, the invention provides a hormonal composition comprising GH (e.g., hGH) linked to PEG, wherein the GH (e.g., hGH) has a mean serum half-life when administered subcutaneously to a mammal of The serum half-life of a composition comprising PEG-free GH (eg, hGH) is at least about 7-fold greater.

Description of drawings

Description of drawings

Figure 1 shows a general structural diagram of a four-helix bundle protein.

Figure 2 shows a general structural diagram of the four-helix bundle protein growth hormone (GH).

Figure 3 shows a general structural diagram of the four-helix bundle protein erythropoietin (EPO).

Figure 4 shows a general structural diagram of the four-helix bundle protein interferon alpha-2 (IFNa-2).

Figure 5 shows a general structural diagram of the four-helix bundle protein granulocyte colony stimulating factor (G-CSF).

Figure 6 shows a Coomassie blue stained SDS-PAGE demonstrating the expression of hGH comprising the non-naturally encoded amino acid p-acetylphenylalanine at each of the following positions: Y35, F92, Y111, G131 , R134, K140, Y143 or K145.

Figure 7, Panel A and Figure 7, Panel B are graphs showing the biological activity of hGH comprising non-naturally encoded amino acids (Panel B) and wild-type hGH (Panel A) in IM9 cells.

Figure 8 shows a Coomassie-stained SDS-PAGE demonstrating the production of hGH comprising non-naturally encoded amino acids to which PEGylated was covalently attached (5 kDa, 20 kDa and 30 kDa).

Figure 9 shows a graph demonstrating the biological activity of various PEGylated forms of hGH comprising non-naturally encoded amino acids in IM9 cells.

Figure 10, Panel A - This figure illustrates the primary structure of hGH with the trypsin cleavage site indicated and arrows illustrating the unnatural amino acid substitution F92pAF (according to Becker et al. Biotechnol Appl Biochem. (1988) 10(4):326 -337 modify the resulting figure). Figure 10, Panel B - shows peptides produced from PEGylated hGH polypeptides comprising non-naturally encoded amino acids (labeled A), peptides produced from hGH polypeptides comprising non-naturally encoded amino acids (labeled B), and peptides produced by WHO rhGH Overlaid tryptic maps of the resulting peptides (labeled C). Figure 10, panel C - shows a magnification of peak 9 from panel B.

Figure 11, panels A and B show Coomassie brilliant blue stained SDS-PAGE analysis of purified PEG-hGH polypeptides.

Figure 12 is a graph showing the biological activity of hGH dimer molecules in IM9 cells.

Figure 13, Panel A - graph showing IM-9 assay data measuring pSTAT5 phosphorylation by hGH antagonists with G120R substitutions. Figure 13, Panel B - graph showing IM-9 assay data measuring phosphorylation of pSTAT5 by a hGH polypeptide with an unnatural amino acid incorporated at the same position (G120).

Figure 14 shows graphs demonstrating that dimers of the hGH antagonists shown in Figure 13, Panel B also lack biological activity in the IM-9 assay.

Figure 15 shows a graph comparing the serum half-life in rats of PEGylated hGH polypeptides comprising non-naturally encoded amino acids with non-PEGylated hGH polypeptides.

Figure 16 shows a graph comparing the serum half-life in rats of PEGylated hGH polypeptides comprising non-naturally encoded amino acids.

Figure 17 shows a graph comparing the serum half-life of PEGylated hGH polypeptides comprising non-naturally encoded amino acids in rats. 2.1 mg/kg was administered once to rats.

Figure 18, Panel A - graph showing the effect on body weight gain in rats following administration of a single dose of PEGylated hGH polypeptide comprising non-naturally encoded amino acids (positions 35, 92). Figure 18, Panel B - graph showing the effect on circulating plasma IGF-1 levels following administration of a single dose of PEGylated hGH polypeptide comprising non-naturally encoded amino acids (positions 35, 92). Figure 18, Panel C - graph showing the effect on body weight gain in rats following administration of a single dose of PEGylated hGH polypeptide comprising non-naturally encoded amino acids (positions 92, 134, 145, 131, 143). Figure 18, Panel D - graph showing the effect on circulating plasma IGF-1 levels following administration of a single dose of PEGylated hGH polypeptide comprising non-naturally encoded amino acids (positions 92, 134, 145, 131, 143). Figure 18, Panel E - graph showing comparison of serum half-life in rats of PEGylated hGH polypeptides comprising non-naturally encoded amino acids (positions 92, 134, 145, 131, 143).

Figure 19 shows a structural diagram of linear, 30 kDa monomethoxy-poly(ethylene glycol)-2-aminooxyethylamine carbamate hydrochloride.

Figure 20 shows a diagram illustrating the synthesis of carbamate-linked oxyamino derivatized PEGs.

Figure 21 presents illustrative, non-limiting examples of PEG-containing reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides.

Figure 22 presents an illustrative, non-limiting example of the synthesis of PEG-containing reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides.

Figure 23 presents an illustrative, non-limiting example of the synthesis of amide-based hydroxylamine-containing PEG reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides.

Figure 24 presents an illustrative, non-limiting example of the synthesis of carbamate-based PEG-containing reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides.

Figure 25 presents an illustrative, non-limiting example of the synthesis of carbamate-based PEG-containing reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides.

Figure 26 presents an illustrative, non-limiting example of the synthesis of simple PEG-containing reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides.

Figure 27 presents illustrative, non-limiting examples of branched PEG-containing reagents that can be used to modify non-natural amino acid polypeptides to form PEG-containing, oxime-linked non-natural amino acid polypeptides and one such reagent to modify a carbonyl-based non-natural amino acid Uses of peptides.

Figure 28 presents graphs illustrating IGF-1 plasma concentrations in hypophysectomized rats treated weekly with placebo or increasing doses of PEG- ^α hGH or daily with placebo or Genotropin.

FIG. 29 presents graphs illustrating tibial length in hypophysectomized rats treated weekly with placebo or PEG- ^α hGH or daily with placebo or glucosamine.

Figure 30 presents a graph illustrating the percent change in body weight in hypophysectomized rats treated weekly with placebo or PEG- ^α hGH or daily with placebo or genpronil.

Figure 31 presents a graph illustrating the plasma concentration versus time of subcutaneously administered PEG- ^α hGH on days 0 and 7.

Figure 32 presents a graph illustrating the plasma concentration versus time of PEG- ^α (met)hGH administered as a single dose subcutaneously or intravenously.

Detailed ways

definition

It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, constructs and reagents described herein and as such may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "hGH" is a reference to one or more such proteins and includes equivalents thereof known to those skilled in the art, and so on.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described.

All publications and patents mentioned herein are incorporated herein by reference to describe and disclose, for example, the constructs and patents described in said publications that might be used in connection with the presently described invention. The purpose of the method. The publications discussed herein are provided for their disclosure prior to the filing date of the present application only. Nothing herein should be construed as an admission that the inventors are not entitled to antedate the disclosure by reason of prior invention or for any other reason.

The term "substantially purified" refers to a GH (e.g., hGH) polypeptide that, in the case of a recombinantly produced GH (e.g., hGH) polypeptide, is substantially or essentially free of the accompanying A protein in its naturally occurring environment (ie, a natural cell or a host cell) or a component that interacts with said protein. A GH (e.g., hGH) polypeptide that can be substantially free of cellular material includes less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about About 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) protein preparation. When the GH (e.g., hGH) polypeptide or variant thereof is recombinantly produced by a host cell, the protein may be about 30%, about 25%, about 20%, about 15%, about 10%, about 5% of the dry weight of the cell %, about 4%, about 3%, about 2%, or about 1% or less. When the GH (e.g., hGH) polypeptide or variant thereof is produced by a host cell by recombinant means, the protein can be about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about An amount of 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weight of cells is present in the medium. Thus, a "substantially purified" GH (e.g., hGH) polypeptide as produced by the methods of the invention may have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least A purity of about 55%, at least about 60%, at least about 65%, at least about 70%, particularly having a purity of at least about 75%, 80%, 85%, and more particularly having a purity of at least about 90% , a purity of at least about 95%, a purity of at least about 99% or greater, as determined by suitable methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

"Recombinant host cell" or "host cell" refers to an exogenous host cell containing inserted regardless of method (e.g., direct uptake, transduction, conjugation, or other methods known in the art to produce recombinant host cells). cells with polynucleotides. The exogenous polynucleotide can be maintained as a non-integrating vector, such as a plasmid, or alternatively, can integrate into the host genome.

The term "medium" as used herein includes any medium, solution, solid, semi-solid or rigid support that can support or contain any host cells and cell contents, including bacterial host cells, Yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli or Pseudomonas host cells. Thus, the term can encompass medium in which the host cell has been grown, eg, medium into which a GH (eg, hGH) polypeptide has been secreted, including medium either before or after the proliferation phase. The term can also encompass a buffer containing a host cell lysate, such as where the GH (e.g., hGH) polypeptide is produced intracellularly and the host cell is lysed or split to release the GH (e.g., hGH) polypeptide or reagents.

A "reducing agent" as used herein in relation to protein refolding is defined as any compound or substance that maintains sulfhydryl groups in a reduced state and reduces intramolecular or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. Those skilled in the art will readily appreciate that a wide variety of reducing agents are suitable for use in the methods and compositions of the invention.

"Oxidizing agent" as used herein in relation to protein refolding is defined as any compound or substance capable of removing electrons from the compound being oxidized. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. It will be readily apparent to those skilled in the art that a wide variety of oxidizing agents are suitable for use in the methods of the invention.

A "denaturant" as used herein is defined as any compound or substance that will cause reversible unfolding of a protein. The strength of the denaturant will be determined by the nature and concentration of the particular denaturant. Suitable denaturants may be chaotropic agents, detergents, organic solvents, water-miscible solvents, phospholipids, or a combination of two or more of these agents. Suitable chaotropic agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium lauryl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), N - Sodium lauryl sarcosinate (sarkosyl), mild nonionic detergents (for example digitonin), mild cationic detergents (such as N->2,3-(dioleoyloxy base)-propyl-N,N,N-trimethylammonium), mild ionic detergents (such as sodium cholate or sodium deoxycholate) or zwitterionic detergents (including but not limited to sulfo Betaine (Zwittergent, zwittergent), 3-(3-cholamidopropyl)dimethylamino-1-propane sulfate (CHAPS) and 3-(3-cholamidopropyl)dimethylamino-2 -Hydroxy-1-propanesulfonate (CHAPSO)). Organic compounds such as acetonitrile, lower alkanols (in particular C _2-4 alkanols such as ethanol or isopropanol) or lower alkanediols (in particular C _2-4 alkanediols such as ethylene glycol) Water-miscible solvents can be used as denaturants. Phospholipids useful in the present invention may be naturally occurring phospholipids (such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol) or synthetic phospholipid derivatives or variants (such as dicaproylphosphatidyl choline or diheptanoylphosphatidylcholine).

"Refolding" as used herein describes any process, reaction or method that converts a disulfide bond-containing polypeptide from an improperly folded or unfolded state to a native or proper folded conformation with respect to disulfide bonds.

"Cofolding" as used herein specifically refers to a refolding process, reaction or method that takes at least two polypeptides that interact with each other and converts an unfolded or improperly folded polypeptide into a native, properly folded polypeptide .

"Somatotropin" or "GH" as used herein, regardless of its biological activity and further regardless of its synthesis or production methods (including but not limited to, in vitro, in vivo by the expression of nucleic acid molecules methods of microinjection to recombine (whether produced from cDNA, genomic DNA, synthetic DNA or from other forms of nucleic acid), synthetic methods, transgenic methods and gene activation methods), which shall include at least one biological activity of growth hormone Those polypeptides and proteins derived from any mammalian animal (including but not limited to human (hGH), bovine (bGH), pig) and other livestock or farm animals (including but not limited to chicken), and Includes its GH analogs, GH isoforms, GH mimetics, GH fragments, hybrid GH proteins, fusion proteins, oligomers and polymers, homologues, glycosylation variants, variants, splice variants bodies and mutant proteins.

The term "hGH polypeptide" encompasses hGH polypeptides comprising one or more amino acid substitutions, additions or deletions. Exemplary substitutions include, for example, substitution of lysine at position 41 or substitution of phenylalanine at position 176 of native hGH. In some cases, the substituent may be an isoleucine or arginine residue if the substitution is at position 41, or a tyrosine residue if position is 176. Position F10 may be substituted with, for example, A, H or I. Position M14 may be substituted with, for example, W, Q or G. Other exemplary substitutions include any substitution comprising, but not limited to, the following substitutions or combinations thereof: R167N, D171S, E174S, F176Y, I179T; R167E, D171S, E174S, F176Y; F10A, M14W, H18D, H21N; F10A, M14W , H18D, H21N, R167N, D171S, E174S, F176Y, I179T; F10A, M14W, H18D, H21N, R167N, D171A, E174S, F176Y, I179T; F10H, M14G, H18N, H21N; , D171A, T175T, I179T; or F10I, M14Q, H18E, R167N, D171S, I179T. See, eg, US Patent No. 6,143,523, which is incorporated herein by reference.

Exemplary substitutions at various amino acid positions in naturally occurring hGH have been described, including substitutions that enhance agonist activity, enhance protease resistance, convert the polypeptide into an antagonist, etc. and are identified by the term "hGH polypeptide" covered.

Agonist GH (eg, hGH) sequences include, for example, naturally occurring hGH sequences comprising the following modifications: H18D, H21N, R167N, D171S, E174S, I179T. See, eg, US Patent No. 5,849,535, which is incorporated herein by reference. Additional agonist hGH sequences include H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; or H18D, Q22A, F25A, D26A, Q29A , E65A, K168A, E174A. See, eg, US Patent No. 6,022,711, which is incorporated herein by reference. hGH polypeptides comprising H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A substitutions increase affinity at site I for the hGH receptor. See, eg, US Patent No. 5,854,026, which is incorporated herein by reference. hGH sequences with enhanced protease resistance include, but are not limited to, hGH polypeptides comprising one or more amino acid substitutions within the C-D loop. In some embodiments, substitutions include, but are not limited to, R134D, T135P, K140A, and any combination thereof. See, eg, Alam et al. (1998) J. Biotechnol. 65:183-190.

Human growth hormone antagonists include, for example, substitutions at G120 (eg, G120R, G120K, G120W, G120Y, G120F, or G120E) and sometimes further include the following substitutions: H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, Those human growth hormone antagonists of E174A. See, eg, US Patent No. 6,004,931, which is incorporated herein by reference. In some embodiments, the hGH antagonist comprises at least one substitution in region 106-108 or 127-129, which allows GH to act as an antagonist. See, eg, US Patent No. 6,608,183, which is incorporated herein by reference. In some embodiments, the hGH antagonist comprises a non-naturally encoded amino acid present in the Site II binding region of the hGH molecule linked to a water soluble polymer. In some embodiments, the hGH polypeptide further comprises the following substitutions: H18D, H21N, R167N, K168A, D171S, K172R, E174S, I179T, and one substitution is at G120 (see, eg, US Pat. No. 5,849,535).

For the complete full-length naturally occurring human GH amino acid sequence as well as the mature naturally occurring GH amino acid sequence and naturally occurring mutants, see herein, respectively, SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, a GH polypeptide (e.g., a hGH polypeptide) of the invention is substantially identical to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or any other sequence of a growth hormone polypeptide. For example, in some embodiments, a GH polypeptide (e.g., hGH polypeptide) of the invention has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% %, at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or any other sequence of a growth hormone polypeptide. In some embodiments, the GH polypeptides (e.g., hGH polypeptides) of the invention have at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 2. A large number of naturally occurring hGH mutants have been identified. These mutants include hGH-V (Seeburg, DNA 1:239 (1982); U.S. Patent Nos. 4,446,235, 4,670,393, and 4,665,180, which are incorporated herein by reference) and hGH containing residues 32- 46 deleted 20-kDa hGH (SEQ ID NO:3) (Kostyo et al., Biochem. Biophys. Acta 925:314 (1987); Lewis, U. et al., J. Biol. Chem., 253:2679- 2687 (1978)). Placental growth hormone is described in Igout, A. et al., Nucleic Acids Res. 17(10):3998 (1989). In addition, numerous hGH variants have been reported resulting from post-transcriptional, post-translational, secretion, metabolic processing, and other physiological processes, including proteolytic cleavage variants or 2-chain variants (Baumann, G., Endocrine Reviews 12: 424 (1991)). Cys- hGH dimer directly linked by Cys disulfide bonds. Nucleic acid molecules encoding hGH mutants and mutant hGH polypeptides are well known and include, but are not limited to, those described in U.S. Pat. U.S. Patent Application Nos. 5,849,535, 5,854,026, 5,962,411, 5,955,346, 6,013,478, 6,022,711, 6,136,563, 6,143,523, 6,428,954, 6,451,561, 6,780, and 0, /0153003, which references are incorporated herein by reference.

Commercial preparations of hGH are sold under the names: Humatrope ^™ (Eli Lilly & Co.), Nutropin ^™ (Genentech), Norditropin ^™ (Novo-Nordisk), Genotropin ^™ (Pfizer) and Saizen/Serostim ^™ (Serono).

The term "hGH polypeptide" also includes pharmaceutically acceptable salts and prodrugs of naturally occurring hGH and prodrugs of salts, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants and stereoisomers variants, as well as naturally occurring hGH agonist, mimetic and antagonist variants and polypeptide fusions thereof. Fusions comprising additional amino acids at the amino-terminus, carboxy-terminus or both are encompassed by the term "hGH polypeptide". Exemplary fusions include, but are not limited to, for example, methionyl somatotropin (wherein methionine is linked to the N-terminus of hGH produced by recombinant expression of a mature form of hGH lacking a secretion signal peptide or a portion thereof ), fusions for purification purposes (including, but not limited to, fusions to polyhistidine or affinity epitopes), fusions to serum albumin-binding peptides, and fusions to serum proteins such as , serum albumin) fusions. US Patent No. 5,750,373, incorporated herein by reference, describes a method for selecting novel protein (such as growth hormone) and antibody fragment variants that have altered binding properties for their corresponding receptor molecules. The method comprises fusing a gene encoding a protein of interest to the carboxy-terminal domain of the gene III coat protein of filamentous bacteriophage M13.

Various references disclose modification of polypeptides by polymer conjugation or glycosylation. The term "hGH polypeptide" includes one or more additional derivatized forms of the polypeptide bound to a polymer such as PEG and which may contain cysteine, lysine or other residues. In addition, the hGH polypeptide may comprise a linker or polymer, wherein the amino acid to which the linker or polymer is bound may be an unnatural amino acid according to the invention, or may be made using techniques known in the art (such as combining lysine acid or cysteine coupling) to naturally encoded amino acids.

Polymer conjugation of hGH polypeptide has been reported. See, eg, US Patent Nos. 5,849,535, 6,136,563, and 6,608,183, which are incorporated herein by reference. US Patent No. 4,904,584 discloses PEGylated lysine-deleted polypeptides in which at least one lysine residue has been deleted or substituted with any other amino acid residue. WO 99/67291 discloses a method of binding a protein to PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to effect binding to the protein. WO99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily in which cysteine residues have been replaced by non-essential amino acid residues located in designated regions of the polypeptide. WO 00/26354 discloses a method of producing glycosylated polypeptide variants with reduced allergenicity comprising at least one additional glycosylation when compared to the corresponding parent polypeptide parts. US Patent No. 5,218,092, incorporated herein by reference, discloses modifications of granulocyte colony stimulating factor (G-CSF) and other polypeptides to introduce at least one additional carbohydrate chain when compared to the native polypeptide.

The term "hGH polypeptide" also includes glycosylated hGH as well as, but not limited to, polypeptides glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered biologically active variants of the hGH polypeptide. In addition, splice variants are also included. The term "hGH polypeptide" also includes any one or more GH (e.g., hGH) polypeptides or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, chemically linked or expressed as a fusion protein, GH (e.g., hGH) polypeptide heterodimers, homodimers, heteromultimers or homomultimers of ligands or other biologically active molecules of any type, and containing, for example, specific deletions or Other modified polypeptide analogs that retain biological activity.

All references to amino acid positions in GH (e.g., hGH) described herein are based on SEQ ID NO: 1, 3, or other hGH sequences unless otherwise stated (i.e., when it is stated that the comparison is based on SEQ ID NO: 1, 3 or other hGH sequences). NO: Position in 2. Those skilled in the art will recognize that amino acid positions corresponding to positions in SEQ ID NO: 1, 2, 3, or any other GH sequence may be fused to any other GH (e.g., hGH) molecule, such as, GH, or hGH species, variants, fragments, etc.) are easily identified. For example, sequence alignment programs such as BLAST can be used to align and identify specific positions in proteins that correspond to positions in SEQ ID NO: 1, 2, 3 or other GH sequences. Amino acid substitutions, deletions or additions described herein with respect to SEQ ID NO: 1, 2, 3 or other GH sequences are also intended to refer to GH or hGH fusions, variations described herein or known in the art Substitutions, deletions or additions at the corresponding positions in variants, fragments, etc., are clearly covered by the present invention.

The term "hGH polypeptide" or "hGH" encompasses hGH polypeptides comprising one or more amino acid substitutions, additions or deletions. The hGH polypeptides of the invention may comprise modifications with one or more natural amino acids in combination with one or more unnatural amino acids. Exemplary substitutions at various amino acid positions in naturally occurring hGH polypeptides have been described, including, but not limited to, modulating one or more biological activities of hGH polypeptides such as, but not limited to ) that enhance agonist activity, increase polypeptide solubility, decrease protease sensitivity, convert polypeptide into an antagonist, etc.), and the term "hGH polypeptide" encompasses such exemplary substitutions.

Human GH antagonists include, but are not limited to, those at positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, Substitutions at 119, 120, 123 and 127 or additions at position 1 (i.e. at the N-terminus) or any combination thereof (SEQ ID NO: 2, SEQ ID NO: 1, 3 or the corresponding in any other GH sequence amino acid) antagonists. In some embodiments, the hGH antagonist is at regions 1-5 (N-terminal), 6-33 (helix A), 34-74 (region between helix A and helix B, A-B loop), 75-96 (helix B), 97-105 (region between helix B and helix C, B-C loop), 106-129 (helix C), 130-153 (region between helix C and helix D, C-D loop), 154-183 (Helix D), 184-191 (C-terminus) contains at least one substitution which allows GH to act as an antagonist. In other embodiments, exemplary incorporation sites for non-naturally encoded amino acids include residues within the amino-terminal region of helix A and a portion of helix C. In another embodiment, G120 is substituted with a non-naturally encoded amino acid such as p-azido-L-phenylalanine or O-propargyl-L-tyrosine. In other embodiments, the substitutions listed above are combined with additional substitutions that render the hGH polypeptide an hGH antagonist. For example, a non-naturally encoded amino acid is substituted at one of the positions identified herein, and a substitution is simultaneously introduced at G120 (eg, G120R, G120K, G120W, G120Y, G120F, or G120E). In some embodiments, the hGH antagonist comprises a non-naturally encoded amino acid present in the receptor binding region of the hGH molecule linked to a water soluble polymer.

In some embodiments, the GH (eg, hGH) polypeptide further comprises an addition, substitution or deletion that modulates the biological activity of the GH or hGH polypeptide. For example, an addition, substitution or deletion can modulate one or more properties or activities of a GH (eg, hGH). For example, additions, substitutions or deletions can modulate affinity for GH (e.g., hGH) polypeptide receptors, modulate (including but not limited to, enhance or decrease) receptor dimerization, stabilize receptor dimers, circulating half-life, Modulating therapeutic half-life, modulating polypeptide stability, modulating cleavage by proteases, modulating dosage, modulating release or bioavailability, facilitating purification, or improving or altering a particular route of administration. Likewise, a GH (e.g., hGH) polypeptide may comprise protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to, FLAG or polyhistidine) or other affinity-based sequences (including but not limited to FLAG, polyhistidine, GST, etc.) or linker molecules (including but not limited to biotin).

The term "hGH polypeptide" also encompasses linked homodimers, heterodimers, homomultimers and heteromultimers, including, but not limited to, directly through non-naturally encoded amino acid side chains with the same Those homodimers, heterodimers, homomultimers and heteromultimers linked to or to different non-naturally encoded amino acid side chains or to naturally encoded amino acid side chains or indirectly through linkers Polymer. Exemplary linkers include, but are not limited to, small organic compounds, water soluble polymers of various lengths such as poly(ethylene glycol) or polydextran, or polypeptides of various lengths.

"Non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid" and "non-naturally occurring amino acid." The term "non-naturally encoded amino acid" also includes, but is not limited to, modification (e.g., translation Amino acids that occur as a result of post-modification) but are not themselves naturally incorporated into the growing polypeptide chain by the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine.

"Amino-terminal modification group" refers to any molecule that can be attached to the amino-terminus of a polypeptide. Likewise, a "carboxy-terminal modification group" refers to any molecule that can be attached to the carboxy-terminus of a polypeptide. Terminal modification groups include, but are not limited to, various water soluble polymers, peptides or proteins such as serum albumin or other moieties that extend the serum half-life of the peptide.

The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive moiety" are used in the art and in is used herein to refer to a distinct, identifiable portion or unit of a molecule. The term has somewhat the same meaning in the chemical arts and is used herein to denote a portion of a molecule that performs some function or has some activity and is readily reactive with other molecules.

The term "bond" or "linker" is used herein to refer to a group or bond, usually formed as a result of a chemical reaction, and usually a covalent bond. A hydrolytically stable bond means a bond that is substantially stable in water and does not react with water for an extended period of time, perhaps even indefinitely, at useful pH values, including but not limited to, under physiological conditions. . A hydrolytically labile or degradable bond means a bond that is degradable in water or in an aqueous solution including, for example, blood. An enzymatically labile or degradable bond means a bond that can be degraded by one or more enzymes. As understood in the art, PEG and related polymers may include degradable linkages in the polymer backbone or in linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule . For example, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on bioactive agents typically hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of amines with aldehydes; phosphate linkages formed by the reaction of alcohols with phosphate groups; the reaction product of hydrazides with aldehydes hydrazone linkages; acetal linkages, which are the reaction products of aldehydes and alcohols; orthoester linkages, which are the reaction products of formate esters and alcohols; through amine groups including, but not limited to, at the end of polymers such as PEG peptide bonds to carboxyl groups of peptides; and oligonucleotide bonds to 5' hydroxyl groups of oligonucleotides through including, but not limited to, phosphoramidite groups at polymer termini.

The term "bioactive molecule", "bioactive moiety" or "bioactive agent" when used herein means any physical or biochemical property that can affect a biological system, pathway, molecule or interact with an organism (including but not limited to) viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals and humans) related to the interaction of any substance. In detail, "biologically active molecules" as used herein include, but are not limited to, molecules intended to diagnose, cure, alleviate, treat or prevent disease in humans or other animals or otherwise enhance the physical or mental health of humans or animals. any substance in good condition. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleosides Acids, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of bioactive agents suitable for use in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antineoplastic agents , cardiovascular agents, anxiolytics, hormones, growth factors, steroidal agents, toxins derived from microorganisms, etc.

"Bifunctional polymer" refers to a polymer comprising two discrete functional groups capable of reacting specifically with other moieties, including but not limited to amino acid side groups, to form covalent or non-covalent bonds. A bifunctional linker having one functional group reactive with a group on a particular bioactive component and another group reactive with a group on a second biological component can be used to form a complex comprising a first bioactive component, A combination of a bifunctional linker and a second biologically active component. Many procedures and linker molecules are known for linking various compounds to peptides. See, eg, European Patent Application No. 188,256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, and 4,569,789, which are incorporated herein by reference. "Multifunctional polymer" refers to a polymer comprising two or more discrete functional groups capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent bonds or non- covalent bond. A bifunctional or multifunctional polymer can be of any desired length or molecular weight, and can be selected to provide a particular desired spacing or conformation between one or more molecules attached to a GH (eg, hGH) molecule.

Where a substituent is specified by its conventional chemical formula written from left to right, it also covers chemically identical substituents that would result from writing the structure from right to left, for example, the structure _-CH2O- is equivalent to in the structure -OCH ₂ -.

The term "substituent" includes, but is not limited to, "non-interfering substituents.""Non-interferingsubstituents" are those groups that result in stable compounds. Suitable non-interfering substituents or groups include, but are not limited to, halo, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C ₁ -C ₁₀ alkoxy C ₁ -C ₁₂ aralkyl, C ₁ -C ₁₂ alkaryl, C ₃ -C ₁₂ cycloalkyl, C ₃ -C ₁₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl , xylyl, biphenyl, C ₂ -C ₁₂ alkoxyalkyl, C ₂ -C ₁₂ alkoxyaryl, C ₇ -C ₁₂ aryloxyalkyl, C ₇ -C ₁₂ oxyaryl group, C ₁ -C ₆ alkylsulfinyl group, C ₁ -C ₁₀ alkylsulfonyl group, -(CH ₂ ) _m -O-(C ₁ -C ₁₀ alkyl) (where m is 1 to 8), Aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclyl, substituted heterocyclyl, nitroalkyl, _-NO2 , -CN, -NRC(O)-( C ₁ -C ₁₀ alkyl), -C(O)-(C ₁ -C ₁₀ alkyl), C ₂ -C ₁₀ alkylsulfanyl, -C(O)O-(C ₁ -C ₁₀ alkane radical), -OH, -SO ₂ , =S, -COOH, -NR ₂ , carbonyl, -C(O)-(C ₁ -C ₁₀ alkyl)-CF ₃ , -C(O)-CF ₃ , -C(O)NR ₂ , -(C ₁ -C ₁₀ aryl)-S-(C ₆ -C ₁₀ aryl), -C(O)-(C ₁ -C ₁₀ aryl), -(CH ₂ ) _m -O-(CH ₂ ) _m -O-(C ₁ -C ₁₀ alkyl) (wherein each m is 1 to 8), -C(O)NR ₂ , -C(S)NR ₂ , - SO ₂ NR ₂ , -NRC(O)NR ₂ , -NRC(S)NR ₂ , groups of salts thereof, and the like. As used herein, each R is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl or alkaryl.

The term "halogen" includes fluorine, chlorine, iodine and bromine.

The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, which may be fully saturated, monounsaturated or polyunsaturated and may include groups having the specified number of carbon atoms ( That is, C ₁ -C ₁₀ means divalent and polyvalent groups of 1 to 10 carbon atoms), linear or branched or cyclic hydrocarbon groups or combinations thereof. Examples of saturated hydrocarbon groups include, but are not limited to, the following groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl ) methyl, cyclopropylmethyl, (eg) n-pentyl, n-hexyl, n-heptyl, n-octyl homologues and isomers, and the like. An unsaturated alkyl group is a group having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, butenyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3 -(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl and higher homologues and isomers. Unless otherwise stated, the term "alkyl" is also intended to include those alkyl derivatives defined in more detail below, such as "heteroalkyl". Alkyl groups limited to hydrocarbyl groups are referred to as "pure alkyl groups".

The term "hydrocarbylene", by itself or as part of another substituent, means derived from an alkane as follows, but not limited to, represented by the structures -CH ₂ CH ₂ - and -CH ₂ CH ₂ CH ₂ CH ₂ - Exemplary divalent groups also include those described below as "heteroalkylene groups". Typically, the alkyl (or hydrocarbylene) group will have from 1 to 24 carbon atoms, with those groups having 10 carbon atoms or fewer are particular embodiments of the methods and compositions described herein. "Lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, typically having 8 or fewer carbon atoms.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense and refer to the connection between a molecule through an oxygen, amino or sulfur atom, respectively. Those alkyl groups to which the rest are attached.

The term "heteroalkyl", by itself or in combination with another term, unless otherwise stated, means comprising a defined number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S A stable linear or branched or cyclic hydrocarbon group or a combination thereof, wherein the nitrogen and sulfur atoms are optionally oxidized and nitrogen heteroatoms are optionally quaternized. The heteroatoms O, N and S and Si can be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH=CH-O-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ and -CH=CH-N(CH ₃ )-CH ₃ . Up to two heteroatoms can be consecutive, such as -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . Similarly, the term "heteroalkylene", by itself or as part of another substituent, means a heteroalkyl group derived from (but not limited to) the group consisting of _-CH2 - _CH2 -S- _CH2 -CH ₂ - and -CH ₂ -S-CH ₂ -CH ₂ -NH-CH ₂ - are exemplified divalent groups. For heteroalkylene groups, the same or different heteroatoms may also be located at either or both ends of the chain (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the direction in which the formula of the linking group is written does not imply the orientation of the linking group. For example, the formula -C(O) ₂ R'- represents -C(O) ₂ R'- and -R'C(O) ₂ -.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, unless otherwise indicated, mean cyclic versions of "alkyl" and "heteroalkyl", respectively. Thus, a cycloalkyl or heterocycloalkyl group can include saturated, partially unsaturated, and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can be located at the position at which the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-indole Linyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, etc. . Additionally, the term encompasses bicyclic and tricyclic structures. Similarly, the term "heterocycloalkylene", by itself or as part of another substituent, means a divalent radical derived from heterocycloalkyl, and the term "cycloalkylene", by itself or as A part of another substituent means a divalent group derived from a cycloalkyl group.

The term "water-soluble polymer" as used herein refers to any polymer that is soluble in an aqueous solvent. Linkage of a water soluble polymer to a GH (e.g., hGH) polypeptide can result in changes including, but not limited to, increased or modulated serum half-life, or increased or modulated therapeutic half-life, relative to the unmodified form, immunogen Properties are modulated, such as the physical association properties of aggregate and multimer formation, altered receptor binding, and altered receptor dimerization or multimerization. The water soluble polymer may or may not have its own biological activity, and may be used as a linker to link GH (e.g., hGH) with other substances, including but not limited to, one or more GH (eg, hGH) polypeptide or one or more biologically active molecules) are linked. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono C ₁ -C ₁₀ alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714, described patent incorporated herein by reference), monomethoxypolyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl) - Methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxypropylene/ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides , polysaccharides, cellulose and cellulose derivatives (including but not limited to methylcellulose and carboxymethylcellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, polyalkylene glycols Copolymers of alkyl glycols and their derivatives, polyvinyl ether and α-β-poly[(2-hydroxyethyl)-DL-asparagine, etc. or mixtures thereof. Examples of such water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

The term "polyalkylene glycol" or "poly(olefin glycol)" as used herein refers to polyethylene glycol (poly(ethylene glycol)), polypropylene glycol, polytetramethylene glycol and derivatives thereof . The terms "polyalkylene glycol" and/or "polyethylene glycol" encompass linear and branched polymers and have an average molecular weight between 0.1 kDa and 100 kDa. For example, other exemplary embodiments are listed in commercial supplier catalogs such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).

Unless otherwise stated, the term "aryl" means a polyunsaturated aromatic hydrocarbon substituent which may be a single ring or multiple rings (including but not limited to 1 to 3 rings) fused together or linked covalently ). The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 4 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally oxidized. Quaternization. A heteroaryl can be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazole Base, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazole Base, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl , 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolinyl, 5-isoquinolinyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl and 6-quinolinyl. Substituents for each of the aforementioned aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For convenience, the term "aryl" when used in combination with other terms including, but not limited to, aryloxy, arylthiooxy, arylalkyl includes aryl and heteroaryl as defined above. Thus, the term "arylalkyl" is intended to include those groups in which an aryl is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl, etc.), which includes carbon atoms ( including, but not limited to, methylene) has been, for example, an oxygen atom (including, but not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propane groups, etc.) replaced by those alkyl groups.

Each of the above terms (including, but not limited to, "alkyl", "heteroalkyl", "aryl" and "heteroaryl") is intended to include both substituted and unsubstituted forms of the indicated groups. Exemplary substituents for each group are provided below.

Alkyl and heteroalkyl (including those groups commonly referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) The substituents of can be one or more groups selected from (but not limited to) the following groups: -OR', =O, =NR', =N-OR', -NR'R",-SR', -halogen, -SiR'R"R, -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R", -OC(O )NR'R", -NR"C(O)R', -NR'-C(O)NR"R, -NR"C(O) ₂ R', -NR-C(NR'R"R )=NR"", -NR-C(NR'R")=NR, -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R", - NRSO ₂ R', -CN and -NO ₂ vary in number from 0 to (2m'+1), where m' is the total number of carbon atoms in the group. R', R", R'', and R"" each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to, via 1-3 halogen atom substituted aryl), substituted or unsubstituted alkyl, alkoxy or thioalkoxy or arylalkyl. For example, when the compound of the present invention includes multiple R groups, As each of the R', R", R'', and R"" groups is independently selected when present, each R group is also independently selected. When R' and R" are attached to the same nitrogen atom, they may combine with said nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but not limited to ) 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will appreciate that the term "alkyl" is intended to include groups comprising carbon atoms bonded to non-hydrogen groups, such as haloalkyl (including but not limited to) _-CF3 and _-CH2CF3 ) and acyl groups (including _but not limited to -C(O) _CH3 , -C(O) _CF3 , -C(O) _{CH2OCH3 ,} etc.).

Similar to the substituents described for alkyl groups, substituents for aryl and heteroaryl groups can vary and are selected from, but not limited to, the following groups: halogen, -OR', =O, =NR', =N -OR', -NR'R", -SR', -halogen, -SiR'R"R', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R",-OC(O)NR'R",-NR"C(O)R',-NR'-C(O)NR"R,-NR"C(O) ₂ R', -NR- C(NR'R"R)-NR"", -NR-C(NR'R")=NR, -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R", -NRSO ₂ R', -CN and -NO ₂ , -R', -N ₃ , -CH(Ph) ₂ , fluoro(C ₁ -C ₄ )alkoxy and fluoro( C ₁ -C ₄ ) alkyl in numbers ranging from 0 to the total number of valences available on the aromatic ring system; and wherein R', R", R'', and R"" are independently selected from hydrogen, alkyl, hetero Alkyl, aryl, and heteroaryl. For example, when a compound of the invention includes multiple R groups, as when multiples of the R', R", R'', and R"" groups are present When each is independently selected, each R group is also independently selected.

The term "modulated serum half-life" as used herein means a positive or negative change in the circulating half-life of modified hGH relative to its unmodified form. Serum half-life was measured by collecting blood samples at various time points after administration of hGH and determining the concentration of the molecule in each sample. The correlation of serum concentration with time allows calculation of serum half-life. An increased serum half-life of at least about two-fold is desired, but a smaller increase may be useful, for example, where it provides a satisfactory dosing regimen or avoids toxic effects. In some embodiments, the elongation is at least about three-fold, at least about five-fold, or at least about ten-fold.

The term "modulated therapeutic half-life" as used herein means a positive or negative change in the half-life of a therapeutically effective amount of modified hGH relative to its unmodified form. Therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. It is hoped that an extended therapeutic half-life will provide a specific beneficial dosing regimen, a specific beneficial total dose, or avoid adverse effects. In some embodiments, the increased therapeutic half-life results from enhanced potency, enhanced or decreased binding of the modified molecule to its target, enhanced or decreased breakdown of the molecule by an enzyme, such as a protease, or another effect of the unmodified molecule. Produced by enhancement (plus) or decrease (minimum) of a parameter or mechanism of action.

The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is free from at least some of the cellular components with which it is naturally associated, or that the nucleic acid or protein has been concentrated to The level of concentration produced in vivo or in vitro. It can be homogeneous. The isolated material may be in a dry or semi-dry state, or in solution, including but not limited to, an aqueous solution. It may be a component of a pharmaceutical composition comprising additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the most predominant species present in the preparation, is substantially purified. Specifically, the isolated gene is separated from open reading frames that flank the gene and encode a protein different from the gene of interest. The term "purified" means that a nucleic acid or protein produces substantially one band in an electrophoretic gel. Specifically, it may mean that the nucleic acid or protein is at least 85%, at least 90%, at least 95%, at least 99% pure or greater.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof, in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs including PNA (peptide nucleic acid), analogs of DNA used in antisense technology (phosphorothioate, phosphoroamidate, etc.) things. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al. People, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)) .

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description for a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. The term as used herein encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine acid, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and pyrrolysine and seleno cysteine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., compounds having an alpha carbon bonded to hydrogen, carboxyl, amino and R groups, such as homoserine, norleucine , Methionine sulfoxide, Methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

Amino acids may be referred to herein by their commonly known 3-letter symbols or by the 1-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, any given protein is encoded by many functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid changes are "static changes," which are conservatively modified changes. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible static variation of the nucleic acid. Those skilled in the art will recognize that individual codons in a nucleic acid (except AUG, which is usually only a codon for methionine, and TGG, which is usually only a codon for tryptophan) can be modified to produce functionally equivalent codons. molecules. Accordingly, various static variations of a nucleic acid encoding a polypeptide are implicit in each sequence described.

With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions or additions to nucleic acid, peptide, polypeptide or protein sequences that alter, add or remove single amino acids or small percentages of amino acids in the encoded sequence are a A "conservatively modified change", wherein the change results in a deletion of an amino acid, an addition of an amino acid, or a substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those of skill in the art. Such conservatively modified variants are variants in addition to and do not exclude polymorphic variants, interspecies homologs and alleles of the invention .

Conservative substitution tables providing functionally similar amino acids are known to those of skill in the art. The following eight groups each contain amino acids that are conservative substitutions for each other:

1) Alanine (A), glycine (G);

2) Aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) serine (S), threonine (T); and

8) Cysteine (C), Methionine (M)

(See, e.g., Creighton, Proteins: Structures and Molecular Properties, W H Freeman &Co.; 2nd Edition (December 1993))

The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences means that two or more sequences or subsequences are identical. When comparing and aligning for maximum correspondence over a comparison window or over a specified region, such as using one of the following sequence comparison algorithms (or others available to those skilled in the art) or by manual alignment and visual Inspection is determined if the sequences have a percentage of amino acid residues or nucleotides that are identical (i.e., at least about 60% identity, at least about 65%, at least about 70%, at least about 75% identity over a defined region , at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity), then the sequences are "substantially identical". This definition also refers to the complement of a test sequence. The identity may exist over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75-100 amino acids or nucleotides in length, or where not specified, across polynucleotides or the entire sequence of the polypeptide.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated (if necessary) and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A "comparison window" as used herein includes segments involving any one contiguous number of digits selected from the group consisting of 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, Wherein after two sequences are optimally aligned, a sequence can be compared to a reference sequence having the same contiguous number of digits. Methods of alignment of sequences for comparison are known to those of skill in the art. Optimal sequence alignments for comparison may include (but are not limited to) the local homology algorithm by Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by Needleman and Wunsch (1970) J. Mol .Biol.48: 443 homology comparison algorithm, by Pearson and Lipman (1988) Proc. Genetics Software Package, Genetics Computer Group, 575 ScienceDr., Madison, WI GAP, BESTFIT, FASTA, and TFASTA), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology ( 1995 Supplement)).

An example of an algorithm suitable for use in determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402 and Altschul et al. ( 1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information, available at www.ncbi.nlm.nih.gov . The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses a wordlength of 3 and an expectation (E) of 10 as defaults, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915) uses The comparison value (B) is 50, the expectation value (E) is 10, M=5, N=-4 and the comparison of two strands are used as default values. The BLAST algorithm is usually performed with the "low complexity" filter turned off.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, eg, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which represents the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

The phrase "selectively (or specifically) hybridizes to" means that when a specific nucleotide sequence is present in a complex mixture, including but not limited to, total cellular or library DNA or RNA, Under hybridization conditions, the molecule binds, complexes or hybridizes only to said nucleotide sequence.

The phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics or combinations thereof under conditions of low ionic strength and high temperature as known in the art. Generally, under stringent conditions, a probe will hybridize to its target subsequence in a complex mixture of nucleic acids (including but not limited to, total cellular or library DNA or RNA), but to no other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize especially at higher temperatures. An extensive guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermodynamic melting point ( _Tm ) for the specific sequence at a defined ionic strength, pH value. The _Tm is the temperature at which 50% of the probes complementary to the target hybridize to the target sequence (occupying 50% of the probes at equilibrium because the target sequence is present in excess at the _Tm ) (at defined ions strength, pH and nucleic acid concentration). Stringent conditions may be at a pH of 7.0 to 8.3, a salt concentration of less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) and for short probes (including but not limited to) 10 up to 50 nucleotides), the temperature is those of at least about 30°C and for long probes (including but not limited to, greater than 50 nucleotides), the temperature is at least about 60°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5X SSC, and 1% SDS, incubated at 42°C, or 5X SSC, 1% SDS, incubated at 65°C, and incubated at 65°C, in 0.2X SSC and wash in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 or more than 120 minutes.

The term "eukaryote" as used herein refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, birds etc.), ciliates, plants (including (but not limited to) monocots, dicots, algae, etc.), fungi, yeasts, flagellates, Microzoa, protists, etc.

The term "non-eukaryotic" as used herein refers to non-eukaryotic organisms. For example, the non-eukaryotic organism can be of the genus Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilics, Bacillus stearothermophilus, Pseudomonas fluorescens Bacteria (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas putida (Pseudomonas putida, etc.) jannaschii), Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus , Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

The term "subject" as used herein refers to an animal, in some embodiments a mammal, and in other embodiments a human, which is the object of treatment, observation or experimentation.

As used herein, the term "effective amount" refers to the amount of a modified non-natural amino acid polypeptide administered which will alleviate to some extent one or both of the disease, condition or disorder being treated. more than one symptom. Compositions containing the modified non-natural amino acid polypeptides described herein can be administered for prophylactic, augmentative, and/or therapeutic treatments.

The term "enhancing" means increasing or prolonging the potency or duration of a desired effect. Thus, in reference to enhancing the effect of therapeutic agents, the term "enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on the system. An "enhancing effective amount" as used herein refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

The term "modified" as used herein refers to any change made to a specified polypeptide, such as altering the length of the polypeptide, the amino acid sequence of the polypeptide, the chemical structure, co-translational modification or post-translational modification. The term "(modified)" form means that the polypeptide in question is optionally modified, ie, the polypeptide in question may or may not be modified.

The term "post-translationally modified" refers to any modification of a natural or unnatural amino acid after it has been incorporated into a polypeptide chain. The term encompasses, by way of example only, co-translational in vivo modification, co-translational in vitro modification (such as in a cell-free translation system), post-translational in vivo modification and post-translational in vitro modification.

In prophylactic applications, compositions containing modified non-natural amino acid polypeptides are administered to a patient predisposed to, or otherwise at risk of, developing a particular disease, disorder or condition. Said amount is defined as "prophylactically effective amount". In such uses, the precise amount will also depend on the patient's state of health, weight, and the like. It is well within the skill of the art to determine such prophylactically effective amounts by routine experimentation (eg, dose escalation clinical trials).

The term "protected" refers to the presence of a "protecting group" or moiety that prevents a chemically reactive functional group from reacting under certain reaction conditions. Protecting groups will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from the group of t-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an orthopyridyl disulfide. If the chemically reactive group is a group of a carboxylic acid such as butanoic or propionic acid, or a hydroxyl group, the protecting group may be benzyl or an alkyl group such as methyl, ethyl or tert-butyl. Other protecting groups known in the art, including photolabile groups such as Nvoc and MeNvoc, can also be used in or with the methods and compositions described herein . Other protecting groups known in the art can also be used in or with the methods and compositions described herein.

By way of example only, capping/protecting groups may be selected from the following groups.

Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York, NY, 1999, which reference is incorporated herein by reference in its entirety.

In therapeutic applications, a composition comprising a modified non-natural amino acid polypeptide is administered to a patient already suffering from a disease, condition or disorder in an amount sufficient to cure or at least to some extent inhibit the progress of the disease, disorder or condition. symptom. Such an amount is defined as a "therapeutically effective amount" and will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. It is well within the skill of the art to determine such a therapeutically effective amount by routine experimentation (eg, dose escalation clinical trials).

The term "treatment" is used to refer to prophylactic and/or therapeutic treatment.

The non-naturally encoded amino acid polypeptides provided herein can include isotopically labeled compounds having one or more atoms replaced by atoms having an atomic mass or mass number different from that found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ^35S , ^18F , ^36Cl . Certain isotopically-labeled compounds described herein, eg, those into which radioactive isotopes such ^{as3H and14C} have been incorporated, are useful in drug and/or matrix tissue distribution assays. Additionally, substitution with isotopes such as deuterium (ie, ^2H ) can afford certain therapeutic advantages resulting from greater metabolic stability, eg, increased in vivo half-life or reduced dosage requirements.

All isomers including, but not limited to, diastereomers, enantiomers and mixtures thereof are considered as part of the compositions described herein. In additional or additional embodiments, a non-naturally encoded amino acid polypeptide is metabolized upon administration to an organism in need thereof to produce metabolites that are subsequently used to produce a desired effect, including a desired therapeutic effect. In other or additional embodiments are active metabolites of non-naturally encoded amino acid polypeptides.

In some cases, non-naturally encoded amino acid polypeptides may exist in tautomeric forms. Furthermore, the non-naturally encoded amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms are also considered disclosed herein. Those skilled in the art will recognize that some of the compounds herein can exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein.

Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, which are within the skill of the art, were employed.

Implementation

I. Introduction

Provided in the present invention are GH (eg, hGH) molecules comprising at least one unnatural amino acid. In certain embodiments of the invention, a GH (eg, hGH) polypeptide having at least one unnatural amino acid includes at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises incorporating a second reactive group using chemistry known to those of skill in the art to be applicable to a particular reactive group including, but not limited to, the following The molecule of the substance is linked to at least one unnatural amino acid comprising a first reactive group: label, dye, polymer, water soluble polymer, derivative of polyethylene glycol, photocrosslinker, radionuclide, Cytotoxic compounds, drugs, affinity tags, photoaffinity tags, reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, carbohydrates, polymers Nucleotides, DNA, RNA, antisense polynucleotides, carbohydrates, water soluble dendrimers, cyclodextrins, inhibitory RNA, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that interact covalently or non-covalently with other molecules, photocaged moiety, actinic radiation excitable moieties, photoisomerization moieties, biotin , biotin derivatives, biotin analogs, moieties incorporating heavy atoms, chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents , aminothioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, chromophores, energy Transfer agents, bioactive agents, detectable labels, small molecules, quantum dots, nanoconductors, radionucleotides, radioconductors, neutron capture agents, or any combination of the foregoing or any other desired compound or substance. For example, the first reactive group is an alkynyl moiety (including, but not limited to, when propargyl is also sometimes referred to as an acetylene moiety, in the unnatural amino acid p-propargyloxyphenylalanine Middle) and the second reactive group is an azido moiety, and utilizes [3+2] cycloaddition chemistry. In another embodiment, the first reactive group is an azido moiety (including but not limited to, in the unnatural amino acid p-azido-L-phenylalanine) and the second reactive group is Alkynyl moiety. In certain embodiments of the modified GH (e.g., hGH) polypeptides of the invention, at least one unnatural amino acid comprising at least one post-translational modification (including but not limited to, an unnatural amino acid containing a keto functional group is used) ), wherein said at least one post-translational modification comprises a carbohydrate moiety. In certain embodiments, post-translational modifications are performed in vivo in eukaryotic cells or in non-eukaryotic cells.

In certain embodiments, the protein includes at least one post-translational modification that is performed in vivo by one host cell, wherein the post-translational modification is not normally performed by another host cell type. In certain embodiments, the protein includes at least one post-translational modification that is performed in vivo by a eukaryotic cell, wherein the post-translational modification is not normally performed by a non-eukaryotic cell. Examples of post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid bond modification, and the like. In one embodiment, the post-translational modification comprises linking the oligosaccharide to asparagine via a GlcNAc-asparagine linkage (including but not limited to, wherein the oligosaccharide comprises (GlcNAc-Man) ₂ -Man-GlcNAc-GlcNAc, etc. etc.). In another embodiment, the post-translational modification comprises making oligosaccharides (including but not limited to, Gal-GalNAc, Gal -GlcNAc, etc.) linked to serine or threonine. In certain embodiments, proteins or polypeptides of the invention may comprise secretory or localization sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, and the like. Examples of secretion signal sequences include, but are not limited to, prokaryotic secretion signal sequences, eukaryotic secretion signal sequences, eukaryotic secretion signal sequences 5' optimized for bacterial expression, novel secretion signal sequences, pectate lyase Secretion signal sequence, OmpA secretion signal sequence and phage secretion signal sequence. Examples of secretion signal sequences include, but are not limited to, STII (prokaryotic), Fd GIII and M13 (phage), Bgl2 (yeast), and the transposon-derived signal sequence bla.

The protein or polypeptide concerned may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or 10 or more unnatural amino acids. The unnatural amino acids may be the same or different, for example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different unnatural amino acids in a protein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different parts. In certain embodiments, at least one (but less than the total) of a particular amino acid present in a naturally occurring form of a protein is substituted with an unnatural amino acid.

The present invention provides methods and compositions based on members of the GH supergene family, in particular hGH, comprising at least one non-naturally encoded amino acid. Introduction of at least one non-naturally encoded amino acid into a member of the GH supergene family may result in specific chemical reactions involving, including but not limited to, with one or more non-naturally encoded amino acids but not with the commonly occurring 20 amino acids The combination chemistry can be applied. In some embodiments, the GH supergene family member comprising a non-naturally encoded amino acid is linked to a water soluble polymer such as polyethylene glycol (PEG) through the side chain of the non-naturally encoded amino acid. The present invention provides a highly efficient method for selectively modifying proteins with PEG derivatives, which involves converting non-genetically encoded amino acids (including, but not limited to, amino acids containing amino acids that are not present in the 20 naturally incorporated amino acids) in response to a selector codon. and including, but not limited to, functional groups or substituents of ketone moieties, azide moieties, or acetylene moieties) are selectively incorporated into proteins, and those amino acids are subsequently modified with appropriately reactive PEG derivatives. Once incorporated, amino acid side chains can then be modified by utilizing chemistries known to those of skill in the art that are applicable to the particular functional group or substituent present in the non-naturally encoded amino acid. A variety of known chemistries are suitable for use in the present invention to incorporate water-soluble polymers into proteins. Such methods include, but are not limited to, Hoo's radical [3+2] cycloaddition of derivatives including, but not limited to, acetylene or azide, respectively (see, e.g., Padwa, A., in Comprehensive Organic Synthesis , Vol. 4 , (1991), Trost, BM, ed., Pergamon, Oxford, pp. 1069-1109; and Huisgen, R., in 1, 3-Dipolar Cycloaddition Chemistry , (1984), Padwa, A. ed., Wiley, New York, pp. 1-176).

Because the Hu's root [3+2] cycloaddition method involves cycloaddition reactions rather than nucleophilic substitution reactions, proteins can be modified with very high selectivity. Its reaction can be carried out with excellent regioselectivity (1,4 > 1,5) by adding a catalytic amount of Cu(I) salt to the reaction mixture under aqueous conditions at room temperature. See, eg, Tomoe et al., (2002) J. Org. Chem. 67:3057-3064; and Rostovtsev et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599; and WO 03/101972 . Molecules that can be added to proteins of the invention via a [3+2] cycloaddition include virtually any molecule with suitable functional groups or substituents, including but not limited to azido or acetylene derivatives. These molecules can be added to unnatural amino acids with ethynyl groups (including but not limited to p-propargyloxyphenylalanine) or unnatural amino acids with azido groups (including but not limited to p-propargyloxyphenylalanine), respectively. azidophenylalanine).

The five-membered rings obtained from the Hu's root [3+2] cycloaddition are generally irreversible under reducing environments and are stable against hydrolysis over long periods of time in aqueous environments. Thus, the physical and chemical properties of a variety of substances can be modified under demanding aqueous conditions with the active PEG derivatives of the present invention. Even more importantly, because the azide and acetylene moieties are specific for each other (and e.g. do not react with any of the 20 common genetically encoded amino acids), they can be found with very high selectivity in A protein is modified at one or more specific sites.

The invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties. PEG polymer derivatives containing acetylene moieties are highly selective for coupling to azide moieties that have been selectively introduced into proteins in response to selector codons. Likewise, PEG polymer derivatives containing azide moieties are highly selective for coupling to acetylene moieties that have been selectively introduced into proteins in response to selector codons.

More specifically, azide moieties include, but are not limited to, alkyl azide moieties, aryl azide moieties, and derivatives of such azide moieties. Derivatives of the alkyl azide moiety and the aryl azide moiety may include other substituents as long as acetylene specific reactivity is maintained. The acetylene moiety includes an alkylacetylene moiety and an arylacetylene moiety and derivatives of each. Derivatives of the alkylacetylene moiety and the arylacetylene moiety may include other substituents as long as the azide-specific reactivity is maintained.

The invention provides combinations of substances having various functional groups, substituents or moieties with other substances including, but not limited to: labels; dyes; polymers; water soluble polymers; derivatives of polyethylene glycol; Photocrosslinkers; radionuclides; cytotoxic compounds; drugs; affinity labels; photoaffinity labels; reactive compounds; resins; second proteins or polypeptides or polypeptide analogs; antibodies or antibody fragments; metal chelators; Cofactors; fatty acids; carbohydrates; polynucleotides; DNA; RNA; antisense polynucleotides; sugars; water-soluble dendrimers; cyclodextrins; inhibitory ribonucleic acids; biomaterials; nanoparticles ; spin labels; fluorophores, metal-containing moieties; radioactive moieties; novel functional groups; groups that interact covalently or non-covalently with other molecules; biotin; biotin derivatives; biotin analogs; moieties incorporating heavy atoms; chemically cleavable groups; photocleavable groups; elongated side chains; carbon-linked Sugars; redox-active agents; aminothioacids; toxic moieties; isotope-labeled moieties; biophysical probes; phosphorescent groups; chemiluminescent groups; electron-dense groups; magnetic groups; intercalating groups ; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; a quantum dot; a nanoconductor; a radionucleotide; a radioconductor; a neutron capture agent; or any combination of the foregoing, or Any other desired compound or substance. Combinations of substances having an azide or acetylene moiety with PEG polymer derivatives having the corresponding acetylene or azide moiety are also encompassed by the present invention. For example, PEG polymers containing azide moieties can be coupled to biologically active molecules at positions in proteins containing non-genetically encoded amino acids with acetylene functionality. Linkages for coupling PEG to biologically active molecules include, but are not limited to, Hu's root [3+2] cycloaddition linkages.

It is well established in the art that PEG can be used to modify the surface of biological materials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar, R., J. Pharm Pharm Sci., 3(1):125-136 (2000), which is incorporated herein by reference). The present invention also includes a surface comprising one or more reactive azide or acetylene sites and one or more of the present invention bonded to said surface via a Hoo's radical [3+2] cycloaddition. Biomaterials of coupled polymers containing azide moieties or acetylene moieties. Biomaterials and other substances can also be derivatized with azide- or acetylene-activated polymers through linkages other than azide or acetylene linkages, such as through linkages containing carboxylic acid, amine, alcohol, or thiol moieties Phase coupling makes the azide or acetylene moiety available for subsequent reactions.

The present invention includes a method of synthesizing the azide moiety-containing and acetylene moiety-containing polymers of the present invention. In the case of PEG derivatives containing an azide moiety, the azide moiety can be directly bonded to a carbon atom of the polymer. Alternatively, PEG derivatives containing an azide moiety can be prepared by linking a linker having an azide moiety at one end to a conventional activated polymer so that the resulting polymer has an azide moiety at its end. In the case of PEG derivatives containing acetylene moieties, the acetylene moieties can be bonded directly to carbon atoms of the polymer. Alternatively, PEG derivatives containing an acetylene moiety can be prepared by linking a linker having an acetylene moiety at one end to a conventional activated polymer so that the resulting polymer has an acetylene moiety at its end.

More specifically, in the case of PEG derivatives containing an azide moiety, a water-soluble polymer having at least one reactive hydroxyl moiety reacts to produce a more reactive moiety thereon (such as mesylate , trifluoroethanesulfonate, tosylate, or halogen-leaving group) substituted polymers. The preparation and use of PEG derivatives containing sulfonyl acid halides, halogen atoms and other leaving groups is known to those skilled in the art. The resulting substituted polymer is then reacted to replace the more reactive moiety at the end of the polymer with an azide moiety. Alternatively, a water-soluble polymer having at least one reactive nucleophilic or electrophilic moiety reacts with a linker having an azide moiety at one end to form a covalent bond between the PEG polymer and the linker and to allow the azide part at the end of the polymer. Nucleophilic and electrophilic moieties including amines, thiols, hydrazides, hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters, and the like are known to those skilled in the art.

More specifically, in the case of PEG derivatives containing an acetylene moiety, a water soluble polymer having at least one active hydroxyl moiety is reacted to displace the halogen or other activated leaving group by the precursor containing the acetylene moiety. Alternatively, a water-soluble polymer having at least one reactive nucleophilic or electrophilic moiety is reacted with a linker having an acetylene moiety at one end to form a covalent bond between the PEG polymer and the linker and position the acetylene moiety end of the polymer. The use of halogen moieties, activated leaving groups, nucleophilic and electrophilic moieties in the context of organic synthesis and in the preparation and use of PEG derivatives is well established to those skilled in the art.

The present invention also provides a method for selectively modifying proteins to incorporate other substances (including but not limited to, water-soluble polymers such as PEG and PEG derivatives, which contain azide and acetylene moieties) to the modified protein. method in protein. PEG derivatives containing azide and acetylene moieties can be used to alter the properties of surfaces and molecules where biocompatibility, stability, solubility, and lack of immunogenicity are important, while at the same time providing A more selective approach than previously known in the art.

II. Growth Hormone Supergene Family

The following proteins include those encoded by genes of the growth hormone (GH) supergene family (Bazan, F., Immunology Today 11:350-354 (1990); Bazan, J.F. Science 257:410-413 (1992); Mott , H.R. and Campbell, I.D., Current Opinion in Structural Biology 5:114-121(1995); Silvennoinen, O. and Ihle, J.N., SIGNALLING BY THE HEMATOPOIETIC CYTOKINERECEPTORS(1996)): growth hormone, prolactin, placental lactogen, Erythropoietin (EPO), Thrombopoietin (TPO), Interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9 , IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, Oncostatin M, Ciliary Neurotrophic Factor (CNTF), Leukemia Inhibitory Factor (LIF), Interferon-α , β interferon, ε interferon, γ interferon, ω interferon, τ interferon, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony Stimulatory factor (M-CSF) and cardiotrophin-1 (CT-1) ("GH supergene family"). It is expected that additional members of this gene family will be identified in the future by gene cloning and sequencing. Although members of the GH supergene family generally have limited amino acid or DNA sequence identities, they have similar secondary and tertiary structures. The shared structural features allow new members of the gene family to be easily identified and allow similar application of the non-natural amino acid methods and compositions described herein. Given the degree of structural homology between members of the GH supergene family, the invention can be utilized to incorporate non-naturally encoded amino acids into any member of the GH supergene family. Each member of this protein family contains a four-helix bundle, the general structure of which is shown in Figure 1 . The general structures of the family members hGH, EPO, IFNα-2 and G-CSF are shown in Figure 2, Figure 3, Figure 4 and Figure 5, respectively.

Including G-CSF (Zink et al., FEBS Lett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill et al., Proc.Natl.Acad.Sci.USA 90:5167 (1993) ), GM-CSF (Diederichs, K. et al., Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol 224: 1075-1085 (1992)), IL-2 (Bazan, J.F. and McKay, D.B., Science 257:410-413 (1992)), IL-4 (Redfield et al., Biochemistry 30:11029-11035 (1991); Powers et al., Science 256:1673-1677 (1992)) and IL-4 5 (Milburn et al., Nature 363:172-176 (1993)) The structures of a large number of cytokines have been determined by X-ray diffraction and NMR studies and despite the lack of significant primary sequence homology, they show that the GH structure surprisingly conservative. Based on modeling and other studies, IFN is considered a member of this family (Lee et al., J. Interferon Cytokine Res. 15:341 (1995); Murgolo et al., Proteins 17:62 (1993); Radhakrishnan et al., Structure 4:1453 (1996); Klaus et al., J. Mol. Biol. 274:661 (1997)). Based on modeling and mutation studies, EPO is considered to be a member of this family (Boissel et al., J. Biol. Chem. 268: 15983-15993 (1993); Wen et al., J. Biol. Chem. 269: 22839-22846 (1994)). All of the aforementioned cytokines and growth factors are now believed to form one large gene family.

In addition to having similar secondary and tertiary structures, members of this family share the property that they necessarily oligomerize cell surface receptors to activate intracellular signaling pathways. Some GH family members, including but not limited to GH and EPO, bind to a single type of receptor and cause it to form homodimers. Other family members including, but not limited to, IL-2, IL-4, and IL-6 bind to more than one type of receptor and cause the receptor to form heterodimers or higher order aggregates (Davis et al. (1993), Science 260: 1805-1808; Paonessa et al., (1995), EMBO J. 14: 1942-1951; Mott and Campbell, Current Opinion in Structural Biology 5: 114-121 (1995)). Mutational studies have shown that these other cytokines and growth factors, like GH, contain multiple receptor binding sites, usually 2, and bind their cognate receptors sequentially (Mott and Campbell, Current Opinion in Structural Biology 5: 114-121 (1995); Matthews et al., (1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, the main receptor binding sites of these other family members are mainly found in the 4 alpha helices and the A-B loop. Specific amino acids in the helical bundles involved in receptor binding differ between family members. Most of the cell surface receptors that interact with members of the GH supergene family are structurally related and constitute a second large multigene family. See, eg, US Patent No. 6,608,183, which is incorporated herein by reference.

A general conclusion drawn from mutational studies of individual members of the GH supergene family is that the loops connecting the alpha helices generally do not tend to be involved in receptor binding. In particular, the short B-C loop appears to be dispensable for receptor binding by most, if not all, family members. To this end, in members of the GH supergene family, the B-C loop can be substituted with a non-naturally encoded amino acid as described herein. The A-B loop, C-D loop (and the D-E loop of interferon/IL-10-like members of the GH superfamily) may also be substituted with non-naturally occurring amino acids. Amino acids closer to helix A and farther from the final helix also tend not to be involved in receptor binding and may also be sites for the introduction of non-naturally occurring amino acids. In some embodiments, within the first 1, 2, 3, 4, 5, 6, 7 or more amino acids including (but not limited to) the A-B, B-C, C-D or D-E loop Any position within the loop structure is substituted with a non-naturally encoded amino acid. In some embodiments, one or more non- Naturally encoded amino acid substitutions.

Including (but not limited to) EPO, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12 p35, IL-13, IL-15 Certain members of the GH family of interferons and beta contain N-linked and/or O-linked sugars. Glycosylation sites in proteins are found almost exclusively in loop regions and not in alpha-helical bundles. Because the loop region is not generally involved in receptor binding and because it is the site for covalent attachment of sugar groups, it can be a useful site for introducing non-naturally occurring amino acid substitutions into proteins. Amino acids comprising N-linked and O-linked glycosylation sites in proteins may be sites for non-naturally occurring amino acid substitutions because these amino acids are surface exposed. Thus, native proteins can tolerate bulky carbohydrate groups attached to the protein at these sites and the sites of glycosylation tend to be located far from the receptor binding site.

It is possible that other members of the GH supergene family will be discovered in the future. New members of the GH supergene family can be identified by computer-aided secondary and tertiary structure analysis of predicted protein sequences and by selection techniques designed to identify molecules that bind to specific targets. Members of the GH supergene family typically have an amphipathic helical structure of 4 or 5 connected by non-helical amino acids (loop regions). Proteins can contain a hydrophobic signal sequence at their N-terminus to facilitate cellular secretion. Members of these later discovered GH supergene families are also included in the present invention. A related application is International Patent Application entitled "Modified Four Helical Bundle Polypeptides and Their Uses" published as WO 05/074650 on 18 August 2005, which is incorporated herein by reference.

Accordingly, the description of the growth hormone supergene family is provided for illustrative purposes and examples only, and not to limit the scope of the methods, compositions, strategies and techniques described herein. Furthermore, references to GH polypeptides in this application are intended to use this general term as an example of any member of the GH supergene family. Accordingly, it is understood that the modifications and chemistries described herein with respect to hGH polypeptides or proteins are equally applicable to any member of the GH supergene family, including those specifically listed herein.

III. General Recombinant Nucleic Acid Methods for Use in the Invention

In many embodiments of the invention, recombinant methods will be used to isolate, clone, and often to alter nucleic acid encoding a GH (eg, hGH) polypeptide of interest. The embodiments are used for, including but not limited to, protein expression or during the generation of variants, derivatives, expression cassettes or other sequences derived from GH (eg, hGH) polypeptides. In some embodiments, a sequence encoding a polypeptide of the invention is operably linked to a heterologous promoter. Isolation of hGH and production of GH in host cells are described, for example, in U.S. Pat. , 6,004,931, 6,022,711, 6,143,523, and 6,608,183, which are incorporated herein by reference.

The nucleotide sequence encoding a hGH polypeptide comprising a non-naturally encoded amino acid can be synthesized based on the amino acid sequence of a parent polypeptide including, but not limited to, having the amino acid sequence set forth in SEQ ID NO: 2 (hGH), and The nucleotide sequence is then altered to effect the introduction (ie, incorporation or substitution) or removal (ie, deletion or substitution) of the relevant amino acid residues. Nucleotide sequences may conveniently be modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis, including, but not limited to, by using an oligonucleotide synthesizer (in which oligonucleotides are designed based on the amino acid sequence of the desired polypeptide) and preferably selected in the sequence that will produce recombinant Those codons that are favorable in the host cell of the polypeptide are prepared. For example, several small oligonucleotides encoding portions of a desired polypeptide can be synthesized and assembled by PCR, ligation or ligation chain reaction. See, eg, Barany et al., Proc. Natl. Acad. Sci. 88:189-193 (1991); US Patent No. 6,521,427, which is incorporated herein by reference.

The present invention utilizes conventional techniques in the field of recombinant genetics. Basic articles disclosing general methods for use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd Edition, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology ( Ausubel et al. eds., 1994).

General articles describing molecular biology techniques include Berger and Kimmel, Guide to Molecular Cloning Techniques. Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology , eds. FMAusubel et al., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (added via 1999) (“Ausubel”). These articles describe mutations, the use of vectors, promoters, and many other related topics related to, including but not limited to, the production of genes or polynucleotides for the production of Selector codons for proteins, orthogonal tRNAs, orthogonal synthetases and their pairs for unnatural amino acids.

Various types of mutations are used in the present invention for various purposes including, but not limited to, generating novel synthetases or tRNAs, mutating tRNA molecules, mutating polynucleotides encoding synthetases, generating tRNA libraries, A library of synthetic enzymes is generated, a selector codon is generated, and a selector codon encoding an unnatural amino acid is inserted into a protein or polypeptide of interest. Such mutations include, but are not limited to, site-directed mutagenesis, random point mutation, homologous recombination, DNA shuffling or other regressive mutation methods, chimeric construction, mutation using uracil-containing templates, oligonucleotide-directed mutagenesis, Phosphoester-modified DNA mutations, mutations using gapped double-stranded DNA, etc., or any combination thereof. Other suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutation by total gene synthesis, double-strand break repair, and the like. Mutations including, but not limited to, those involving chimeric constructions are also encompassed by the invention. In one embodiment, mutations may be guided by known information about the naturally occurring molecule or the naturally occurring molecule that has been altered or mutated, including but not limited to sequence, sequence comparison, physical properties, secondary , tertiary or quaternary structure, crystal structure, etc. information.

The articles and examples found in this article describe these programs. Additional information is found in the following publications and references cited therein: Ling et al., Approaches to DNA mutagenesis: an overview, Anal Biochem. 254(2):157-178 (1997); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method, Methods Mol.Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann.Rev.Genet. 19:423-462 (1985); Botstein & Shortle, Strategies and applications of in vitro mutagenesis, Science 229: 1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237: 1-7 (1986); Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. MJ. Ed., Springer Verlag, Berlin) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol. 154,367-382(1987); Bass et al., Mutant Trp repressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, Nucleic Acids Res. 10: 6487-6500 (1982); Zoller & Smith, Oligonucleotide-directed mutagenesis of DNA frag vectors, Methods in Enzymol .100:468-500(1983); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol. 154:329-3750)( Taylor et al., The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepared DNA, Nucl.Acids Res. 13:8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide-directed mutations at high frequency rousing pho -modified DNA, Nucl.Acids Res. 13:8765-8785 (1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenests, Nucl.Acids Res. 14:9689 ( 1986); Sayers et al., 5'-3'Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802 (1988); Sayers et al., Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucl. Acids Res. 16: 803-814; Kramer et al., The gapped duplexDNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res. 12: 9441-9456 (1984) ; Kramer & Fritz Oligonucleotide-directed construction of mutations viagapped duplex DNA, Methods in Enzymol. 154:350-367 (1987); Kramer et al., Improved in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction . Acids Res. 16: 7207 (1988); Fritz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, Nucl. Acids Res. 16: 6987-6999 (1988); Kramer et al., Difierentbase /base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using Ml3 vectors, Methods in Enzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate large deletions, Nucl. : 5115 (1986); Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloning of a gene coding for the ribonuclease Sprotein, Science 223:1299-1301 (1984); Sakmar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl .Acids Res. 14:6361-6372(1988); Wells et al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34:315-323(1985); Grundström et al., Oligonucleotide-directed Mutagenesis by microscale'shot-gun'gene synthesis, Nucl. Acids Res. 13:3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repairin plasmamids of Escherichia coli: amethod for site-specific mutagenesis, Proc. Natl. Acad. Sci. USA , 83: 7177-7181 (1986); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology 4: 450-455 (1993); Sieber et al., Nature Biotechnology, 19: 456-460 (2001); WPC Stemmer, Nature 370, 389-91 (1994); and IALorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8 (1995). Additional detailed descriptions of many of the above methods can be found in Methods in Enzymology Volume 154, which also describes efficient controls for troubleshooting problems using various mutation methods.

(For example) mutated oligonucleotides of the invention useful for e.g. mutating synthetase repertoires or altering tRNAs are generally solids according to Beaucage and Caruthers, Tetrahedron Letts. 22(20):1859-1862, (1981). Phosphoramidite triester methods are chemically synthesized, eg, using an automated synthesizer, as described in Needham-Van Devanter et al., Nucleic Acids Res., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic host cells and organisms that incorporate unnatural amino acids in vivo via orthogonal tRNA/RS pairs. A host cell is genetically engineered with a polynucleotide of the invention or a construct comprising a polynucleotide of the invention (including but not limited to a vector of the invention, which may be, for example, a cloning vector or an expression vector). Modification (including but not limited to transformation, transduction or transfection). For example, the coding regions of the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivatized are operably linked to gene expression control elements functional in the desired host cell. Vectors can be, for example, in the form of plasmids, cosmids, phage, bacteria, viruses, naked polynucleotides, or conjugated polynucleotides. Vectors are introduced into cells and/or microorganisms by standard methods including electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), infection by viral vectors, on beads or High velocity ballistic penetration of small particles with nucleic acids in their matrix or on their surface (Klein et al., Nature 327.70-73 (1987)) and/or the like.

The engineered host cells can be cultured in conventional nutrient media modified as appropriate for purposes such as screening procedures, activation of promoters or selection of transformants for activity. These cells can optionally be cultured in transgenic organisms. Other useful references, including but not limited to, on cell isolation and culture (e.g., on subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique , 3rd Edition, Wiley-Liss, New York and references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture ; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

Several well-known methods for introducing target nucleic acids into cells are available, any of which can be used in the present invention. These methods include fusion of recipient cells with DNA-containing bacterial protoplasts, electroporation, projectile bombardment, and infection with viral vectors (discussed further below), among others. Bacterial cells can be used to increase the number of plasmids containing the DNA constructs of the invention. The bacteria are grown to log phase and the plasmids in the bacteria can be isolated by various methods known in the art (see, eg, Sambrook). In addition, kits for purification of plasmids from bacteria are commercially available (see, eg, EasyPrep ^™ , FlexiPrep ^™ , all from Pharmacia Biotech; StrataClean ^™ from Stratagene; and QIAprep ^™ from Qiagen). The isolated and purified plasmids are then further processed to generate other plasmids that are used to transfect cells or incorporated into related vectors to infect organisms. A typical vector contains transcriptional and translational terminators, transcriptional and translational initiation sequences, and a promoter (useful to regulate the expression of a particular target nucleic acid). The vector optionally comprises a gene expression cassette containing at least one independent terminator sequence, sequences that allow the cassette to replicate in eukaryotes or prokaryotes, or both (including but not limited to, shuttle vectors), and suitable Selectable markers for prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81 (1979); Roberts et al., Nature , 328:731 (1987); Schneider, E. et al., Protein Expr. Purif. 6(1):10-14 (1995) ; Ausubel, Sambrook, Berger (all ibid.). Catalogs of bacteria and phages useful for cloning are provided, for example, by the ATCC, eg The ATCC Catalog of Bacteria and Bacteriophage (1992) Ghema et al. (eds.), published by ATCC. Other basic procedures and fundamental theoretical perspectives on sequencing, cloning, and other aspects of molecular biology are also found in Watson et al. (1992) Recombinant DNA (2nd Ed.) Scientific American Books, NY. In addition, essentially any nucleic acid (and almost any labeled nucleic acid, whether standard or non-standard) may be ordered as a routine or standard from any of a variety of commercial sources, such as Midland Certified Reagent Company (Midland, TX, available at www.mcrc.com ), The Great American Gene Company (Ramona, CA, available at www.genco.com ), ExpressGen Inc. (Chicago, IL, available at www. . expressgen.com ), Operon Technologies Inc. (Alameda, CA), and many other commercial sources.

(a) selector codon

The selector codons of the present invention expand the genetic codon framework of the protein biosynthetic machinery. For example, selector codons include, but are not limited to, single three-base codons, nonsense codons such as, but not limited to, amber codons (UAG), ocher codons, or opal codons (UGA). stop codon), unnatural codons, 4 or more base codons, rare codons, etc. Those skilled in the art will readily appreciate that the number of selector codons that can be introduced into a desired gene or polynucleotide ranges over a wide range, including but not limited to, in a single polynucleotide encoding at least part of a hGH polypeptide. Among them, there are 1 or more, 2 or more, 3 or more, 4, 5, 6, 7, 8, 9, 10 or more choices a.

In one embodiment, the method involves the use of a selector codon, which is a stop codon for the in vivo incorporation of one or more unnatural amino acids in a cell. For example, O-tRNAs that recognize stop codons including, but not limited to, UAG are generated and aminoacylated by O-RS with the desired unnatural amino acid. The O-tRNA is not recognized by naturally occurring host aminoacyl-tRNA synthetases. Routine site-directed mutagenesis can be used to introduce stop codons including, but not limited to, TAG at sites of interest in a polypeptide of interest. See, eg, Sayers, JR et al. (1988), 5'-3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA, and nucleic acid encoding the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon to result in a polypeptide containing the unnatural amino acid at the specified position.

In vivo incorporation of unnatural amino acids can be accomplished without significant disturbance of eukaryotic host cells. For example, since the suppressive efficacy of the UAG codon is considered to include (but not limited to) the amber suppressor tRNA O-tRNA and eukaryotic release factors (including but not limited to eRF) (which bind to the stop codon and initiate growth Depends on the competition between the release of peptides from ribosomes in ), so inhibitory efficacy can be modulated by, including but not limited to, increasing the expression level of O-tRNA and/or inhibiting tRNA.

Unnatural amino acids can also be encoded by rare codons. For example, the rare arginine codon AGG has been shown to be efficient for insertion of Ala by a synthetic tRNA acylated with alanine when the concentration of arginine in in vitro protein synthesis reactions is reduced. See, eg, Ma et al., Biochemistry , 32:7939 (1993). In this case, the synthetic tRNA competes with the naturally occurring tRNAArg that is present as a lesser species in E. coli. Some organisms do not use all triplet codons. The unassigned codon AGA in Micrococcus luteus has been used to insert amino acids in in vitro transcription/translation extracts. See, eg, Kowal and Oliver, Nucl. Acid. Res., 25:4685 (1997). Components of the invention can be produced to use these rare codons in vivo.

Selector codons also include extension codons, including but not limited to, 4 or more base codons, such as 4, 5, 6 or more base codons. Examples of four base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU, and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, and the like. Features of the invention include the use of extended codons based on frameshift suppression. A codon of 4 or more bases allows insertion of one or more unnatural amino acids into the same protein, including but not limited to. For example, 4 or More than 4 base codons can be read as a single amino acid. In other embodiments, the anticodon loop can decode including, but not limited to, at least four base codons, at least five base codons, or at least six base codons or at least six or more base codons. Because there are 256 possible four-base codons, multiple unnatural amino acids can be encoded in the same cell using four or more base codons. See, Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology , 9: 237-244; Magliery, (2001) Expanding the Genetic Code: Selection of Efficient Suppressors of Four-base Codons and Identification of "Shifty "Four-base Codons with a Library Approach in Escherichia coli, J. Mol. Biol. 307:755-769.

For example, 4-base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, eg, Ma et al., (1993) Biochemistry , 32:7939; and Hohsaka et al., (1999) J. Am. Chem. Soc, 121:34. Simultaneous in vitro incorporation of NBD derivatives of 2-naphthylalanine and lysine into streptavidin by two chemically acylated frameshift suppressor tRNAs using CGGG and AGGU. See, eg, Hohsaka et al. (1999) J. Am. Chem. Soc, 121:12194. In an in vivo study, Moore et al. studied the ability of tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (N can be U, A, G, or C) and found that the quadruplet UAGA could be inhibited by UCUA anticodons. The tRNALeu codons were decoded with efficiencies ranging from 13 to 26%, while decoding rarely occurred in 0 or -1 frames. See, Moore et al., (2000) J. Mol. Biol., 298:195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the present invention, which can reduce missense reads and frameshift suppression at otherwise unwanted sites.

For a given system, the selector codon may also include one of the natural three-base codons for which no (or very little) natural base codons are used by the endogenous system. Such systems include, for example, systems lacking tRNAs that recognize natural three-base codons and/or systems in which the three-base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. One extra base pair increases the number of triplet codons from 64 to 125. Properties of third base pairs include stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by polymerases, and efficient continuous primer extension following synthesis of nascent unnatural base pairs. Descriptions of unnatural base pairs that may be suitable for methods and compositions include, for example, Hirao et al., (2002) An unnatural base pair for incorporating aminoacid analogues into protein, Nature Biotechnology , 20:177-182. See also Wu, Y. et al., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevant publications are listed below.

For in vivo use, unnatural nucleosides are membrane permeable and phosphorylated to form the corresponding triphosphates. Furthermore, the increased genetic information is stable and not destroyed by cellular enzymes. Benner and others have earlier attempted to exploit hydrogen bonding patterns different from those in typical Watson-Crick pairs, the most notable example of which is the iso-C:iso-G pair. See, eg, Switzer et al., (1989) J.Am.Chem.Soc , 111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000) Curr.Opin.Chem.Biol. , 4:602. These bases are generally mismatched to some extent with natural bases and cannot be enzymatically replicated. Kool and colleagues demonstrated that hydrophobic stacking interactions between bases can replace hydrogen bonding to facilitate base pair formation. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl. , 36, 2825. In an effort to develop unnatural base pairs that meet all of the above requirements, Schultz, Romesberg, and colleagues have systematically synthesized and investigated a series of unnatural hydrophobic bases. It was found that PICS:PICS self-pairing is more stable than natural base pairs, and can be effectively incorporated into DNA by the Klenow fragment (Klenow fragment, KF) of Escherichia coli DNA polymerase I. See, eg, McMinn et al., (1999) J.Am.Chem.Soc, 121:11585-6; and Ogawa et al., (2000) J.Am.Chem.Soc , 122:3274. 3MN:3MN self-pairing Can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, eg, Ogawa et al. (2000) J. Am. Chem. Soc, 122:8803. However, two bases act as chain terminators for further replication. More recently, mutant DNA polymerases have been developed that can be used to replicate the PICS self-pair. In addition, 7AI's own pairing can be replicated. See, eg, Tae et al., (2001) J. Am. Chem. Soc, 123:7439. A novel metallobase pairing, Dipic:Py, has also been developed that forms a stable pair upon copper(II) binding. See, Meggers et al. (2000) J. Am. Chem. Soc, 122:10714. Because extended codons and unnatural codons are inherently orthogonal to natural codons, the methods of the invention can take advantage of this property to generate their orthogonal tRNAs.

Translational bypass systems can also be used to incorporate unnatural amino acids into desired polypeptides. In translation bypass systems, large sequences are incorporated into genes but not translated into proteins. The sequence contains structures that act as cues that induce ribosomes to skip the sequence and resume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions of the invention is encoded by a nucleic acid. Typically, the nucleic acid comprises at least 1 selector codon, at least 2 selector codons, at least 3 selector codons, at least 4 selector codons, at least 5 selector codons, at least 6 selector codons, at least 7 selector codons codons, at least 8 selector codons, at least 9 selector codons, 10 or more selector codons.

A gene encoding a protein or polypeptide of interest can be mutated to include, for example, one or more selector codons for the incorporation of an unnatural amino acid using methods well known to those of skill in the art and described herein. For example, the nucleic acid of a protein of interest is mutated to include one or more selector codons for the incorporation of one or more unnatural amino acids. The invention includes any such variants, including but not limited to mutants, eg, any protein form that includes at least one unnatural amino acid. Likewise, the invention also includes corresponding nucleic acids, ie, any nucleic acid having one or more selector codons encoding one or more unnatural amino acids.

Nucleic acid molecules encoding proteins of interest, such as hGH polypeptides, can readily be mutated to introduce cysteines at any desired position in the polypeptide. Cysteine is widely used to introduce reactive molecules, water soluble polymers, proteins or various other molecules onto proteins of interest. Suitable methods for incorporating cysteines into desired positions in polypeptides are known to those skilled in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated herein by reference and standard mutation techniques.

IV. Non-Naturally Encoded Amino Acids

There is a wide variety of non-naturally encoded amino acids suitable for use in the present invention. Any number of non-naturally encoded amino acids can be introduced into a GH (eg, hGH) polypeptide. In general, relative to the 20 common, genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and In other words, the introduced non-naturally encoded amino acid is substantially chemically inert. In some embodiments, the non-naturally encoded amino acid includes functional groups that can efficiently and selectively interact with functional groups that are not present in the 20 common amino acids, including but not limited to azido, keto, aldehyde, and aminooxy groups. ) react to form side chain functional groups of stable conjugates. For example, GHs (e.g., hGH) that include non-naturally encoded amino acids that contain azido functional groups because azide functional groups and alkyne functional groups selectively react to form Hu's radical [3+2] cycloaddition products A polypeptide can be reacted with a polymer containing an alkyne moiety, including but not limited to poly(ethylene glycol), or a second polypeptide to form a stable conjugate.

The general structure of α-amino acids is illustrated below (Formula I).

A non-naturally encoded amino acid is generally any structure having the formula listed above, wherein the R group is any substituent other than that used in the 20 naturally occurring amino acids, and may be suitable for use in the present invention. Because the non-naturally encoded amino acids of the invention generally differ from natural amino acids only in the structure of the side chain, the non-naturally encoded amino acids form amide bonds with other amino acids including, but not limited to, naturally or non-naturally encoded amino acids, so The amide bond is formed in the same manner as it is formed in naturally occurring polypeptides. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally includes alkyl-, aryl-, acyl-, keto-, azido-, hydroxy-, hydrazino, cyano-, halo-, hydrazide, alkenyl, alkyne group, ether group, thiol group, selenium group-, sulfonyl group-, boronic acid group, yopentyl acid group, phosphoric acid group, phosphono group, phosphino group, heterocyclic group, enone group, imine group, aldehyde group, ester group, thioacid group, hydroxylamine group, amino group, etc. or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising photoactivatable cross-linkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, amino acids containing Metallic amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and/or photoisomerizable amino acids, containing biotin or biotin-like amino acids, glycosylated amino acids such as sugar-substituted serine, amino acids modified with other carbohydrates, ketogenic amino acids, amino acids containing polyethylene glycol or polyethers, amino acids substituted with heavy atoms, chemically cleavable and/or photocleavable amino acids having extended side chains (including but not limited to) polyethers or long chain hydrocarbons including but not limited to greater than about 5 or greater than about 10 when compared to natural amino acids carbon), carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids containing one or more toxic moieties.

Exemplary non-naturally encoded amino acids that may be suitable for use in the present invention and that may be used to react with water soluble polymers include, but are not limited to, those having carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, and alkyne reactivity group of those amino acids. In some embodiments, the non-naturally encoded amino acid comprises a carbohydrate moiety. Examples of the amino acid include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosamine-L-serine, Threonine, N-Acetyl-L-Glucosaminyl-L-Asparagine, and O-Mannosyl-L-Serine. Examples of the amino acid also include those in which the naturally occurring N-bond or O-bond between the amino acid and the carbohydrate is a covalent bond not commonly found in nature (including but not limited to, an olefin bond, an oxime bond, a thioether bond , amide bond, etc.) replaced examples. Examples of such amino acids also include carbohydrates not commonly found in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, and the like.

Many of the non-naturally encoded amino acids provided herein are commercially available, for example, from Sigma-Aldrich (St. Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany) or Peptech (Burlington, MA, USA) . Those amino acids that are not commercially available are optionally synthesized as provided herein or using standard methods known to those of skill in the art. For organic synthesis techniques, see, for example, Fessendon and Fessendon's Organic Chemistry , (1982, 2nd edition, Willard Grant Press, Boston Mass.); March's Advanced Organic Chemistry (3rd edition, 1985, Wiley and Sons, New York); and Carey and Sundberg, Advanced Organic Chemistry (3rd Edition, Parts A and B, 1990, Plenum Press, New York). See also US Patent Application Publications 2003/0082575 and 2003/0108885, which are incorporated herein by reference. In addition to unnatural amino acids containing novel side chains, unnatural amino acids that may be suitable for use in the present invention also optionally include modified backbone structures, including (but not limited to) as illustrated by the structures of Formula II and Formula III,

where Z typically comprises OH, _NH2 , SH, NH-R' or SR'; X and Y may be the same or different and generally comprise S or O, and R and R' are optionally the same or different and are usually selected From the same compositional entries and hydrogens for the R group described above for the unnatural amino acid of formula I. For example, the unnatural amino acids of the invention optionally comprise substitutions at the amino or carboxyl groups as illustrated by Formula II and Formula III. Unnatural amino acids of this type include (but are not limited to) α-hydroxy acids, α-thioacids, α-aminothiocarboxylates, including (but not limited to) those with sidewalls corresponding to the common 20 natural amino acids chains or those with unnatural side chains. In addition, substitutions at the alpha-carbon optionally include, but are not limited to, L-amino acids such as D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like , D-amino acid or α-α-disubstituted amino acid. Other structural substitutes include cyclic amino acids such as proline analogs and 3, 4, 6, 7, 8 and 9 membered ring proline analogs, such as substituted beta-alanine and gamma-aminobutyric acid β amino acids and γ amino acids.

Many unnatural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, etc., and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, wherein substituted tyrosines include (including, but not limited to) ) Keto group (including (but not limited to) acetyl group), benzoyl group, amino group, hydrazine group, hydroxylamine group, thiol group, carboxyl group, isopropyl group, methyl group, C ₆ -C ₂₀ straight chain or branched hydrocarbon group , saturated or unsaturated hydrocarbon group, O-methyl group, polyether group, nitro group, alkynyl group, etc. In addition, multiply substituted aryl rings are also contemplated. Glutamine analogs that may be suitable for use in the present invention include, but are not limited to, alpha-hydroxy derivatives, gamma-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Examples of phenylalanine analogs that may be suitable for use in the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine, wherein the substituent Including (including but not limited to) hydroxyl, methoxy, methyl, allyl, aldehyde, azido, iodo, bromo, keto (including but not limited to acetyl), benzyl Acyl, alkynyl, etc. Specific examples of unnatural amino acids that may be suitable for use in the present invention include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl) Alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-Dopa, Fluorinated Phenylalanine, Isopropyl-L-Phenylalanine, Para-Azido-L-Phenylalanine, Para-Acyl-L-Phenylalanine, Para-Benzoyl-L -Phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L -Phenylalanine and p-propargyloxy-phenylalanine and more. Examples of structures of various unnatural amino acids that may be suitable for use in the present invention are provided, for example, in WO2002/085923 entitled "In vivoincorporation of unnatural amino acids." For other methionine analogs, see also Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated herein by reference.

In one embodiment, compositions of GH (eg, hGH) polypeptides comprising an unnatural amino acid, such as p-(propargyloxy)-phenylalanine, are provided. Also provided are various compositions comprising p-(propargyloxy)-phenylalanine and including, but not limited to, proteins and/or cells. In one aspect, a composition comprising the p-(propargyloxy)-phenylalanine unnatural amino acid further comprises an orthogonal tRNA. The unnatural amino acid can be bound to the orthogonal tRNA, including but not limited to covalently, including but not limited to, covalently bound to the orthogonal tRNA through an amino-acyl bond, covalently bound to the The 3'OH or 2'OH of the terminal ribose of the orthogonal tRNA is bonded and so on.

Various advantages and manipulations are provided to proteins via chemical moieties of unnatural amino acids that can be incorporated into proteins. For example, the unique reactivity of the keto functional group allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Heavy atom unnatural amino acids, for example, can be used for phased X-ray structure data. Site-specific introduction of heavy atoms using unnatural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms. Photoreactive unnatural amino acids (including, but not limited to, amino acids with benzophenone and aryl azide (including, but not limited to, phenyl azide) side chains) allow for efficient activity of proteins, for example. Photocrosslinking in vivo and in vitro. Examples of photoreactive unnatural amino acids include, but are not limited to, p-azidophenylalanine and p-benzoylphenylalanine. Proteins with photoreactive unnatural amino acids can thus be cross-linked at will through excitation of photoreactive groups - providing transient control. In one example, the methyl group of the unnatural amino acid can be substituted with an isotopically labeled (including but not limited to) methyl group as (including but not limited to) )) Probes of local structure and dynamics for use with NMR and vibrational spectroscopy. Alkynyl or azido functional groups, for example, allow the selective modification of proteins with molecules through [3+2] cycloaddition reactions.

Unnatural amino acids incorporated into polypeptides at the amino terminus may contain R groups that are any of the substituents different from those used in the 20 natural amino acids, and different from the R groups normally found in α-amino acids (see Formula I). The second reactive group of the _NH2 group. Similar unnatural amino acids can be incorporated at the carboxyl terminus with a second reactive group different from the COOH group normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention can be selected or designed to provide additional characteristics that are difficult to obtain among the 20 natural amino acids. For example, unnatural amino acids can be designed or selected as desired to alter the biological properties of the protein into which the unnatural amino acid is incorporated. For example, the following properties can optionally be altered by including unnatural amino acids in proteins: toxicity, biodistribution, solubility, stability (e.g., thermal stability, hydrolytic stability, oxidative stability, resistance to enzymatic degradation stability, etc.), ease of purification and handling, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic activity, redox potential, half-life, interaction with other molecules (e.g., covalently or noncovalently ) ability to react, etc.

Structures of unnatural amino acids (containing carbonyl, carbonyl-like, masked carbonyl, protected carbonyl and hydroxylamine groups) and synthesis

In some embodiments, the present invention provides a GH (eg, hGH) linked to a water soluble polymer (eg, PEG) via an oxime linkage.

Many types of non-naturally encoded amino acids are suitable for forming oxime bonds. These amino acids include, but are not limited to, non-naturally encoded amino acids containing a carbonyl, dicarbonyl, or hydroxylamine group. Such amino acids are described in U.S. Patent Application Nos. 60/638,418, 60/638,527, and 60/639,195 (titled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides", 2004 filed December 22, 2010), the entirety of which is incorporated herein by reference. Such amino acids are also described in U.S. Patent Application Nos. 60/696,210, 60/696,302, and 60/696,068 (titled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides", 2005 filed July 1), the entirety of which is incorporated herein by reference. Non-naturally encoded amino acids are also described in U.S. Patent Application No. 10/126,931, filed April 19, 2002, and U.S. Patent Application No. 10/126,927, filed April 19, 2002, the Incorporated herein by reference in its entirety.

Some embodiments of the invention utilize GH (eg, hGH) polypeptides substituted with the amino acid p-acetylphenylalanine at one or more positions. The synthesis of p-acetyl-(+/-)-phenylalanine and m-acetyl-(+/-)-phenylalanine is described in Zhang, Z., et al. , Biochemistry 42:6735-6746 (2003). Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared by those skilled in the art. In addition, non-limiting exemplary syntheses of the unnatural amino acids included herein are provided in Figure 4, Figures 24-34, and Figures 36-39 of U.S. Patent Application Serial No. 10/126,931, which is incorporated in its entirety at Incorporated herein by reference.

Amino acids with electrophilic reactive groups allow various reactions to attach to molecules, especially by nucleophilic addition reactions. Such electrophilic reactive groups include carbonyls (including keto and dicarbonyls), carbonyl-like groups (which have similar reactivity and are structurally similar to carbonyls (including keto and dicarbonyls), masked carbonyls (which can be easily converted to a carbonyl (including keto and dicarbonyl)) or a protected carbonyl (which has similar reactivity to a carbonyl (including keto and dicarbonyl) after deprotection). The amino acids include amino acids having the structure of formula (IV),

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(RR') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R') -, wherein each R' is independently H, alkyl, or substituted alkyl;

J for

或

or

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

each R" is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R" optionally form a heterocycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

Each of R _{and R} is independently H, halogen, lower alkyl _, or substituted lower alkyl, or R and _R or two _R groups optionally form a cycloalkyl or hetero Cycloalkyl;

or - the A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl group comprising at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl) or Heterocycloalkyl;

or -J-R groups together form a monocyclic or bicyclic cycloalkyl group comprising at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl) or Heterocycloalkyl;

with the proviso that when A is phenylene and each _R3 is H, B is present; and when A is -( _CH2 ) _4- and each _R3 is H, B is not -NHC(O) ( _CH2CH2 )-; and when A and B are absent and each _{R3 is} H, R is other than methyl.

In addition, including amino acids having the structure of formula (V),

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R') -, wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

with the proviso that when A is phenylene, B is present; and when A is -(CH ₂ ) ₄ -, B is not -NHC(O)(CH ₂ CH ₂ )-; and when A and In the absence of B, R is not methyl.

Furthermore, including amino acids having the structure of formula (VI),

in:

B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene Hydrocarbyl, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or substituted hydrocarbylene)-,- S(O) _k -(where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted hydrocarbylene)-, -C(O)-, -C(O)-(alkylene Hydrocarbyl or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')-, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-, -CSN(R')-, -CSN (R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N( R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-,- C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')-, wherein each R' is independently H, alkyl or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

Each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2, or 3), -C(O)N(R') ₂ , -OR', and -S(O) _kR ', wherein each R' is independently H, alkyl, or substituted alkyl.

Additionally, the following amino acids are included:

Wherein the compound is optionally an amino group protected group, a carboxy protected compound or a salt thereof. In addition, any of the following unnatural amino acids can be incorporated into a non-natural amino acid polypeptide.

In addition, the following amino acids having the structure of formula (VII) are included,

in:

B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R'), -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')- , wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

Each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _k R', wherein each R' is independently H, alkyl or substituted alkyl; And n is 0 to 8;

The limitation is that when A is -(CH ₂ ) ₄ -, B is not -NHC(O)(CH ₂ CH ₂ )-.

Additionally, the following amino acids are included:

and

The compound wherein the compound is optionally an amino-protected compound, optionally a carboxyl-protected compound, optionally an amino-protected and carboxyl-protected compound, or a salt thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

In addition, the following amino acids having the structure of formula (VIII) are included,

wherein A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted Lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene radical, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R'), -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R')- , wherein each R' is independently H, alkyl, or substituted alkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R2 is optional and when present is OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide. In addition, the following amino acids having the structure of formula (IX) are included,

B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R') -, wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

wherein each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _k R', wherein each R' is independently H, alkyl or substituted alkyl .

Additionally, the following amino acids are included:

, wherein the compound is optionally an amino-protected compound, optionally a carboxyl-protected compound, optionally an amino-protected and carboxyl-protected compound, or a salt thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

In addition, the following amino acids having the structure of formula (X) are included,

wherein B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Carboalkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O) -, -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R' )-, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-, -CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N( R')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S) N(R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-,-C(R')=NN=,-C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R' )-, wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

Each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _k R', wherein each R' is independently H, alkyl or substituted alkyl; And n is 0 to 8.

Additionally, the following amino acids are included:

and

其中所述化合物视需要为氨基受保护的化合物，视需要为羧基受保护的化合物，视需要为氨基受保护且羧基受保护的化合物，或其盐。此外，可将这些非天然氨基酸和以下非天然氨基酸中的任一种并入非天然氨基酸多肽中。

The compound wherein the compound is optionally an amino-protected compound, optionally a carboxyl-protected compound, optionally an amino-protected and carboxyl-protected compound, or a salt thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

In addition to single carbonyl structures, the unnatural amino acids described herein can include groups such as dicarbonyls, quasi-dicarbonyls, masked dicarbonyls, and protected dicarbonyls.

For example, include the following amino acids having the structure of formula (XI),

wherein A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted Lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene radical, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R') -, wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R2 is optional and when present is OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.

In addition, the following amino acids having the structure of formula (XII) are included,

B is optional and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or Substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O) _k (hydrocarbylene or substituted alkylene)-, -C(O)- , -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R') -, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-,- CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N(R ')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N (R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R') =NN(R')-, -C(R')=NN=, -C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R') -, wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

wherein each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _k R', wherein each R' is independently H, alkyl or substituted alkyl .

Additionally, the following amino acids are included:

和

其中所述化合物视需要为氨基受保护的化合物，视需要为羧基受保护的化合物，视需要为氨基受保护且羧基受保护的化合物，或其盐。此外，可将这些非天然氨基酸和以下非天然氨基酸中的任一种并入非天然氨基酸多肽中。

and

The compound wherein said compound is optionally an amino-protected compound, optionally a carboxyl-protected compound, optionally an amino-protected and carboxyl-protected compound, or a salt thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

In addition, the following amino acids having the structure of formula (XIII) are included,

wherein B is optional and, when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower Carboalkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(hydrocarbylene or substituted hydrocarbylene)-, -S-, -S-(hydrocarbylene or substituted alkylene)-, -S(O) _k- (where k is 1, 2 or 3), -S(O)k(hydrocarbylene or substituted alkylene)-, -C(O) -, -C(O)-(hydrocarbylene or substituted hydrocarbylene)-, -C(S)-, -C(S)-(hydrocarbylene or substituted hydrocarbylene)-, -N(R' )-, -NR'-(hydrocarbylene or substituted hydrocarbylene)-, -C(O)N(R')-, -CON(R')-(hydrocarbylene or substituted hydrocarbylene)-, -CSN(R')-, -CSN(R')-(hydrocarbylene or substituted hydrocarbylene)-, -N(R')CO-(hydrocarbylene or substituted hydrocarbylene)-, -N( R')C(O)O-, -S(O) _k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S) N(R')-, -N(R')S(O) _k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-,-C(R')=NN=,-C(R') ₂ -N=N- and -C(R') ₂ -N(R')-N(R' )-, wherein each R' is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

Each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2 or 3), -C(O)N(R') ₂ , -OR' and -S(O) _k R', wherein each R' is independently H, alkyl or substituted alkyl; And n is 0 to 8.

Additionally, the following amino acids are included:

and

其中所述化合物视需要为氨基受保护的化合物，视需要为羧基受保护的化合物，视需要为氨基受保护且羧基受保护的化合物，或其盐。此外，可将这些非天然氨基酸和以下非天然氨基酸中的任一种并入非天然氨基酸多肽中。

The compound wherein the compound is optionally an amino-protected compound, optionally a carboxyl-protected compound, optionally an amino-protected and carboxyl-protected compound, or a salt thereof. In addition, any of these unnatural amino acids and the following unnatural amino acids can be incorporated into unnatural amino acid polypeptides.

In addition, the following amino acids having the structure of formula (XIV) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

_X is C, S, or S(O); and L is hydrocarbylene, substituted hydrocarbylene, N(R')(hydrocarbylene), or N(R')(substituted hydrocarbylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of formula (XIV-A) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

L is hydrocarbylene, substituted hydrocarbylene, N(R')(hydrocarbylene) or N(R')(substituted hydrocarbylene), where R' is H, alkyl, substituted alkyl, ring Alkyl or substituted cycloalkyl.

In addition, the following amino acids having the structure of formula (XIV-B) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

L is hydrocarbylene, substituted hydrocarbylene, N(R')(hydrocarbylene) or N(R')(substituted hydrocarbylene), where R' is H, alkyl, substituted alkyl, ring Alkyl or substituted cycloalkyl.

In addition, the following amino acids having the structure of formula (XV) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

X ₁ is C, S, or S(O); and n is O, 1, 2, 3, 4, or 5; and each of R ⁸ and R ⁹ on each CR ⁸ R ⁹ group is independently selected from Groups consisting of: H, alkoxy, alkylamino, halogen, alkyl, aryl, or any of which ^R8 and ^R9 may together form =O or cycloalkyl, or any of which is combined with Adjacent R groups may together form a ^cycloalkyl group.

In addition, the following amino acids having the structure of formula (XV-A) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

n ^is 0, 1, 2, 3, 4 or 5; and R and ^R on each CR ⁸ R ⁹ group are each independently selected from the group consisting of the following groups: H, alkoxy, Alkylamino, halogen, alkyl, aryl, or any of which ^R8 and ^R9 may together form =0 or cycloalkyl, or any of these groups together with adjacent ^R8 groups may form cycloalkyl.

In addition, the following amino acids having the structure of formula (XV-B) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

n ^is 0, 1, 2, 3, 4 or 5; and R and ^R on each CR ⁸ R ⁹ group are each independently selected from the group consisting of the following groups: H, alkoxy, Alkylamino, halogen, alkyl, aryl, or any of which ^R8 and ^R9 may together form =0 or cycloalkyl, or any of these groups together with adjacent ^R8 groups may form cycloalkyl.

In addition, the following amino acids having the structure of formula (XVI) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

_X is C, S, or S(O); and L is hydrocarbylene, substituted hydrocarbylene, N(R')(hydrocarbylene), or N(R')(substituted hydrocarbylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of formula (XVI-A) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

L is hydrocarbylene, substituted hydrocarbylene, N(R')(hydrocarbylene) or N(R')(substituted hydrocarbylene), where R' is H, alkyl, substituted alkyl, ring Alkyl or substituted cycloalkyl.

In addition, the following amino acids having the structure of formula (XVI-B) are included,

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

L is hydrocarbylene, substituted hydrocarbylene, N(R')(hydrocarbylene) or N(R')(substituted hydrocarbylene), where R' is H, alkyl, substituted alkyl, ring Alkyl or substituted cycloalkyl.

Furthermore, including amino acids having the structure of formula (XVII),

in:

A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower Carboalkenylene, Alkynylene, Lower Heteroalkylene, Substituted Heteroalkylene, Lower Heterocycloalkylene, Substituted Lower Heterocycloalkylene, Arylene, Substituted Arylene , heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

M for

wherein (a) represents a bond to the A group, and (b) represents a bond to the corresponding carbonyl, R _{and R} are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkane or substituted cycloalkyl, or R _and R _or two _R groups or two _R groups optionally form a cycloalkyl or heterocycloalkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_T is a bond, C(R)(R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R2 is optional and when present is OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.

Furthermore, including amino acids having the structure of formula (XVIII),

in:

M is -C(R ₃ )-,

wherein (a) represents a bond to the A group, and (b) represents a bond to the corresponding carbonyl, R _{and R} are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkane or substituted cycloalkyl, or R _and R _or two _R groups or two _R groups optionally form a cycloalkyl or heterocycloalkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

_T is a bond, C(R)(R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

_R is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and

_R is optional and when present is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide;

Each R _a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R') ₂ , -C(O) _k R' (where k is 1, 2, or 3), -C(O)N(R') ₂ , -OR', and -S(O) _kR ', wherein each R' is independently H, alkyl, or substituted alkyl.

Furthermore, including amino acids having the structure of formula (XIX),

in:

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and

T ₃ is O or S.

In addition, including amino acids having the structure of formula (XX),

in:

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of formula (XXI) are included:

和

and

The synthesis of p-acetyl-(+/-)-phenylalanine and m-acetyl-(+/-)-phenylalanine is described in Zhang, Z. et al., incorporated by reference. Biochemistry 42:6735-6746 (2003). Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared by those skilled in the art. Additionally, non-limiting exemplary syntheses of the unnatural amino acids included herein are provided in Figure 4, Figures 24-34, and Figures 36-39.

In some embodiments, a polypeptide comprising an unnatural amino acid is chemically modified to generate a reactive carbonyl or dicarbonyl functional group. For example, aldehyde functional groups useful for conjugation reactions can arise from functional groups having adjacent amino and hydroxyl groups. Where the biologically active molecule is a polypeptide, for example, an N-terminal serine or threonine (which may be normally present or may be exposed by chemical or enzymatic digestion) may be used under mild oxidative cleavage conditions using periodate. ) to generate the aldehyde functional group. See, e.g., Gaertner et al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3: 138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are limited to amino acids at the N-terminus of peptides or proteins.

In the present invention, unnatural amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functions. For example, 5-hydroxylysine has a hydroxyl group adjacent to an ε amine. Reaction conditions for production of aldehydes generally involve the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves the addition of about 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, eg, US Patent No. 6,423,685.

Carbonyl or dicarbonyl functional groups can selectively react with hydroxylamine-containing reagents in aqueous solution under mild conditions to form corresponding oxime linkages that are stable under physiological conditions. See, eg, Jencks, W.P., J.Am.Chem.Soc. 81, 475-481 (1959); Shao, J. and Tam, J.P., J.Am.Chem.Soc. 117:3893-3899 (1995) . Furthermore, the unique reactivity of the carbonyl or dicarbonyl group allows selective modification in the presence of other amino acid side chains. See, e.g., Cornish, V.W. et al., J.Am.Chem.Soc. 118:8150-8151 (1996); Geoghegan, K.F. & Stroh, J.G., Bioconjug.Chem.3:138-146 (1992) ; Mahal, L K. et al., Science 276:1125-1128 (1997).

Structure and Synthesis of Unnatural Amino Acids (Amino Acids Containing Hydroxylamine)

The entirety of US Provisional Patent Application No. 60/638,418 is incorporated herein by reference. Thus, in Section V of U.S. Provisional Patent Application No. 60/638,418 (titled "Non-natural Amino Acids"), Part B (titled "Structure and Synthesis of Non-Natural Amino Acids: Hydroxylamine-Containing Amino Acids" ) is fully applicable to the methods, compositions (including Formulas I-XXXV), techniques, and and policy, to the extent that the disclosure is provided entirely herein.

Chemical Synthesis of Unnatural Amino Acids

Many unnatural amino acids suitable for use in the present invention are commercially available, eg, from Sigma (USA) or Aldrich (Milwaukee, WI, USA). Those amino acids that are not commercially available are optionally synthesized as provided herein or as provided in various publications or using standard methods known to those of skill in the art. For techniques of organic synthesis, see, e.g., Fessendon and Fessendon, Organic Chemistry (1982, 2nd ed., Willard Grant Press, Boston Mass.); March, Advanced Organic Chemistry (3rd ed., 1985, Wiley and Sons, New York) and Carey and Sundberg, Advanced Organic Chemistry (3rd Edition, Parts A and B, 1990, Plenum Press, New York). Other publications describing the synthesis of unnatural amino acids include, for example, WO2002/085923 entitled "In vivo incorporation of Unnatural Amino Acids"; Matsoukas et al., (1995) J. Med. Chem. , 38, 4660-4669; King, FE & Kidd, DAA (1949) A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates. J. Chem. Soc , 3315-3319; Friedman, OM & Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J.Am. Chem.Soc. 81,3750-3752; Craig, JC et al. (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[-(diethylamino)-1- methylbutyl]amino]quinoline (Chloroquine). J.Org.Chem .53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J.Med.Chem 26 , 201-5; Koskinen, AMP & Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J.Org. Chem. 54, 1859-1866; Christie, BD & Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates from L-Asparagine. Application to the TotalSynthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium IonCyclization. J.Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha -Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of L-andD-alpha-Amino-Adipfc Acids, L-alpha-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and Subasinghe et al., (1992) Quisqualic acid analogues : synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site. J. Med. Chem. 35: 4602-7. See also US Patent Publication No. US 2004/0198637, entitled "Protein Arrays," which is incorporated herein by reference.

A. Carbonyl Reactive Group

Amino acids with carbonyl-reactive groups allow a variety of reactions to attach to molecules, including but not limited to PEG or other water-soluble molecules, particularly by nucleophilic addition or aldol condensation reactions.

其中n为0-10；R₁为烷基、芳基、经取代的烷基或经取代的芳基；R₂为H、烷基、芳基、经取代的烷基和经取代的芳基；并且R₃为H、氨基酸、多肽或氨基端修饰基团，并且R₄为H、氨基酸、多肽或羧基端修饰基团。在一些实施例中，n为1，R₁为苯基，并且R₂为简单烷基(即，甲基、乙基或丙基)，并且酮部分是位于相对于烷基侧链的对位上。在一些实施例中，n为1，R₁为苯基，并且R₂为简单烷基(即，甲基、乙基或丙基)，并且酮部分是位于相对于烷基侧链的间位上。Exemplary carbonyl-containing amino acids can be represented as follows,

wherein n is 0-10; R is alkyl, aryl, substituted alkyl, or substituted aryl; _R is H, _alkyl , aryl, substituted alkyl, and substituted aryl and R ₃ is H, an amino acid, a polypeptide, or an amino-terminal modification group, and R ₄ is H, an amino acid, a polypeptide, or a carboxyl-terminal modification group. In some embodiments, n is ₁ , R is phenyl, and _R is simple alkyl (i.e., methyl, ethyl, or propyl), and the ketone moiety is in the para position relative to the alkyl side chain superior. In some embodiments, n is ₁ , R is phenyl, and _R is a simple alkyl (i.e., methyl, ethyl, or propyl), and the ketone moiety is located in the meta position relative to the alkyl side chain superior.

The synthesis of p-acetyl-(+/-)-phenylalanine and/m-acetyl-(+/-)-phenylalanine is described in Zhang, Z. et al., Biochemistry 42:6735-6746 (2003), which is incorporated herein by reference. Other carbonyl-containing amino acids can be similarly prepared by those skilled in the art.

In some embodiments, a polypeptide comprising a non-naturally encoded amino acid is chemically modified to generate a reactive carbonyl functionality. For example, aldehyde functional groups useful for conjugation reactions can arise from functional groups having adjacent amino and hydroxyl groups. Where the biologically active molecule is a polypeptide, for example, an N-terminal serine or threonine (which may be normally present or may be exposed by chemical or enzymatic digestion) may be used under mild oxidative cleavage conditions using periodate. ) to generate the aldehyde functional group. See, e.g., Gaertner et al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3: 138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are limited to amino acids at the N-terminus of peptides or proteins.

In the present invention, non-naturally encoded amino acids with adjacent hydroxyl and amino groups can be incorporated into polypeptides as "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to an ε amine. Reaction conditions for production of aldehydes generally involve the addition of a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves the addition of about 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide, followed by incubation in the dark for about 10 minutes. See, eg, US Patent No. 6,423,685, which is incorporated herein by reference.

The carbonyl functional group can selectively react with reagents containing hydrazine, hydrazide, hydroxylamine or semicarbazide in aqueous solution under mild conditions to form corresponding hydrazone linkages, oxime linkages or semicarbazone linkages which are stable under physiological conditions, respectively. See, e.g., Jencks, W.P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, J.P., J. Am. Chem. Soc. 117:3893-3899 (1995) . Furthermore, the unique reactivity of the carbonyl allows selective modification in the presence of other amino acid side chains. See, e.g., Cornish, V.W. et al., J.Am.Chem.Soc. 118:8150-8151 (1996); Geoghegan, K.F. & Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, L. K. et al., Science 276: 1125-1128 (1997).

B. Hydrazine, hydrazide or semicarbazide reactive groups

Non-naturally encoded amino acids containing nucleophilic groups such as hydrazine, hydrazide, or semicarbazide allow reaction with various electrophilic groups to form conjugates (including, but not limited to, reactions with PEG or other water-soluble polymers). form a bond).

Exemplary amino acids containing hydrazine, hydrazide or semicarbazide moieties can be represented as follows,

wherein n is 0-10; R is _alkyl , aryl, substituted alkyl or substituted aryl or absent; X is 0, N or S or absent; _R is H, amino acid, polypeptide or an amino-terminal modification group, and R ₃ is H, an amino acid, a polypeptide or a carboxy-terminal modification group.

In some embodiments, n is 4, _R is absent, and X is N. In some embodiments, n is 2, _R is absent, and X is absent. In some embodiments, n is ₁ , R is phenyl, X is O, and the oxygen atom is para to the aliphatic group on the aryl ring.

Amino acids containing hydrazide, hydrazine and semicarbazide moieties are available from commercial sources. For example, L-glutamic acid-γ-hydrazide can be purchased from Sigma Chemical (St. Louis, MO). Other amino acids that are not commercially available can be prepared by those skilled in the art. See, eg, US Patent No. 6,281,211, which is incorporated herein by reference.

Polypeptides containing non-naturally encoded amino acids with hydrazide, hydrazine, or semicarbazide functional groups react efficiently and selectively with a variety of molecules containing aldehyde or other functional groups with similar chemical reactivity. See, eg, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995). The unique reactivity of the hydrazide, hydrazine, and semicarbazide functional groups makes them suitable for use with nucleophilic groups present on 20 common amino acids (including, but not limited to, serine or threonine hydroxyl groups or lysine and N-terminal amino groups) Compared to aldehydes, ketones and other electrophilic groups have more significant reactivity.

C. Amino acids containing aminooxy groups

Non-naturally encoded amino acids containing aminooxy groups (also known as hydroxylamine groups) allow for reaction with various electrophilic groups to form conjugates (including, but not limited to, reaction with PEG or other water-soluble polymers to form conjugates ). Like hydrazines, hydrazides, and semicarbazides, the enhanced nucleophilicity of aminooxy groups allows them to react efficiently and selectively with a variety of molecules containing aldehydes or other functional groups with similar chemical reactivity. See, eg, Shao, J. and Tam, J., J. Am. Chem. Soc, 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). While the product of the reaction with a hydrazino group is the corresponding hydrazone, oximes generally result from the reaction of an aminooxy group with a carbonyl-containing group such as a keto group.

Exemplary amino acids containing aminooxy groups can be represented as follows,

wherein n is 0-10; R is _alkyl , aryl, substituted alkyl or substituted aryl or absent; X is O, N, S or absent; m is 0-10; Y is C(O) or absent; _R2 is H, an amino acid, a polypeptide, or an amino-terminal modification group, and _R3 is H, an amino acid, a polypeptide, or a carboxy-terminal modification group. In some embodiments, n is ₁ , R is phenyl, X is O, m is 1, and Y is present. In some embodiments, n is ₂ , R and X are absent, m is 0, and Y is absent.

Aminooxy-containing amino acids can be prepared from readily available amino acid precursors (homoserine, serine and threonine). See, eg, M. Carrasco and R. Brown, J. Org. Chem. 68:8853-8858 (2003). Certain aminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butanoic acid, have been isolated from natural sources (Rosenthal, G, Life Sci. 60:1635-1641 (1997)) . Other aminooxy-containing amino acids can be prepared by those skilled in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes them ideal for the selective modification of peptides and other biomolecules. Organic azides (especially aliphatic azides) and alkynes are generally stable to common reactive chemical conditions. In particular, azide and alkyne functional groups are inert to the side chains (ie, R groups) of the 20 common amino acids found in naturally occurring polypeptides. However, upon further investigation, it was shown that the azido and alkynyl groups are "spring-loaded" and that they can be selectively and efficiently added via the Hoo radical [3+2] cycloaddition The reaction proceeds to produce the corresponding triazole. See, for example, Chin J. et al., Science 301:964-7 (2003); Wang, Q. et al., J.Am.Chem.Soc.125, 3192-3193 (2003); Chin, J.W. et al., J . Am. Chem. Soc. 124:9026-9027 (2002).

Because the Hu's radical cycloaddition involves a selective cycloaddition (see, for example, Padwa, A., in COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4 (Trost, BM Ed., 1991), pp. 1069-1109; Huisgen , R., in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (Padwa, A. Ed., 1984), pp. 1-176) instead of nucleophilic substitution, so non- The incorporation of a naturally encoded amino acid allows the resulting polypeptide to be selectively modified at the position of the non-naturally encoded amino acid. Cycloaddition reactions involving GH (e.g., hGH) polypeptides containing azide or alkyne moieties can be achieved by reducing Cu(II) to Cu(I) in situ under catalytic amounts under aqueous conditions at room temperature. The addition of Cu(II) in the presence of an additive (including, but not limited to, in the form of CuSO ₄ in catalytic amounts) was performed. See, eg, Wang, Q. et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, CW et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev et al., Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agents include, including but not limited to, ascorbate, copper metal, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe2 ⁺ , Co2 ⁺ and the applied potential.

In some cases, where a Hooggen [3+2] cycloaddition reaction between an azide and an alkyne is desired, the GH (e.g., hGH) polypeptide comprises a non-naturally encoded amino acid that includes an alkyne moiety, and will The water soluble polymer linked to the amino acid contains an azide moiety. Alternatively, the reverse reaction (ie, via the reaction of an azide moiety on the amino acid with an alkyne moiety present on the water soluble polymer) can also be performed.

Azide functional groups can also optionally be reacted with water soluble polymers containing aryl ester moieties and suitably functionalized with arylphosphine moieties to generate amide linkages. The arylphosphino group reduces the azide in situ and the resulting amine then efficiently reacts with the adjacent ester linkage to generate the corresponding amide. See, eg, E. Saxon and C. Bertozzi, Science 287, 2007-2010 (2000). Amino acids containing an azide group can be alkyl azides (including but not limited to, 2-amino-6-azido-1-hexanoic acid) or aryl azides (p-azido-phenylpropanoid acid).

Exemplary water soluble polymers containing aryl ester and phosphine moieties can be represented as follows,

Where X can be O, N, S or absent, Ph is phenyl, W is a water soluble polymer and R can be H, alkyl, aryl, substituted alkyl and substituted aryl. Exemplary R groups include, but are not limited to, _-CH2 , -C( _CH3 ) ₃ , -OR', -NR'R", -SR', -halogen, -C(O)R', -CONR 'R', -S(O) _2R ', -S(O) _2NR'R ", -CN, and _-NO2 . R', R", R'', and R"" each independently refer to hydrogen , substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1-3 halogens), substituted or unsubstituted Alkyl, alkoxy or thioalkoxy or arylalkyl. For example, when a compound of the invention includes multiple R groups, as when multiples of the R', R", R'', and R"" groups are present, each is independently selected That way, each R group is also independently selected. When R' and R" are attached to the same nitrogen atom, they can combine with said nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will appreciate that the term "alkyl" is intended to Include groups comprising _carbon atoms bonded to non-hydrogen groups, such as haloalkyl groups (including but not limited to _-CF3 and _-CH2CF3 ) and acyl groups (including but not limited to -C( O) _CH3 , -C(O) _CF3 , -C(O) _{CH2OCH3 ,} etc.).

Azide functional groups can also optionally be reacted with water soluble polymers containing thioester moieties and suitably functionalized with arylphosphine moieties to generate amide linkages. The arylphosphino group reduces the azide in situ and the resulting amine then efficiently reacts with the thioester linkage to generate the corresponding amide. Exemplary water soluble polymers containing thioester and phosphine moieties can be represented as follows,

wherein n is 1-10; X can be O, N, S or absent, Ph is phenyl and W is a water-soluble polymer. An exemplary amino acid containing an alkyne moiety can be represented as follows,

wherein n is 0-10; R is _alkyl , aryl, substituted alkyl or substituted aryl or absent; X is O, N, S or absent; m is _0-10 , R is H, an amino acid, a polypeptide, or an amino-terminal modification group, and _R is H, an amino acid, a polypeptide, or a carboxy-terminal modification group. In some embodiments, n is 1, R is _phenyl , X is absent, m is 0 and the acetylene moiety is in the para position relative to the alkyl side chain. In some embodiments, n is ₁ , R is phenyl, X is O, m is 1, and propargyloxy is in the para position relative to the alkyl side chain (i.e., O-propargyl -tyrosine). In some embodiments, n is 1, R and X are absent and m is 0 (ie, propargylglycine) _.

Amino acids containing alkyne moieties are commercially available. For example, propargylglycine is commercially available from Peptech (Burlington, MA). Alternatively, amino acids containing alkyne moieties can be prepared according to standard methods. For example, p-propargyloxyphenylalanine can be synthesized, for example, as described in Deiters, A. et al., J. Am. Chem. Soc. 125:11782-11783 (2003), and 4 -Alkynyl-L-phenylalanine can be synthesized as described in Kayser, B. et al., Tetrahedron 53(7):2475-2484 (1997). Other amino acids containing alkyne moieties can be prepared by those skilled in the art.

Exemplary amino acids containing azide moieties can be represented as follows,

wherein n is 0-10; R is _alkyl , aryl, substituted alkyl, substituted aryl or absent; X is O, N, S or absent; m is _0-10 ; R is H, an amino acid, a polypeptide, or an amino-terminal modification group, and _R is H, an amino acid, a polypeptide, or a carboxy-terminal modification group. In some embodiments, n is ₁ , R is phenyl, X is absent, m is 0, and the azide moiety is para to the side chain of the alkyl. In some embodiments, n is 0-4, and R ₁ and X are absent, and m is 0. In some embodiments, n is 1, R ₁ is phenyl, X is 0, m is 2, and the β-azidoethoxy moiety is in the para position relative to the alkyl side chain.

Amino acids containing azide moieties can be obtained from commercial sources. For example, 4-azidophenylalanine is available from Chem-Impex International, Inc. (Wood Dale, IL). For those amino acids containing an azide moiety that are not commercially available, the azido group can be prepared relatively easily using standard methods known to those skilled in the art, including, but not limited to, via a suitable leaving group ( Displacement including, but not limited to, halide, mesylate, tosylate groups) or by ring opening of an appropriately protected lactone is relatively easy. See, eg, March, Advanced Organic Chemistry (3rd Ed., 1985, Wiley and Sons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of β-substituted aminothiol functional groups makes them highly suitable for the selective modification of aldehyde-containing polypeptides and other biomolecules through the formation of thiazolidines. See, eg, J. Shao and J. Tam, J. Am. Chem. Soc. 1995, 117(14) 3893-3899. In some embodiments, a β-substituted aminothiol amino acid can be incorporated into a GH (eg, hGH) polypeptide and then reacted with a water soluble polymer comprising an aldehyde functional group. In some embodiments, a water soluble polymer, drug conjugate or other cargo can be coupled to a GH (eg, hGH) polypeptide comprising a β-substituted aminothiol amino acid by forming a thiazolidine.

Cellular uptake of unnatural amino acids

Uptake of unnatural amino acids by cells is an issue generally considered in the design and selection of, including but not limited to, unnatural amino acids for incorporation into proteins. For example, the high charge density of α-amino acids suggests that these compounds are unlikely to be cell permeable. Natural amino acids are taken up into eukaryotic cells by a number of protein-based transport systems. A rapid screen can be performed which assesses which unnatural amino acids, if any, are taken up by cells. See, e.g., in, e.g., U.S. Patent Publication No. US 2004/0198637 entitled "Protein Arrays," which is incorporated herein by reference, and Liu, DR & Schultz, PG (1999) Progress toward the evolution of an organism with an expanded genetic code. Toxicity assay in PNAS United States 96:4780-4785. While absorption is readily analyzed by various assays, another option for designing unnatural amino acids to undergo cellular uptake pathways is to provide biosynthetic pathways to produce amino acids in vivo.

(i) Biosynthesis of unnatural amino acids

Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. Although methods for the biosynthesis of particular unnatural amino acids may not exist in nature, including, but not limited to, in cells, the present invention provides such methods. For example, biosynthetic pathways for unnatural amino acids are optionally generated in host cells by adding new enzymes or altering existing host cell pathways. Other novel enzymes are optionally naturally occurring enzymes or artificially developed enzymes. For example, the biosynthesis of p-aminophenylalanine (as presented in an example of WO 2002/085923 entitled "In vivoincorporation of unnatural amino acids") relies on the addition of a combination of known enzymes from other organisms . Genes for these enzymes can be introduced into eukaryotic cells by transforming the cells with a plasmid containing the genes. A gene, when expressed in a cell, provides an enzymatic pathway to synthesize a desired compound. Examples of the types of enzymes that are optionally added are provided in the Examples below. Additional enzyme sequences can be found, for example, in Genbank. Artificially developed enzymes are also optionally added to cells in the same manner. In this way, the cell's organelles and resources are manipulated to produce unnatural amino acids.

Various methods are available to generate novel enzymes for use in biosynthetic pathways or for the development of existing pathways. For example, recursive recombination including, but not limited to, as developed by Maxygen, Inc. (available at www.maxygen.com ) may be used to develop novel enzymes and pathways as desired. See, e.g., Stemmer (1994), Rapid evolution of aprotein in vitro by DNA shuffling, Nature 370(4):389-391; and Stemmer, (1994), DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc .Natl.Acad.Sci.USA. , 91:10747-10751. Likewise, DesignPath™ developed by Genencor (available at www.genencor.com ) is used optionally for metabolic pathway engineering, including, but not limited to, for the production of O-methyl-L- Pathway engineering of tyrosine. This technique uses a combination of novel genes (including but not limited to those identified by functional genomics) and molecular evolution and design to recreate existing pathways in a host organism. Diversa Corporation (available at www.diversa.com ) also provides technologies for rapid screening of gene libraries and gene pathways, including but not limited to, for generating new pathways.

Typically, the unnatural amino acids produced by the engineered biosynthetic pathways of the invention are in concentrations including, but not limited to, natural cellular amounts sufficient to achieve efficient protein biosynthesis but not to a degree that affects other amino acids or depletes cellular resources concentration produced. Typical concentrations produced in vivo in this manner are from about 10 mM to about 0.05 mM. Once cells have been transformed with plasmids containing genes for the enzymes required for a particular pathway and unnatural amino acids are produced, in vivo selection is used to further optimize the availability of unnatural amino acids for ribosomal protein synthesis and cell growth, if necessary. produce.

(b) Polypeptides with unnatural amino acids

Incorporation of unnatural amino acids can be performed for a variety of purposes including, but not limited to: modulating changes in protein structure and/or function, altering size, acidity, nucleophilicity, hydrogen bonding, hydrophobicity , accessibility of protease target sites, targeting a portion (including (but not limited to) portions of protein arrays), adding bioactive molecules, linking polymers, linking radionuclides, regulating serum half-life, regulating tissue penetration rate ( For example, tumors), modulation of active transport, modulation of tissue, cell or organ specificity or distribution, modulation of immunogenicity, modulation of protease resistance, etc. Proteins that include unnatural amino acids can have enhanced or even novel catalytic or biophysical properties. For example, the following properties can optionally be altered by including unnatural amino acids in proteins: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic ability, half-life (including but not limited to ) serum half-life), the ability to react with other molecules (including but not limited to, covalently or non-covalently), and the like. Compositions comprising proteins comprising at least one unnatural amino acid are useful in, including but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to, antibodies), and (including But not limited to)) the study of protein structure and function. See, eg, Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology , 4:645-652.

In one aspect of the invention, the composition comprises at least one protein having at least 1 unnatural amino acid, including but not limited to having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 or more unnatural amino acids. The unnatural amino acids can be the same or different, including (but not limited to) 1, 2, 3, 4, 5, 6, 7, 8, 9 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different positions of 1 or 10 or more different unnatural amino acids . In another aspect, a composition includes a protein in which at least one (but less than all) of the specified amino acids present in the protein are substituted with an unnatural amino acid. For a given protein with multiple unnatural amino acids, the unnatural amino acids can be the same or different (including but not limited to, proteins can include 2 or more different types of unnatural amino acids, or may include 2 identical unnatural amino acids). For a specified protein with more than 2 unnatural amino acids, the unnatural amino acids may be the same, different, or a combination of multiple unnatural amino acids of the same type and at least one different unnatural amino acid.

Contemplated proteins or polypeptides having at least one unnatural amino acid are a feature of the invention. The invention also includes polypeptides or proteins having at least one unnatural amino acid produced using the compositions and methods of the invention. Excipients, including but not limited to pharmaceutically acceptable excipients, may also be present with the protein.

To the extent that a protein or polypeptide of interest having at least 1 unnatural amino acid is produced in a eukaryotic cell, the protein or polypeptide will typically include a eukaryotic post-translational modification. In certain embodiments, the protein comprises at least 1 unnatural amino acid and at least 1 post-translational modification by eukaryotic cells in vivo, wherein the post-translational modification is not by prokaryotic cells. For example, post-translational modifications include, including but not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid linkages modification, glycosylation, and more. In one aspect, the post-translational modification comprises attachment of an oligosaccharide (including but not limited to, (GlcNAc-Man)2-Man-GlcNAc-GlcNAc) to asparagine via a GlcNAc-asparagine bond. See Table 1, which lists some examples of N-linked oligosaccharides of eukaryotic proteins (other residues may also be present, not shown in the table). In another aspect, the post-translational modification includes the conversion of oligosaccharides (including but not limited to Gal-GalNAc, Gal-GlcNAc, Gal-GalNAc, Gal-GlcNAc, etc.) etc.) linked to serine or threonine.

Table 1: Examples of GlcNAc-bonded oligosaccharides

In another aspect, post-translational modifications include modification of precursors, including but not limited to, precalcitonin, calcitonin gene-related peptide precursor, preproparathyroid hormone, preproinsulin, insulin Pro-opiomelanocortin (prepro-opiomelanocortin, pro-opiomelanocortin, etc.) undergoes proteolytic processing, assembles into multi-subunit proteins or undergoes macromolecular assembly, and translates into another cell in the cell A site (including, but not limited to, translation into organelles such as the endoplasmic reticulum, Golgi apparatus, nucleus, lysosome, peroxisome, mitochondria, chloroplast, vacuole, etc., or via the secretory pathway) . In certain embodiments, proteins comprise secretory or localization sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, and the like. US Patent Nos. 4,963,495 and 6,436,674, incorporated herein by reference, describe in detail constructs designed to improve secretion of GH (eg, hGH) polypeptides.

One advantage of unnatural amino acids is that they provide additional chemical moieties that can be used to add other molecules. These modifications can be made in vivo or in vitro in eukaryotic or non-eukaryotic cells. Thus, in certain embodiments, post-translational modifications are achieved by unnatural amino acids. For example, post-translational modifications can be achieved through nucleophilic-electrophilic reactions. Most reactions currently used to selectively modify proteins involve the formation of covalent bonds between nucleophilic and electrophilic reaction partners, including (but not limited to) reactions of α-haloketones with histidine or cysteine side chains . Selectivity in these cases is determined by the number and accessibility of nucleophilic residues in the protein. In proteins of the invention, other more selective reactions may be employed, such as the reaction of unnatural keto-amino acids with hydrazides or aminooxy compounds in vitro and in vivo. See, eg, Cornish et al., (1996) J. Am. Chem. Soc , 118:8150-8151; Mahal et al., (1997) Science , 276:1125-1128; Wang et al., (2001) Science 292: 498-500; Chin et al., (2002) J.Am.Chem.Soc. 124:9026-9027; Chin et al., (2002) Proc.Natl.Acad.Sci . , 99:11020-11024; Wang et al. , (2003) Proc.Natl.Acad.Sci ., 100:56-61; Zhang et al., (2003) Biochemistry, 42:6735-6746; and Chin et al., (2003) Science, 301:964-7, All documents are incorporated herein by reference. This allows the selective labeling of virtually any protein with a number of reagents including fluorophores, cross-linkers, carbohydrate derivatives and cytotoxic molecules. See also US Patent No. 6,927,042 entitled "Glycoprotein synthesis," which is incorporated herein by reference. Post-translational modifications, including but not limited to, via azido amino acids can also be performed via Staudinger linkages (including, but not limited to, with triarylphosphine reagents). See, eg, Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24.

The present invention provides another highly efficient method of selectively modifying proteins, which involves the genetic incorporation of unnatural amino acids, including but not limited to, containing azide or alkynyl moieties, into proteins in response to a selector codon. These amino acid side chains can then be synthesized separately by reactions including, but not limited to, Hu's root [3+2] cycloaddition (see, e.g., Padwa, A., in Comprehensive Organic Synthesis, Vol. 4, (1991), Trost , BM, ed., Pergamon, Oxford, pp. 1069-1109; and Huisgen, R., in 1,3-Dipolar Cycloaddition Chemistry , (1984) Padwa, A. ed., Wiley, New York, pp. 1-176 ) are modified with including, but not limited to, alkynyl or azide derivatives. Because this method involves cycloaddition rather than nucleophilic substitution, proteins can be modified with extremely high selectivity. This reaction can be carried out with excellent regioselectivity (1,4 > 1,5) at room temperature under aqueous conditions by adding catalytic amounts of Cu(I) salts to the reaction mixture. See, eg, Tornoe et al., (2002) J.Org.Chem. 67:3057-3064; and Rostovtsev et al., (2002) Angew.Chem.Int.Ed. 41:2596-2599. Another method that can be used is ligand exchange on diarsenic compounds using the tetracysteine motif, see, eg, Griffin, et al., (1998) Science 281:269-272.

Molecules that can be added to proteins of the invention via [3+2] cycloaddition include virtually any molecule with an azide or alkynyl derivative moiety. Molecules include (but are not limited to) dyes, fluorophores, crosslinkers, carbohydrate derivatives, polymers (including but not limited to derivatives of polyethylene glycol), photocrosslinkers, cytotoxic compounds, hydrophilic and labels, derivatives of biotin, resins, beads, second proteins or polypeptides (or more), polynucleotides (including but not limited to DNA, RNA, etc.), metal chelators, cofactors, Fatty acids, carbohydrates, etc. These molecules can be added to unnatural amino acids with alkynyl groups (including but not limited to, p-propargyloxyphenylalanine) or unnatural amino acids with azido groups (including but not limited to, p-azido phenylalanine).

V. In Vivo Production of GH (e.g., hGH) Polypeptides Comprising Non-Genetically Encoded Amino Acids

GH (eg, hGH) polypeptides of the invention can be produced in vivo using modified tRNAs and tRNA synthetases to add or substitute amino acids that are not encoded in naturally occurring systems.

Methods for generating tRNAs and tRNA synthetases that utilize amino acids that are not encoded in naturally occurring systems are described, for example, in U.S. Patent Application Publication Nos. 2003/0082575 (No. 10/126,927) and 2003/0108885 (No. 10/126,931), said reference is incorporated herein by reference. These methods involve the generation of translation machinery that functions independently of synthetases and tRNAs that are endogenous to the translation system (and are therefore sometimes referred to as "orthogonal"). Typically, a translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, an O-RS preferentially aminoacylates an O-tRNA with at least one non-naturally occurring amino acid in a translation system, and the O-tRNA recognizes at least one alternative that cannot be recognized by other tRNAs in the system a. The translation system thus inserts the non-naturally encoded amino acid into the protein produced in the system in response to the encoded selector codon, thereby "substituting" the amino acid into its position in the encoded polypeptide.

Various orthogonal tRNA and aminoacyl tRNA synthetases have been described in the art for inserting specific synthetic amino acids into polypeptides and are generally suitable for use in the present invention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetases are described in Wang, L. et al., Proc. Natl. Acad. Sci. USA 100:56-61 (2003) and Zhang, Z. et al. People, Biochem. 42(22):6735-6746 (2003). Exemplary O-RSs, or portions thereof, are encoded by polynucleotide sequences and include the amino acid sequences disclosed in U.S. Patent Application Publications 2003/0082575 and 2003/0108885, each of which is incorporated by reference In this article. Corresponding O-tRNA molecules for use with O-RS are also described in U.S. Patent Application Publications 2003/0082575 (Serial No. 10/126,927) and 2003/0108885 (Serial No. 10/126,931), which are incorporated by reference in way incorporated into this article.

An example of an azido-specific O-tRNA/aminoacyl-tRNA synthetase system is described in Chin, J.W. et al., J. Am. Chem. Soc. 124:9026-9027 (2002). Exemplary O-RS sequences for p-azido-L-phenylalanine include, but are not limited to, the nucleotide sequence SEQ ID as disclosed in U.S. Patent Application Publication 2003/0108885 (Serial No. 10/126,931) NO: 14-16 and 29-32 and amino acid sequence SEQ ID NO: 46-48 and 61-64, said publication is incorporated herein by reference. Exemplary O-tRNA sequences suitable for use in the present invention include, but are not limited to, the nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. Patent Application Publication 2003/0108885 (No. 10/126,931), which discloses The case is incorporated herein by reference. Other examples of O-tRNA/aminoacyl-tRNA synthetase pairs specific for particular non-naturally encoded amino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No. 10/126,927), which Incorporated herein by reference. Incorporation of keto- and azide-containing amino acids into O-RS and O-tRNA in S. cerevisiae is described in Chin, J.W. et al., Science 301:964-967 (2003) .

Some other orthogonal pairs have been reported. Glutaminyl groups derived from Saccharomyces cerevisiae tRNAs and synthetases have been described (see, e.g., Liu, DR and Schultz, PG (1999) Proc. Natl. Acad. Sci. USA 96:4780-4785), aspartyl groups (see, for example, Pastrnak, M. et al., (2000) Helv. Chim. Acta 83:2277-2286) and tyrosyl (see, for example, Ohno, S. et al., (1998) J.Biochem.( Tokyo , Jpn.) 1 124:1065-1068; and Kowal, AK et al., (2001) Proc. middle. Glutaminyl (see, for example, Kowal, AK et al. (2001) Proc. Natl. Acad. Sci. USA 98:2268-2273) and tyrosyl (see, for example, Edwards, H. and Schimmel, P. (1990) Mol. Cell. Biol. 10: 1633-1641) The system of synthetases is applicable to Saccharomyces cerevisiae. The E. coli tyrosyl system has been used to incorporate 3-iodo-L-tyrosine into mammalian cells in vivo. See, Sakamoto, K. et al. (2002) Nucleic Acids Res. 30:4692-4699.

The use of O-tRNA/aminoacyl-tRNA synthetases involves the selection of specific codons encoding non-naturally encoded amino acids. While any codon can be used, it is often desirable to select codons that are rarely or never used in cells expressing the O-tRNA/aminoacyl tRNA synthetase. Exemplary codons include, for example, nonsense codons, such as stop codons (amber, ocher, and opal); four or more base codons; and rarely or never used codons. Other natural 3 base codons used.

A specific selector codon can be introduced into the GH (e.g., hGH) polynucleus using mutagenesis methods known in the art, including but not limited to site-specific mutagenesis, cassette mutagenesis, restriction selective mutagenesis, etc. appropriate position in the nucleotide coding sequence.

Methods for generating protein biosynthesis machinery components (such as O-RS, O-tRNA, and orthogonal O-tRNA/O-RS pairs) that can be used to incorporate non-naturally encoded amino acids are described in Wang, L. et al. People, Science 292: 498-500 (2001); Chin, J.W. et al., J. Am. Chem. Soc. 124: 9026-9027 (2002); Zhang, Z. et al., Biochemistry 42: 6735-6746 (2003 )middle. Methods and compositions for incorporation of non-naturally encoded amino acids in vivo are described in US Patent Application Publication 2003/0082575 (Serial No. 10/126,927), which is incorporated herein by reference. Methods for selecting orthogonal tRNA-tRNA synthetase pairs suitable for use in an organism's in vivo translation system are also described in U.S. Patent Application Publication Nos. 2003/0082575 (No. 10/126,927) and 2003/0108885 (No. No.), said publication is incorporated herein by reference. PCT Publication No. WO 04/035743, entitled "Site Specific Incorporation of Keto Amino Acids into Proteins," which is hereby incorporated by reference in its entirety, describes orthogonal RS and tRNA pairs for incorporation of keto amino acids. PCT Publication No. WO 04/094593, entitled "Expanding the Eukaryotic Genetic Code," which is incorporated herein by reference in its entirety, describes an orthogonal method for incorporating non-naturally encoded amino acids into eukaryotic host cells. RS and tRNA pairs.

The method for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase (O-RS) comprises: (a) producing at least one aminoacyl-tRNA synthetase (RS) derived from a first organism A library of (optionally mutant) RSs from first organisms including, but not limited to, such as Methanococcus jannaschii, Yokogawa virus, Halobacter, Escherichia coli, Archaeoglobus fulgii (A Prokaryotes such as .fulgidus), P.furiosus, P horikoshii, A. pernix, T. thermophilus, etc. , or eukaryotes; (b) select (and/or screen) members of a library of RS (and optionally mutant RS) that render an orthogonal tRNA (O - tRNA) aminoacylation, thereby providing a set of active (if necessary mutant) RS; and/or (c) selecting (optionally by negative selection) active RS (including but not limited to) mutant RS) of the set , the active RS preferentially aminoacylates the O-tRNA in the absence of a non-naturally encoded amino acid, thereby providing at least one recombinant O-RS; wherein the at least one recombinant O-RS is preferentially expressed in a non-naturally encoded Naturally encoded amino acids aminoacylate the O-tRNA.

In one embodiment, the RS is an inactive RS. Inactive RSs can be produced by mutating active RSs. For example, an inactive RS can be mutated by at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, or at least about 10 or more amino acids. Changed to produce different amino acids including, but not limited to, alanine.

Libraries of mutant RSs can be generated using various techniques known in the art including, but not limited to, rational design based on protein three-dimensional RS structures, or mutation of RS nucleotides in random or rational design techniques. For example, mutant RS can be generated by site-specific mutagenesis, random mutagenesis, differentially generated recombination mutagenesis, chimeric construction, rational design and by other methods described herein or known in the art.

In one embodiment, selection (and/or screening) of a library of RSs (and optionally mutant RSs) includes, but is not limited to, rendering orthogonal tRNAs (O-tRNAs) in the presence of non-naturally encoded amino acids and naturally occurring amino acids. Aminoacylated active members include the introduction into a plurality of cells of positive selection or selection markers including, but not limited to, antibiotic resistance genes and the like, and (optionally mutated) RS repertoires, wherein the positive selection and and/or selectable markers comprising at least one selector codon including, but not limited to, amber codons, ocher codons, or opal codons; growing said plurality of cells in the presence of a selection agent; Cells that survive (or exhibit a specific response) in the presence of an agent by inhibiting at least one selector codon in a positive selection or selection marker, thereby providing positively selected cells containing a collection of active (and optionally mutated) RSs subset of . Selection and/or screening agent concentrations can be varied as desired.

In one aspect, the positive selectable marker is the chloramphenicol acetyltransferase (CAT) gene and the selector codon is the amber stop codon in the CAT gene. Optionally, the positive selectable marker is the β-lactamase gene and the selector codon is the amber stop codon in the β-lactamase gene. In another aspect, positive-positive selection markers comprise fluorescent or luminescent selection markers or affinity-based selection markers (including but not limited to, cell surface markers).

In one embodiment, negatively selecting or screening a set of active RSs (optionally mutated) that preferentially aminoacylate O-tRNAs in the absence of non-naturally encoded amino acids comprises: negatively selecting or screening for A marker is introduced into a plurality of cells of a second organism together with a collection of active (and optionally mutated) RSs from positive selection or selection, wherein the negative selection or selection marker comprises at least one selector codon (comprising ( but not limited to) antibiotic resistance genes including but not limited to chloramphenicol acetyltransferase (CAT) gene); and identification of survival in a first medium supplemented with non-naturally encoded amino acids and selection or selection agents Or cells that exhibit a specific selection response but fail to survive or exhibit a specific response in a second medium that is not supplemented with non-naturally encoded amino acids and selection or selection agents, thereby providing cells with at least one recombinant O-RS Viable or screened cells. For example, the CAT identification protocol acts as a positive and/or negative selection in the identification of appropriate O-RS recombinants, as desired. For example, pools of clones are optionally replicated on growth plates containing a CAT comprising at least one selector codon, with or without one or more non-naturally encoded amino acids. It was therefore believed that only colonies grown on plates containing non-naturally encoded amino acids contained recombinant O-RS. In one aspect, the concentration of the selection (and/or screening) agent can be varied. In some aspects, the first organism and the second organism are different. Thus, the first organism and/or the second organism optionally includes: prokaryotes, eukaryotes, mammals, Escherichia coli, fungi, yeasts, archaea, eubacteria, plants, insects, protists, etc. . In other embodiments, the selection marker comprises a fluorescent or luminescent selection marker or an affinity-based selection marker.

In another embodiment, screening or selecting (including but not limited to, negative selection) a pool of active (optionally mutated) RSs comprises: isolating the pool of active mutant RSs from positive selection step (b) introducing a collection of negative selection or selection markers and active (optionally mutated) RSs into a plurality of cells of a second organism, wherein said negative selection or selection markers comprise at least one selector codon (including but without limitation) a virulence marker gene comprising at least one selector codon, including but not limited to ribonuclease barnase (barnase) gene); Cells that survive or exhibit a specific selection response but fail to survive or exhibit a specific selection response in a second medium supplemented with a non-naturally encoded amino acid, thereby providing surviving cells with at least one recombinant O-RS or via Cells screened wherein at least one recombinant O-RS is specific for a non-naturally encoded amino acid. In one aspect, at least one selector codon comprises about 2 or more selector codons. Such embodiments can optionally include wherein at least one selector codon comprises 2 or more selector codons and wherein the first organism and the second organism are different (including, but not limited to, each organism is optionally (including but not limited to) prokaryotes, eukaryotes, mammals, Escherichia coli, fungi, yeasts, archaea, eubacteria, plants, insects, protists, etc.). Additionally, some aspects include wherein the negative selection marker comprises the ribonuclease barnase gene comprising at least one selector codon. Other aspects include wherein the selection marker optionally comprises a fluorescent or luminescent selection marker or an affinity based selection marker. In the embodiments herein, screening and/or selection optionally includes variations in the stringency of the screening and/or selection.

In one embodiment, the method of producing at least one recombinant orthogonal aminoacyl-tRNA synthetase (O-RS) may further comprise: (d) isolating the at least one recombinant O-RS; (e) producing a second A set of O-RSs (optionally mutated) derived from said at least one recombinant O-RS; and (f) repeating steps (b) and (c) until obtaining the ability to preferentially aminoacylate O-tRNA comprising The mutation O-RS so far. Steps (d)-(f) are repeated as desired, including, but not limited to, at least about two times. In one aspect, the second set of mutant O-RS derived from at least one recombinant O-RS can be generated by mutation including, but not limited to, random mutation, site-specific mutation, recombination, or combinations thereof.

In the above method, comprising (but not limited to) positive selection/screening step (b), negative selection/screening step (c) or both positive and negative selection/screening steps (b) and (c) The stringency of the selection/screening step optionally includes varying the stringency of the selection/screening. In another embodiment, the positive selection/screening step (b), negative selection/screening step (c) or both positive and negative selection/screening steps (b) and (c) comprise the use of a reporter, wherein the reporter is detected by fluorescence activated cell sorting (FACS) or wherein the reporter is detected by luminescence. Optionally, the reporter is displayed on the cell surface, on a phage display system, etc., and is selected based on affinity or catalytic activity involving the non-naturally encoded amino acid or analog. In one embodiment, the mutated synthetase is displayed on the cell surface, on a phage display system, or the like.

A method of producing a recombinant orthogonal tRNA (O-tRNA) comprising: (a) producing a library of mutant tRNAs derived from at least one tRNA from a first organism including, but not limited to, suppressor tRNAs; (b) Selecting (including but not limited to) negative selection) or screening the library for aminoacylation by RS from a second organism in the absence of an aminoacyl-tRNA synthetase (RS) from the first organism (optionally mutated) tRNAs, thereby providing a collection of tRNAs (optionally mutants); and (c) the introduced orthogonal RS (O- RS) aminoacylated member, thereby providing at least one recombinant O-tRNA; wherein said at least one recombinant O-tRNA recognizes a selector codon and is not efficiently recognized by RS from a second organism, and is recognized by O -RS is preferentially aminoacylated. In some embodiments, at least one tRNA is a suppressor tRNA and/or comprises a single 3-base codon of natural and/or unnatural bases, or a nonsense codon, an unusual codon, an unnatural codon, Codons containing at least 4 bases, amber codons, ocher codons or opal stop codons. In one embodiment, the recombinant O-tRNA has improved orthogonality. It will be appreciated that in some embodiments the O-tRNA may optionally be imported from the second organism into the first organism without modification. In various embodiments, the first organism and the second organism are the same or different, and are optionally selected from, including but not limited to, prokaryotes, including but not limited to, Methanococcus jannaschii , Yokogawa virus, Escherichia coli, Halobacterium, etc.), eukaryotes, mammals, fungi, yeasts, archaea, eubacteria, plants, insects, protists, etc. In addition, recombinant tRNAs are optionally aminoacylated with non-naturally encoded amino acids that are biosynthesized in vivo either naturally or through genetic manipulation. The non-naturally encoded amino acid is optionally added to the growth medium of at least the first organism or the second organism.

In one aspect, selecting (including but not limited to, negative selection) or screening a library for tRNAs aminoacylated (optionally mutated) by an aminoacyl-tRNA synthetase (step (b)) comprises: Genes and (optionally mutated) tRNA repertoires are introduced into a plurality of cells from a second organism, wherein the toxicity marker gene comprises at least one selector codon (or a gene or gene that results in the production of a toxic or tranquilizing agent or organism is an essential gene, wherein the marker gene comprises at least one selector codon); and selection of surviving cells, wherein the surviving cells contain at least one orthogonal tRNA or non-functional tRNA (optionally Mutated) collection of tRNAs. For example, surviving cells can be selected by using a cell density comparison ratio assay.

In another aspect, a toxic marker gene can include 2 or more selector codons. In another embodiment of the method, the toxicity marker gene is the ribonuclease barnase gene, wherein the ribonuclease barnase gene comprises at least one amber codon. Optionally, the ribonuclease barnase gene may include 2 or more amber codons.

In one embodiment, selecting or screening (optionally mutated) members of a pool of (optionally mutated) tRNAs aminoacylated by an introduced orthogonal RS (O-RS) may comprise adding a positive selection or screening marker gene together with an O-RS - A collection of RS and (optionally mutated) tRNAs is introduced into a plurality of cells from a second organism, wherein positive marker genes include drug resistance genes (including but not limited to β-lactamase genes, which comprising at least one selector codon, such as at least one amber stop codon) or a gene essential to the organism or a gene that results in the detoxification of a toxic agent; and identifying growth in the presence of selection or screening agents including, but not limited to, antibiotics surviving cells or cells screened, thereby providing a collection of cells with at least one recombinant tRNA, wherein in response to at least one selector codon, at least one recombinant tRNA is aminoacylated by an O-RS and inserts an amino acid into a normal in the translation products encoded by sex marker genes. In another embodiment, the concentration of the selection and/or screening agent is variable.

Methods for generating specific O-tRNA/O-RS pairs are provided. The method comprises: (a) generating a library of mutant tRNAs derived from at least one tRNA from a first organism; (b) negatively selecting or screening said library in the absence of an aminoacyl- (optionally mutated) tRNAs aminoacylated by RS from a second organism in the case of tRNA synthetase (RS), thereby providing a collection of (optionally mutated) tRNAs; (c) selecting or screening ( Optionally a member of the pool of mutated) tRNAs that is aminoacylated by the introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA. The at least one recombinant O-tRNA recognizes the selector codon and is not efficiently recognized by the RS from the second organism, and is preferentially aminoacylated by the O-RS. The method also includes (d) generating a library of (optionally mutated) RSs derived from at least one aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting or screening for mutant RSs Members of the library that preferentially aminoacylate at least one recombinant O-tRNA in the presence of the non-naturally encoded amino acid and the natural amino acid, thereby providing a collection of active (optionally mutated) RSs; and (f) negative selection Or screen the collection for an active (optionally mutated) RS that preferentially aminoacylates at least one recombinant O-tRNA in the absence of a non-naturally encoded amino acid, thereby providing at least one specific O-tRNA /O-RS pair, wherein at least one specific O-tRNA/O-RS pair comprises at least one recombinant O-RS specific for a non-naturally encoded amino acid and at least one recombinant O-tRNA. Specific O-tRNA/O-RS pairs generated by the method are included. For example, specific O-tRNA/O-RS pairs may include, including but not limited to, mutRNATyr-mutTyrRS pairs, such as, mutRNATyr-SS12TyrRS pairs, mutRNALeu-mutLeuRS pairs, mutRNAThr-mutThrRS pairs, mutRNAGlu-mutGluRS pairs etc. Additionally, the method includes wherein the first organism and the third organism are the same (including, but not limited to, Methanococcus jannaschii).

Also included in the invention are methods for selecting orthogonal tRNA-tRNA synthetase pairs suitable for use in an in vivo translation system of a second organism. The method comprises: introducing a marker gene, tRNA, and an aminoacyl-tRNA synthetase (RS) isolated or obtained from a first organism into a first set of cells from a second organism; introducing the marker gene and tRNA into from a set of replicating cells from a second organism; and selecting cells that survived in the first set but not in the set of replicating cells, or screening for cells that exhibit a specific selection response but do not produce said response in the set of replicating cells Cells wherein the first set and the set of replicating cells are grown in the presence of a selection or screening agent, wherein the surviving cells or the screened cells comprise an orthogonal tRNA-tRNA synthetase pair suitable for use in an in vivo translation system of a second organism . In one embodiment, the comparison and selection or screening comprises an in vivo complementation assay. Concentrations of selection or screening agents can be varied.

Organisms of the present invention include various organisms and various combinations. For example, the first and second organisms of the methods of the invention may be the same or different. In one embodiment, the organism is optionally a prokaryote, including, but not limited to, Methanococcus jannaschii, Yokogawa virus, Halobacterium, Escherichia coli, A. Pyrococcus (P. furiosus), Pyrococcus horikoshii (P. horikoshii), thermophilic bacteria Aerothermus agile (A. pernix), thermophilic Thermus and the like. Alternatively, organisms optionally include eukaryotes, including but not limited to plants (including but not limited to complex plants such as monocots or dicots), algae, protists, fungi (including but not limited to ) yeast, etc.), animals (including (but not limited to) mammals, insects, arthropods, etc.) and the like. In another embodiment, the second organism is a prokaryote including, but not limited to, Methanococcus jannaschii, Yokogawa virus, Halobacter, Escherichia coli, A. fulgidus, Halobacterium, P.furiosus, P.horikoshii, A.pernix, Thermus thermophiles, etc. Alternatively, the second organism can be a eukaryote including, but not limited to, yeast, animal cells, plant cells, fungi, mammalian cells, and the like. In various embodiments, the first organism and the second organism are different.

VI. Location of Non-Naturally Occurring Amino Acids in GH (e.g., hGH) Polypeptides

The invention contemplates the incorporation of one or more non-naturally occurring amino acids into GH (eg, hGH) polypeptides. One or more non-naturally occurring amino acids can be incorporated at specific positions that do not disrupt the activity of the polypeptide. This can be achieved by making "conservative" substitutions including, but not limited to, substitution of hydrophobic amino acids for hydrophobic amino acids, bulky amino acids for bulky amino acids, hydrophilic amino acids for hydrophilic amino acids and/or insertion of non-naturally occurring amino acids in positions not required for activity.

Regions of GH (e.g., hGH) can be illustrated as follows, where the amino acid positions in hGH are indicated in the middle row (SEQ ID NO: 2).

Helix A Helix B Helix C Helix D

[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184 -191]

N end A-B B-C ring C-D ring C end

Various biochemical and structural approaches can be employed to select desired sites for substitution with non-naturally encoded amino acids within a GH (eg, hGH) polypeptide. Those skilled in the art will readily appreciate that any position of the polypeptide chain is suitable for selection to incorporate a non-naturally encoded amino acid, and that selection may be based on rational design, for any particular desired purpose or not. Or by random selection. Selection of desired sites can be used to generate GH (e.g., hGH) molecules having any desired property or activity (including, but not limited to, agonists, superagonists, inverse agonists, antagonists, receptor binding modulators, agent, receptor activity modulator), for dimer or multimer formation, does not alter the activity or properties compared to the natural molecule, or modulates any physical or chemical property of the polypeptide (such as solubility, aggregation, or stability) sex). For example, point mutation analysis, alanine scanning, or homolog scanning methods known in the art can be used to identify polypeptide positions required for biological activity of a GH (eg, hGH) polypeptide. See, e.g., Cunningham, B. and Wells, J., Science, 244:1081-1085 (1989) (identification of 14 residues critical for GH (e.g., hGH) biological activity) and Cunningham, B. et al. Human, Science 243: 1330-1336 (1989) (identification of antibody and receptor epitopes using homolog scanning mutagenesis). U.S. Patent Nos. 5,580,723, 5,834,250, 6,013,478, 6,428,954, and 6,451,561 describe methods for systematically analyzing the structure and function of polypeptides (such as hGH) by using target substances to identify active domains that affect the activity of the polypeptide , said patent is incorporated herein by reference. Residues other than those identified by alanine or homolog scanning mutagenesis as critical to biological activity may be good candidates for substitution with non-naturally encoded amino acids, depending on the nature of the polypeptide sought. Depends on the desired activity. Alternatively, sites identified as critical to biological activity may also be good candidates for substitution with non-naturally encoded amino acids, again depending on the desired activity of the polypeptide being sought. Another alternative would be to simply make serial substitutions at various positions on the polypeptide chain with non-naturally encoded amino acids and observe the effect on polypeptide activity. It will be readily apparent to those skilled in the art that any means, technique or method for selecting a position to substitute an unnatural amino acid into any polypeptide is suitable for use in the present invention.

The structure and activity of naturally occurring mutants containing deleted hGH polypeptides can also be studied to identify regions of the protein that might allow substitution with non-naturally encoded amino acids. Regarding hGH, see, eg, Kostyo et al., Biochem. Biophys. Acta, 925:314 (1987); Lewis, U. et al., J. Biol. Chem., 253:2679-2687 (1978). In a similar manner, protease digestion and monoclonal antibodies can be used to identify regions of hGH responsible for binding hGH receptors. See, eg, Cunningham, B. et al., Science 243:1330-1336 (1989); Mills, J. et al., Endocrinology, 107:391-399 (1980); Li, C., Mol. Cell. Biochem. , 46:31-41 (1982) (showing that amino acids between residues 134 and 149 can be deleted without loss of activity). Once the residues that may not tolerate substitution with a non-naturally encoded amino acid have been removed, the effect of proposed substitutions at each remaining position can be studied from the three-dimensional crystal structure of hGH and its binding protein. For hGH, see de Vos, A. et al., Science, 255:306-312 (1992); all crystal structures of hGH are available at the Protein Data Bank (including 3HHR, 1AXI, and 1HWG) (PDB, available at www.rcsb.org ), a centralized database containing three-dimensional structure data of protein and nucleic acid macromolecules. If three-dimensional structural data are not available, models can be built to study the secondary and tertiary structure of the polypeptide. Thus, one of skill in the art can readily identify amino acid positions that may be substituted with a non-naturally encoded amino acid.

In some embodiments, a GH (eg, hGH) polypeptide of the invention comprises one or more non-naturally occurring amino acids located in a region of the protein that does not disrupt the helical or beta-sheet secondary structure of the polypeptide.

Exemplary residues for incorporation of non-naturally encoded amino acids may be those that are excluded from potential receptor binding regions (including, but not limited to, Site I and Site II), and may be those that are fully or partially exposed Solvent-dependent residues, those with minimal or no hydrogen-bonding interactions with neighboring residues, may be those with minimal exposure to neighboring reactive residues and may be those that are highly flexible (including but not limited to the C-D loop) as predicted by the three-dimensional crystal structure, secondary, tertiary or quaternary structure of hGH bound to its receptor or not. or residues in structurally rigid (including, but not limited to, B-helix) regions.

In some embodiments, one or more non-naturally encoded amino acids are incorporated at any position in one or more of the following regions corresponding to hGH secondary structure: corresponding to 1- from SEQ ID NO:2 5 (N terminal), 6-33 (helix A), 34-74 (region between helix A and helix B, A-B loop), 75-96 (helix B), 97-105 (between helix B and helix C region between, B-C loop), 106-129 (helix C), 130-153 (region between helix C and helix D, C-D loop), 154-183 (helix D), 184-191 (C-terminal) Location. In other embodiments, a GH polypeptide (e.g., hGH polypeptide) of the invention comprises at least one non-naturally occurring amino acid that substitutes for at least one amino acid located in at least one region of GH (e.g., hGH) , said region is selected from the group consisting of regions corresponding to the following regions: N-terminus (1-5) of SEQ ID NO: 2, N-terminus of A-B loop (32-46), B-C loop (97-105) , C-D loop (132-149) and C-terminus (184-191). In some embodiments, one or more non-naturally encoded amino acids are incorporated in GH (e.g., hGH) at one or more positions corresponding to: before position 1 (i.e., at the N-terminus ), position 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97 ,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130 ,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155 . ID NO: the corresponding amino acid of 1 or 3).

Exemplary positions for incorporating one or more non-naturally encoded amino acids include positions corresponding to positions 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186 and 187 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3 ).

A subset of exemplary positions incorporating one or more non-naturally encoded amino acids includes positions corresponding to positions 29, 33, 35, 37, 39, 49, 57, 69, 70, 71, 74, 88 ,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145 , 147, 154, 155, 156, 186 and 187 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). Studies of the crystal structure of GH (e.g., hGH) and its interaction with GH (e.g., hGH) receptors have shown that the side chains of these amino acid residues are fully or partially solvent accessible and non-naturally encoded. The side chains of amino acids can be directed away from the surface of the protein and into the solvent.

Exemplary positions for incorporating one or more non-naturally encoded amino acids include positions corresponding to positions 35, 88, 91, 92, 94, 95, 99, 101, 103, 111, 131, 133, 134, 135, 16, 139, 140, 143, 145 and 155 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). Studies of the crystal structure of GH (eg, hGH) and its interaction with the GH (eg, hGH) receptor indicate that the side chains of these amino acid residues are completely solvent exposed and that the side chains of the native residues point into the solvent .

A subset of exemplary positions incorporating one or more non-naturally encoded amino acids include positions corresponding to positions 30, 74, 103, or any combination thereof (from SEQ ID NO: 2, or SEQ ID NO: 1 or the corresponding amino acid of 3). Another subset of exemplary positions incorporating one or more non-naturally encoded amino acids includes positions corresponding to positions 35, 92, 143, 145, or any combination thereof (from SEQ ID NO: 2, or SEQ ID NO: 2, or SEQ ID NO: ID NO: the corresponding amino acid of 1 or 3). Yet another subset of exemplary positions incorporating one or more non-naturally encoded amino acids includes positions corresponding to positions 35, 92, 131, 134, 143, 145, or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). Yet another subset of exemplary positions incorporating one or more non-naturally encoded amino acids includes positions corresponding to positions 30, 35, 74, 92, 103, 145, or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). Yet another subset of exemplary positions incorporating one or more non-naturally encoded amino acids includes positions corresponding to positions 35, 92, 143, 145, or any combination thereof (from SEQ ID NO: 2, or SEQ ID NO: the corresponding amino acid of 1 or 3). In certain embodiments, the site that incorporates one or more non-naturally encoded amino acids includes the site corresponding to position 35 (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3).

In some embodiments, at least one non-naturally encoded amino acid incorporated into the GH (eg, hGH) contains a carbonyl group (eg, a keto group). In certain embodiments, at least one non-naturally encoded amino acid incorporated into the GH (eg, hGH) is p-acetylphenylalanine. In some embodiments where the GH (eg, hGH) contains non-naturally encoded amino acids, the non-naturally encoded amino acid incorporated into the GH (eg, hGH) is p-acetylphenylalanine. In some embodiments where the GH (eg, hGH) contains multiple non-naturally encoded amino acids, substantially all of the non-naturally encoded amino acid incorporated into the GH (eg, hGH) is p-acetylphenylalanine.

In some embodiments, the non-naturally occurring amino acid is linked to the water soluble polymer at one or more positions including, but not limited to, positions corresponding to: before position 1 (i.e., at the N-terminus) , position 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 2, or SEQ ID NO: the corresponding amino acid of 1 or 3). In some embodiments, the non-naturally occurring amino acid is linked to the water soluble polymer at positions including, but not limited to, positions corresponding to 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3) one or more positions in these positions. In some embodiments, the non-naturally occurring amino acid is linked to the water soluble polymer at positions including, but not limited to, those corresponding to 35, 92, 143, 145 (SEQ ID NO: 2, or SEQ ID NO: 2, or SEQ ID NO: ID NO: the corresponding amino acid of 1 or 3) the position of one or more positions in these positions. In some embodiments, the non-naturally occurring amino acid is attached to the water soluble polymer at positions including, but not limited to, positions corresponding to 35, 92, 131, 134, 143, 145, or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3) the position of one or more positions in these positions. In some embodiments, the non-naturally occurring amino acid is attached to the water soluble polymer at positions including, but not limited to, positions corresponding to 30, 35, 74, 92, 103, 145, or any combination thereof ( A position at one or more of these positions from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurring amino acid is attached to the water soluble polymer at positions including, but not limited to, positions corresponding to 35, 92, 143, 145, or any combination thereof (from SEQ ID NO : 2, the corresponding amino acid of SEQ ID NO: 1 or 3) the position of one or more positions in these positions. In some embodiments, the non-naturally occurring amino acid is linked to the water soluble polymer at a position corresponding to, but not limited to, position 35 (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3) .

In some embodiments, the water soluble polymer linked to GH (eg, hGH) includes one or more molecules of polyethylene glycol (PEG). Polymers (eg, PEG) can be linear or branched. Typically, linear polymers (eg, PEG) useful in the present invention can have a molecular weight of about 0.1 kDa to about 100 kDa, or have a molecular weight of about 1 kDa to about 60 kDa, or have a molecular weight of about 20 kDa to about 40 kDa, or have a molecular weight of about Molecular weight of 30kDa. Typically, branched polymers (eg, PEG) useful in the present invention can have a molecular weight of about 1 kDa to about 100 kDa, or have a molecular weight of about 30 kDa to about 50 kDa, or have a molecular weight of about 40 kDa. Polymers such as PEG are further described herein. In certain embodiments, the bond between the GH (eg, hGH) and the water soluble polymer (eg, PEG) is an oxime bond.

Certain embodiments of the invention encompass compositions comprising GH (eg, hGH) linked by a covalent bond to at least one water-soluble polymer, wherein the covalent bond is an oxime bond. In some embodiments, the water soluble polymer is PEG, eg, linear PEG. In some embodiments encompassing at least one linear PEG linked to GH (e.g., hGH) via an oxime bond, the PEG can have a molecular weight of about 0.1 kDa to about 100 kDa, or have a molecular weight of about 1 kDa to about 60 kDa, or Has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa. In certain embodiments encompassing linear PEG attached to GH (eg, hGH) via an oxime bond, the PEG has a molecular weight of about 30 kDa. In some embodiments encompassing at least one branched PEG linked to GH (e.g., hGH) via an oxime linkage, the PEG may have a molecular weight of about 1 kDa to about 100 kDa, or have a molecular weight of about 30 kDa to about 50 kDa, or Has a molecular weight of about 40 kDa. In certain embodiments encompassing a branched PEG linked to GH (eg, hGH) via an oxime linkage, the PEG has a molecular weight of about 40 kDa. In some embodiments, the GH is a GH such as hGH, and in certain of these embodiments, the GH (e.g., hGH) has a sequence at least about 80% identical to SEQ ID NO: 2; in some embodiments In one example, GH (e.g., hGH) has a sequence that is the sequence of SEQ ID NO:2. In some embodiments, the GH (e.g., hGH) contains at least one non-naturally encoded amino acid; in some of these embodiments, at least one oxime linkage is between the non-naturally encoded amino acid and at least one water-soluble polymer between. In some embodiments, the non-naturally encoded amino acid contains a carbonyl group, such as a keto group; in some embodiments, the non-naturally encoded amino acid is p-acetylphenylalanine. In some embodiments, the acetylphenylalanine is substituted at a position corresponding to position 35 of SEQ ID NO:2.

Accordingly, in some embodiments, the present invention provides a GH (eg, hGH) linked to at least one water-soluble polymer (eg, PEG) by a covalent bond, wherein the covalent bond is an oxime bond. In certain embodiments, the water soluble polymer is PEG, and the PEG is linear PEG. In these embodiments, the linear PEG has a molecular weight of about 0.1 kDa to about 100 kDa, or has a molecular weight of about 1 kDa to about 60 kDa, or has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa. In certain embodiments encompassing linear PEG attached to GH (eg, hGH) via an oxime bond, the PEG has a molecular weight of about 30 kDa. In certain embodiments, the water soluble polymer is PEG, which is branched PEG. In these embodiments, the branched PEG has a molecular weight of about 1 kDa to about 100 kDa, or has a molecular weight of about 30 kDa to about 50 kDa, or has a molecular weight of about 40 kDa. In certain embodiments encompassing a branched PEG attached to GH (eg, hGH) via an oxime linkage, the PEG has a molecular weight of about 40 kDa.

In some embodiments, the invention provides a GH (e.g., hGH), wherein the GH (e.g., hGH) comprises a non-naturally encoded amino acid, wherein the GH is covalently bonded to at least one water-soluble polymer (e.g., , PEG), and wherein the covalent bond is an oxime bond between the non-naturally encoded amino acid and a water-soluble polymer (eg, PEG). In some embodiments, a non-naturally encoded amino acid is incorporated into GH (e.g., hGH) at a position corresponding to position 35 of SEQ ID NO: 2. In certain embodiments where the water soluble polymer is PEG, the PEG is linear PEG. In these embodiments, the linear PEG has a molecular weight of about 0.1 kDa to about 100 kDa, or has a molecular weight of about 1 kDa to about 60 kDa, or has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa. In certain embodiments encompassing linear PEG attached to GH (eg, hGH) via an oxime bond, the PEG has a molecular weight of about 30 kDa. In certain embodiments where the water soluble polymer is PEG, the PEG is a branched PEG. In these embodiments, the branched PEG has a molecular weight of about 1 kDa to about 100 kDa, or has a molecular weight of about 30 kDa to about 50 kDa, or has a molecular weight of about 40 kDa. In certain embodiments encompassing a branched PEG linked to GH (eg, hGH) via an oxime linkage, the PEG has a molecular weight of about 40 kDa.

In some embodiments, the invention provides a GH (e.g., hGH), wherein the GH (e.g., hGH) comprises a non-naturally encoded amino acid that is a carbonyl-containing non-naturally encoded amino acid, wherein the GH is covalently The bond is to at least one water soluble polymer (eg, PEG), and the covalent bond is an oxime bond between the non-naturally encoded carbonyl-containing amino acid and the water soluble polymer (eg, PEG). In some embodiments, a non-naturally encoded carbonyl-containing amino acid is incorporated into GH (e.g., hGH) at a position corresponding to position 35 of SEQ ID NO: 2. In certain embodiments where the water soluble polymer is PEG, the PEG is linear PEG. In these embodiments, the linear PEG has a molecular weight of about 0.1 kDa to about 100 kDa, or has a molecular weight of about 1 kDa to about 60 kDa, or has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa. In certain embodiments encompassing linear PEG attached to GH (eg, hGH) via an oxime bond, the PEG has a molecular weight of about 30 kDa. In certain embodiments where the water soluble polymer is PEG, the PEG is a branched PEG. In these embodiments, the branched PEG has a molecular weight of about 1 kDa to about 100 kDa, or has a molecular weight of about 30 kDa to about 50 kDa, or has a molecular weight of about 40 kDa. In certain embodiments encompassing a branched PEG linked to GH (eg, hGH) via an oxime linkage, the PEG has a molecular weight of about 40 kDa.

In some embodiments, the invention provides a GH (e.g., hGH) comprising a non-naturally encoded amino acid comprising a keto group, wherein the GH is covalently bonded to at least one water-soluble polymer (e.g., PEG) and wherein the covalent bond is an oxime bond between a non-naturally encoded amino acid containing a keto group and a water-soluble polymer (eg, PEG). In some embodiments, a non-naturally encoded amino acid containing a keto group is incorporated into GH (e.g., hGH) at a position corresponding to position 35 of SEQ ID NO: 2. In certain embodiments where the water soluble polymer is PEG, the PEG is linear PEG. In these embodiments, the linear PEG has a molecular weight of about 0.1 kDa to about 100 kDa, or has a molecular weight of about 1 kDa to about 60 kDa, or has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa. In certain embodiments encompassing linear PEG attached to GH (eg, hGH) via an oxime bond, the PEG has a molecular weight of about 30 kDa. In certain embodiments where the water soluble polymer is PEG, the PEG is a branched PEG. In these embodiments, the branched PEG has a molecular weight of about 1 kDa to about 100 kDa, or has a molecular weight of about 30 kDa to about 50 kDa, or has a molecular weight of about 40 kDa. In certain embodiments encompassing a branched PEG attached to GH (eg, hGH) via an oxime linkage, the PEG has a molecular weight of about 40 kDa.

In some embodiments, the invention provides a GH (e.g., hGH) comprising a non-naturally encoded amino acid that is p-acetylphenylalanine, wherein the GH is covalently bonded to at least one water-soluble polymer ( For example, PEG) is linked, and wherein the covalent bond is an oxime bond between p-acetylphenylalanine and a water-soluble polymer (eg, PEG). In some embodiments, p-acetylphenylalanine is incorporated into GH (e.g., hGH) at a position corresponding to position 35 of SEQ ID NO:2. In certain embodiments where the water soluble polymer is PEG, the PEG is linear PEG. In these embodiments, the linear PEG has a molecular weight of about 0.1 kDa to about 100 kDa, or has a molecular weight of about 1 kDa to about 60 kDa, or has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa. In certain embodiments encompassing linear PEG attached to GH (eg, hGH) via an oxime bond, the PEG has a molecular weight of about 30 kDa. In certain embodiments where the water soluble polymer is PEG, the PEG is a branched PEG. In these embodiments, the branched PEG has a molecular weight of about 1 kDa to about 100 kDa, or has a molecular weight of about 30 kDa to about 50 kDa, or has a molecular weight of about 40 kDa. In certain embodiments encompassing branched PEG attached to GH (eg, hGH) via an oxime linkage, the PEG has a molecular weight of about 40 kDa.

In certain embodiments, the invention provides a GH (e.g., hGH) comprising SEQ ID NO: 2, and wherein said GH (e.g., hGH) is at position 35 corresponding to SEQ ID NO: 2 The position was substituted with p-acetylphenylalanine linked by an oxime bond to a linear PEG with a molecular weight of about 30 kDa.

In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions including, but not limited to, positions corresponding to : Before position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88 ,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122 ,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150 , 151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein ) (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions including, but not limited to, positions corresponding to : positions 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions including, but not limited to, positions corresponding to : position 35, 92, 143, 145 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions including, but not limited to, positions corresponding to : position 35, 92, 131, 134, 143, 145 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions including, but not limited to, positions corresponding to : position 30, 35, 74, 92, 103, 145 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions including, but not limited to, positions corresponding to : position 35, 92, 143, 145 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid substituted at one or more positions, including but not limited to, corresponding to position 35 (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3) position. In embodiments where the PEG is a linear PEG, the PEG can have a molecular weight of about 0.1 kDa to about 100 kDa, or have a molecular weight of about 1 kDa to about 60 kDa, or have a molecular weight of about 20 kDa to about 40 kDa, or have a molecular weight of about 30 kDa.

In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions including, but not limited) to positions corresponding to the following positions: anterior to position 1 (i.e., at the N-terminus), positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69 ,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115 ,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146 ,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions including, but not limited to) positions corresponding to the following positions: positions 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions including, but not limited to) a position corresponding to the following positions: position 35, 92, 143, 145 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions including, but not limited to) a position corresponding to position 35, 92, 131 , 134, 143, 145 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions including, but not limited to) a position corresponding to position 30, 35, 74, 92, 103, 145 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the invention provides a hormone composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQ The amino acid sequence of ID NO: 2, and wherein the GH (e.g., hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions including, but not limited to) a position corresponding to position 35, 92, 143, 145 or any combination thereof (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In some embodiments, the present invention provides a hormonal composition comprising GH (eg, hGH) linked by an oxime bond to at least one PEG (eg, linear PEG), wherein the GH (eg, hGH) comprises SEQID The amino acid sequence of NO: 2, and wherein GH (eg, hGH) contains at least one non-naturally encoded amino acid that is p-acetylphenylalanine substituted at one or more positions, including but not limited to ) corresponds to position 35 (from SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or 3). In embodiments where the PEG is a linear PEG, the PEG can have a molecular weight of about 0.1 kDa to about 100 kDa, or have a molecular weight of about 1 kDa to about 60 kDa, or have a molecular weight of about 20 kDa to about 40 kDa, or have a molecular weight of about 30 kDa.

In some embodiments, the invention provides a GH (e.g., hGH), wherein the GH (e.g., hGH) comprises at least one non-naturally encoded amino acid, wherein the GH is covalently bonded to a plurality of water-soluble polymers (eg, multiple PEGs), wherein one or more covalent bonds are oxime bonds between at least one non-naturally encoded amino acid and a water-soluble polymer (eg, PEG). GH (eg, hGH) can be linked to about 2-100 water-soluble polymers (eg, PEG), or to about 2-50 water-soluble polymers (eg, PEG), or to about 2-25 A water-soluble polymer (eg, PEG) linked, or about 2-10 water-soluble polymers (eg, PEG), or about 2-5 water-soluble polymers (eg, PEG) , or with about 5-100 water-soluble polymers (eg, PEG), or with about 5-50 water-soluble polymers (eg, PEG), or with about 5-25 water-soluble polymers (eg, PEG), or with about 5-10 water-soluble polymers (eg, PEG), or with about 10-100 water-soluble polymers (eg, PEG), or with about 10 - 50 water-soluble polymers (eg, PEG) linked, or about 10-20 water-soluble polymers (eg, PEG), or about 20-100 water-soluble polymers (eg, PEG) Linked, or linked with about 20-50 water-soluble polymers (eg, PEG), or linked with about 50-100 water-soluble polymers (eg, PEG). One or more non-naturally encoded amino acids can be incorporated into GH (eg, hGH) at any position described herein. In some embodiments, at least one non-naturally encoded amino acid is incorporated into GH (e.g., hGH) at a position corresponding to position 35 of SEQ ID NO: 2. In some embodiments, the non-naturally encoded amino acid includes at least one non-naturally encoded amino acid that is a non-naturally encoded amino acid that contains a carbonyl group, for example, a non-naturally encoded amino acid that contains a keto group, such as, p-acetylphenylalanine . In some embodiments, the GH (eg, hGH) comprises p-acetylphenylalanine. In some embodiments, p-acetylphenylalanine is incorporated into GH (e.g., hGH) at a position corresponding to position 35 of SEQ ID NO: 2, wherein p-acetylphenylalanine is polymerized via an oxime bond. One of the objects (for example, one of the PEGs) is connected. In some embodiments, at least one water soluble polymer (eg, PEG) is linked to GH (eg, hGH) via a covalent bond to at least one non-naturally encoded amino acid. In some embodiments, the covalent bond is an oxime bond. In some embodiments, the plurality of water soluble polymers (eg, PEG) is linked to the GH (eg, hGH) through covalent bonds to the plurality of non-naturally encoded amino acids. In some embodiments, at least one covalent bond is an oxime bond; in some embodiments, a plurality of covalent bonds are oxime bonds; in some embodiments, substantially all of the bonds are oxime bonds. The plurality of water soluble polymers (eg, PEG) can be linear polymers, branched polymers, or any combination thereof. In embodiments incorporating one or more linear PEGs, the linear PEG has a molecular weight of about 0.1 kDa to about 100 kDa, or has a molecular weight of about 1 kDa to about 60 kDa, or has a molecular weight of about 20 kDa to about 40 kDa, or has a molecular weight of about 30 kDa molecular weight. In embodiments incorporating one or more branched PEGs, the branched PEG has a molecular weight of about 1 kDa to about 100 kDa, or has a molecular weight of about 30 kDa to about 50 kDa, or has a molecular weight of about 40 kDa. It should be understood that embodiments employing multiple water soluble polymers (eg, PEG) will generally employ said polymers at a lower molecular weight than embodiments employing a single PEG. Thus, in some embodiments, the plurality of PEGs have a total molecular weight of about 0.1-500 kDa, or about 0.1-200 kDa, or about 0.1-100 kDa, or about 1-1000 kDa, or about 1-500 kDa, or about 1-200 kDa, or about 1-100 kDa, or about 10-1000 kDa, or about 10-500 kDa, or about 10-200 kDa, or about 10-100 kDa, or about 10-50 kDa, or about 20-1000 kDa, or about 20-500 kDa, or about 20-200 kDa, or about 20-100 kDa, or about 20-80 kDa, about 20-60 kDa, about 5-100 kDa, about 5-50 kDa, Or about 5-20 kDa.

Human GH antagonists include, but are not limited to, those at positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, Substitution at 119, 120, 123 and 127 or addition at position 1 (i.e. at the N-terminus) or any combination thereof (SEQ ID NO: 2, or SEQ ID NO: 1, 3 or any other GH sequence those antagonists of the corresponding amino acids).

Various non-naturally encoded amino acids can be substituted or incorporated at defined positions in a GH (eg, hGH) polypeptide. In general, based on studies of the three-dimensional crystal structures of GH (e.g., hGH) polypeptides and their receptors, the preference for conservative substitutions (i.e., aryl-based non-naturally encoded amino acids such as p-acetylphenylalanine or O- propargyl tyrosine substitution of Phe, Tyr or Trp) and those skilled in the art wish to introduce specific binding chemistry into GH (e.g., hGH) polypeptides (e.g., if one of skill in the art wants to achieve Partial Hu's radical [3+2] cycloaddition of the water soluble polymer or amide bond formation with the water soluble polymer having an aryl ester which in turn incorporates a phosphine moiety then introduces the 4-azido group phenylalanine), the specific non-naturally encoded amino acid is selected for incorporation.

In one embodiment, the method further comprises: incorporating an unnatural amino acid into a protein, wherein the unnatural amino acid comprises a first reactive group; and contacting the protein with a molecule comprising a second reactive group (comprising (but not limited to) labels, dyes, polymers, water soluble polymers, polyethylene glycol derivatives, photocrosslinkers, radionuclides, cytotoxic compounds, drugs, affinity tags, photoaffinity tags, Reactive compounds, resins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides , carbohydrates, water-soluble dendrimers, cyclodextrins, inhibitory ribonucleic acids, biomaterials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, covalently or non- Groups that covalently interact with other molecules, photocaging moieties, actinic radiation excitable moieties, photoisomerization moieties, biotin, derivatives of biotin, biotin analogs, moieties incorporating heavy atoms , chemically cleavable groups, photocleavable groups, extended side chains, carbon-linked sugars, redox active agents, aminothioacids, toxic moieties, isotopically labeled moieties, biophysical Probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, chromophores, energy transfer agents, bioactive agents, detectable labels, small molecules, quantum dots, nanoconductors, radionucleotides, radioconductors, neutron capture agents, or any combination of the foregoing or any other desired compound or substance). The first reactive group reacts with the second reactive group to link the molecule to an unnatural amino acid via a [3+2] cycloaddition. In one embodiment, the first reactive group is an alkynyl or azido moiety and the second reactive group is an azido or alkynyl moiety. For example, the first reactive group is an alkynyl moiety (including, but not limited to, in the unnatural amino acid p-propargyloxyphenylalanine), and the second reactive group is an azido part. In another example, the first reactive group is an azido moiety (including, but not limited to, in the unnatural amino acid p-azido-L-phenylalanine), and the second reactive group The group is an alkynyl moiety.

In some cases, non-naturally encoded amino acid substitutions will be combined with other additions, substitutions or deletions within the GH (eg, hGH) polypeptide to affect other biological properties of the GH (eg, hGH) polypeptide. In some cases, other additions, substitutions, or deletions may increase the stability of the GH (e.g., hGH) polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the resistance of the GH (e.g., hGH) polypeptide to its body affinity. In some embodiments, the GH (e.g., hGH) polypeptide comprises an amino acid substitution selected from the group consisting of each of the following substitutions: F10A, F10H, F10I; M14W, M14Q, M14G; H18D; H21N; G120A in SEQ ID NO: 2 ; R167N; D171S; E174S; F176Y, I179T or any combination thereof. In some cases, other additions, substitutions, or deletions may increase the solubility of a GH (eg, hGH) polypeptide (including, but not limited to, when expressed in E. coli or other host cells). In some embodiments, additions, substitutions or deletions can increase the solubility of the polypeptide after expression in E. coli or other recombinant host cells. In some embodiments, in addition to sites suitable for incorporation of non-natural amino acids that result in increased polypeptide solubility upon expression in E. Other sites for amino acid substitutions. In some embodiments, the GH (e.g., hGH) polypeptide comprises another addition, substitution or deletion that modulates affinity for the GH (e.g., hGH) polypeptide receptor, modulation (including but not limited to, enhancement or attenuation) by Dimerization of receptors, stabilization of receptor dimers, regulation of circulating half-life, regulation of release or bioavailability, promotion of purification, or improvement or modification of specific routes of administration. For example, in addition to introducing one or more non-naturally encoded amino acids as described herein, one or more of the following substitutions are introduced to increase the affinity of a GH (e.g., hGH) variant for its receptor: F10A, F10H or F10I; M14W, M14Q or M14G; H18D; H21N; R167N; D171S; E174S; F176Y and I179T. Likewise, a GH (e.g., hGH) polypeptide may comprise a chemical or enzymatic cleavage sequence, a protease cleavage sequence, a reactive group, an antibody binding domain (including but not limited to, FLAG or polyhistidine), or other affinity-based sequences (including, but not limited to, FLAG, polyhistidine, GST, etc.) or linker molecules (including, but not limited to, biotin), which improve detection (including, but not limited to, GFP), purification, passage through tissue or cell membranes transport, prodrug release or activation, hGH size reduction, or other properties of the polypeptide.

In some embodiments, substitution of a non-naturally encoded amino acid results in a GH (eg, hGH) antagonist. A subset of exemplary positions suitable for incorporation of one or more non-naturally encoded amino acids include: positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103 , 109, 112, 113, 115, 116, 119, 120, 123, 127 or an addition before position 1 (SEQ ID NO: 2, SEQ ID NO: 1, 3 or the corresponding amino acid of any other GH sequence). In some embodiments, the GH (e.g., hGH) antagonist comprises at least one substitution in the following regions such that GH acts as an antagonist: 1-5 (N-terminal), 6-33 (helix A), 34-74 ( Region between helix A and helix B, A-B loop), 75-96 (helix B), 97-105 (region between helix B and helix C, B-C loop), 106-129 (helix C), 130- 153 (region between helix C and helix D, C-D loop), 154-183 (helix D), 184-191 (C-terminus). In other embodiments, exemplary sites for incorporation of non-naturally encoded amino acids include residues within the amino-terminal region of helix A and a portion of helix C. In another embodiment, G120 is substituted with a non-naturally encoded amino acid such as p-azido-L-phenylalanine or O-propargyl-L-tyrosine. In other embodiments, the above-listed substitutions are combined with other substitutions such that the GH (eg, hGH) polypeptide is a GH (eg, hGH) antagonist. For example, a non-naturally encoded amino acid is substituted at one of the positions identified herein, and a substitution is simultaneously introduced at G120 (eg, G120R, G120K, G120W, G120Y, G120F, or G120E). In some embodiments, the GH (eg, hGH) antagonist comprises a non-naturally encoded amino acid present in the receptor binding region of the GH (eg, hGH) molecule linked to a water soluble polymer.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are modified by one or more non-naturally encoded Amino acid substitutions. In some instances, the GH (e.g., hGH) polypeptide additionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than one or Substitution of one or more non-naturally encoded amino acids for naturally occurring amino acids. For example, in some embodiments, one or more residues in the following regions of GH (e.g., hGH) are substituted with one or more non-naturally encoded amino acids: 1-5 (N-terminal); 32-46 (N-terminal of the A-B loop); 97-105 (B-C loop); and 132-149 (C-D loop); and 184-191 (C-terminal). In some embodiments, one or more residues in the following regions of GH (e.g., hGH) are substituted with one or more non-naturally encoded amino acids: 1-5 (N-terminal), 6-33 (helix A) , 34-74 (region between helix A and helix B, A-B loop), 75-96 (helix B), 97-105 (region between helix B and helix C, B-C loop), 106-129 (helix C), 130-153 (region between helix C and helix D, C-D loop), 154-183 (helix D), 184-191 (C-terminus). In some cases, one or more non-naturally encoded residues are linked to one or more lower molecular weight linear or branched PEGs (mass approximately 5-20 kDa or less), compared to single, smaller High molecular weight PEG-linked substances resulted in enhanced binding affinity and comparable serum half-lives.

In some embodiments, up to two of the following GH (e.g., hGH) residues are substituted with one or more non-naturally encoded amino acids at the following positions: positions 29, 30, 33, 34, 35, 37 ,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129 , 130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187. In some cases, any of the following pairs of substitutions are made: K38X ^* and K140X ^* ; K41X ^* and K145X ^* ; Y35X ^* and E88X ^* ; Y35X ^* and F92X ^* ; Y35X ^* and Y143X ^* ; F92X ^* and Y143X ^* , where X ^* represents a non-naturally encoded amino acid. Preferred positions suitable for incorporation of two or more non-naturally encoded amino acids include combinations of the following residues: positions 29, 33, 35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 186 and 187. Particularly preferred positions suitable for incorporation of two or more non-naturally encoded amino acids include combinations of the following residues: positions 35, 88, 91, 92, 94, 95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145 and 155.

Preferred sites suitable for incorporation of two or more non-naturally encoded amino acids into GH (e.g., hGH) include combinations of residues from before position 1 (i.e., at the N-terminus) of SEQ ID NO: 2 , position 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, or any combination thereof.

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high-level expression of a cloned GH (e.g., hGH) polynucleotide, one skilled in the art typically subclones a polynucleotide encoding a GH (e.g., hGH) polypeptide of the invention into an expression vector, The expression vector contains a strong promoter directing transcription, a transcription/translation terminator and, in the case of a nucleic acid encoding a protein, a ribosome binding site for translation initiation. Suitable bacterial promoters are known to those skilled in the art and are described, for example, in Sambrook et al. and Ausubel et al.

Bacterial expression systems for expressing GH (e.g., hGH) polypeptides of the invention can be used in, including but not limited to, Escherichia coli, Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa bacteria, Pseudomonas putida and Salmonella (Palva et al., Gene: 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are known to those of skill in the art and are commercially available. Where orthogonal tRNAs and aminoacyl tRNA synthetases (described above) are used to express GH (eg, hGH) polypeptides of the invention, host cells for expression are selected based on their ability to use the orthogonal components. Exemplary host cells include Gram-positive bacteria (including, but not limited to, B. brevis, B. subtilis, or Streptomyces) and gram-positive bacteria. Gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida) and yeast and other eukaryotic cells. Cells comprising O-tRNA/O-RS pairs can be used as described herein.

The eukaryotic or non-eukaryotic host cells of the invention provide the ability to synthesize proteins comprising large quantities of useful unnatural amino acids. In one aspect, the composition optionally comprises (including but not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 micrograms Milligrams, at least 100 milligrams, at least 1 gram or more of protein comprising unnatural amino acids, or including amounts obtainable by in vivo protein production methods (details on recombinant protein production and purification are provided herein). In another aspect, the protein is optionally prepared in a liquid suspension including, but not limited to, a cell lysate, buffer, pharmaceutical buffer, or other liquid suspension, including but not limited to, for including, but not limited to, about 1 nl to about 100 L) including, but not limited to, at least 10 micrograms per liter, at least 50 micrograms per liter, at least 75 micrograms per liter, at least 100 micrograms per liter , present in the composition at a concentration of at least 200 micrograms per liter, at least 250 micrograms per liter, at least 500 micrograms per liter, at least 1 milligram per liter, or at least 10 milligrams per liter or more . The production of proteins comprising at least one unnatural amino acid in eukaryotic cells in large quantities (including but not limited to amounts greater than would normally be possible by other methods including but not limited to in vitro translation) is an inherent A feature of the invention.

The eukaryotic or non-eukaryotic host cells of the invention provide the ability to biosynthesize proteins comprising large quantities of useful unnatural amino acids. For example, proteins comprising unnatural amino acids can be produced in cell extracts, cell lysates, media, buffers, etc. at concentrations including, but not limited to, at least 10 μg/L, at least 50 μg/L , at least 75 µg/L, at least 100 µg/L, at least 200 µg/L, at least 250 µg/L, or at least 500 µg/L, at least 1 mg/L, at least 2 mg/L, at least 3 mg/L, at least 4 mg/L, at least 5 mg/L, at least 6 mg/L, at least 7 mg/L, at least 8 mg/L, at least 9 mg/L, at least 10 mg/L, at least 20 mg/L, at least 30 mg /L, at least 40 mg/L, at least 50 mg/L, at least 60 mg/L, at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 200 mg/L , at least 300 mg/L, at least 400 mg/L, at least 500 mg/L, at least 600 mg/L, at least 700 mg/L, at least 800 mg/L, at least 900 mg/L, 1 g/L, 5 g /L, 10 g/L or more than 10 g/L of protein.

I. Expression System, Culture and Isolation

GH (eg, hGH) polypeptides can be expressed in a number of suitable expression systems including, for example, yeast, insect cells, mammalian cells, and bacteria. A description of exemplary expression systems is provided below.

Yeast The term "yeast" as used herein includes any of a variety of yeasts capable of expressing a gene encoding a GH (eg, hGH) polypeptide. Such yeasts include, but are not limited to, ascospore-forming yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the group of incomplete fungi (Blastomycetes). Ascospore-forming yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae. The latter comprises four subfamilies, Schizosaccharomycoideae (eg, genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomyces ( Saccharomycoideae) (eg, genera Pichia, Kluyveromyces, and Saccharomyces). Basidiosporidium-producing yeasts include the basidiomycetes genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, and Pseudosporidium ( Filobasidiella). Yeasts belonging to the group of incomplete fungi (Blastomycetaceae) are divided into two families, Sporobolomycetaceae (for example, genera Sporobolomyces and Bullera) and Cryptospora. Cryptococcaceae (eg, genus Candida).

Of particular interest for use in the present invention are species within the following species: Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hansenula, Torulopsis (Torulopsis) and Candida species including (but not limited to) P. pastoris, P. guillerimondii, S. cerevisiae, Karlsberg Saccharomyces (S.carlsbergensis), Saccharomyces saccharification (S.diastaticus), Douglas yeast (S.douglasii), Kluver's yeast (S.kluyveri), Nobens yeast (S.norbensis), oval yeast (S.oviformis ), Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), Candida albicans (C.albicans), Candida maltosa (C.maltosa) and Hansenula polymorpha ( H. polymorpha).

Selection of suitable yeast for expressing a GH (eg, hGH) polypeptide is within the skill of those in the art. In selecting yeast hosts for expression, suitable hosts may include those shown to have, for example, good secretion capacity, low proteolytic activity, good secretion capacity, good soluble protein production, and overall robustness. Yeast is generally available from a variety of sources including, but not limited to, the Yeast GeneticStock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA), and the American Type Culture Collection ("ATCC") (Manassas, VA).

The term "yeast host" or "yeast host cell" includes yeast that can be used or has been used as a recipient of recombinant vectors or other transferred DNA. The term includes progeny of the original yeast host cell that have received the recombinant vector or other transferred DNA. It is understood that the progeny of a single parental cell may not necessarily be identical in morphology or in genomic or total DNA that is complementary to the original parent, due to accidental or deliberate mutation. Progeny of a parental cell that closely resembles the parent to be characterized by a relevant property such as the presence of a nucleotide sequence encoding a GH (eg, hGH) polypeptide are included within the meaning of this definition.

Expression and transformation vectors, including extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeast hosts. For example, expression vectors have been developed for Saccharomyces cerevisiae (Sikorski et al., GENETICS (1989) 122:19; Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC.NATL.ACAD .SCI.USA(1978)75:1929); for the expression vector of Candida albicans (Kurtz et al., _{MOL.CELL.BIOL} .(1986)6:142); for the expression vector of Candida maltosa ( Kunze et al., J.BASIC M _ICROBIOL . (1985) 25:141); Expression vectors for Hansenula polymorpha (Gleeson et al., JG _{EN.M ICROBIOL} . (1986) 132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302); expression vector for Kluyveromyces fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); expression for K. lactis Vectors (De Louvencourt et al., J. BACTERIOL. (1983) 154:737; Van denBerg et al., BIOTECHNOLOGY (NY) (1990) 8:135); expression vectors for Pichia lemorti (Kunze et al. , J.BASIC M _ICROBIOL .(1985) 25:141); expression vectors for Pichia pastoris (US Patent Nos. 5,324,639, 4,929,555 and 4,837,148; Gregg et al., M _{OL . .BIOL} . (1985) 5:3376); expression vector for Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and for Yarrowia lipolytica (Y. lipolytica) expression vector; expression vector for Aspergillus nidulans (A. nidulans) (Ballance et al., B _IOCHEM . B _IOPHYS . R _ES . _COMMUN . (1983) 112:284-89; Tilburn et al. People, G _ENE (1983) 26:205-221; With Yelton et al., PROC.N _ATL .A _CAD .S _CI .USA (1984) 81:1470-74); For black mold (A. niger) Expression vectors (Kelly and Hynes, EMBO J. (1985) 4:475-479); expression vectors for Trichoderma reesei (T. reesia) (EP 0 244 234); Expression vectors for filamentous fungi of the genera Neurospora, Penicillium, Tolypocladium (WO 91/00357), each reference is incorporated herein by reference.

Control sequences for yeast vectors are known to those skilled in the art and include, but are not limited to, promoter regions from genes such as: alcohol dehydrogenase (ADH) (EP 0 284 044); Enolase; Glucokinase; Glucose-6-phosphate isomerase; Glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); Hexokinase; Phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase (PyK) (EP 0 329 203). The yeast PHO5 gene encoding acid phosphatase may also provide a useful promoter sequence (Miyanohara et al., PROC.N _{ATL.A CAD.SCI.USA} (1983) 80:1). Other suitable promoter sequences for use with yeast hosts may include 3-phosphoglycerate kinase (Hitzeman et al., J. BIOL. _CHEM . (1980) 255:12073) and promoters such as pyruvate decarboxylase, triose phosphate isomer Promoters for other glycolytic enzymes of the enzyme and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY (1978) 17:4900; Hess et al., JA _DV . E _NZYME R _EG . (1969) 7:149). Inducible yeast promoters with additional advantages for controlling transcription by growth conditions may include alcohol dehydrogenase 2, isocytochrome c, acid phosphatase, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, nitrogen metabolism-related Promoter regions for degrading enzymes and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters suitable for yeast expression are further described in EP 0 073 657.

Yeast enhancers can also be used with yeast promoters. In addition, synthetic promoters can also function as yeast promoters. For example, the upstream activating sequence (UAS) of a yeast promoter can be joined to the transcriptional activation region of another yeast promoter to create a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcriptional activation region. See, US Patent Nos. 4,880,734 and 4,876,197, which are incorporated herein by reference. Other examples of hybrid promoters include promoters consisting of the regulatory sequence of the ADH2, GAL4, GAL10 or PHO5 gene combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK. See, EP 0 164 556. In addition, yeast promoters can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of a yeast expression vector include, for example, terminators from the GAPDH or enolase genes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). Alternatively, the origin of replication from the 2μ plasmid source is suitable for use in yeast. A suitable selection gene for use in yeast is the trp1 gene present in yeast plasmids. See, Tschumper et al., GENE (1980) 10:157; Kingsman et al., GENE (1979) 7:141. The trp1 gene provides a selection marker for mutant strains of yeast unable to grow in tryptophan. Likewise, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids carrying the Leu2 gene.

Methods for introducing exogenous DNA into yeast hosts are known to those of skill in the art and generally include, but are not limited to, transformation of whole yeast host cells or spheroplasts treated with basic cations. For example, the transformation of yeast can be performed according to Hsiao et al., _{PROC.N ATL.A CAD.SCI.USA} (1979) 76:3829 and Van Solingen et al., JB _ACT .(1977) 130:946. method to proceed. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation or protoplast fusion can also be used as generally described in SAMBROOK et al., MOLECULAR CLONING: AL _AB . MANUAL (2001). Yeast host cells can then be cultured using standard techniques known to those of skill in the art.

Other methods for expressing heterologous proteins in yeast host cells are known to those of skill in the art. See generally, US Patent Publication No. 20020055169, US Patent Nos. 6,361,969; 6,312,923; 6,183,985; 6,083,723; 6,017,731; Reexamined Patent Nos. RE37,343 and RE35,749; PCT Published Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080; European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480; WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274 and EP 0 164 556. See also Gellissen et al., _ANTONIE V _AN L _EEUWENHOEK (1992) 62(1-2): 79-93; Romanos et al., YEAST (1992) 8(6): 423-488; Goeddel, METHODS IN ENZYMOLOGY (1990 ) 185:3-7, each reference is incorporated herein by reference.

Yeast host strains can be grown in fermentors during the expansion phase using standard fed-batch fermentation methods known to those skilled in the art. Due to differences in the carbon utilization pathway or mode of expression control of a particular yeast host, the fermentation method can be adapted. For example, fermentation of a Saccharomyces yeast host may require a single glucose feed, a complex nitrogen source (eg, casein hydrolyzate), and multiple vitamin supplements. In contrast, the methylotrophic yeast Pichia pastoris may require glycerol, methanol, and trace mineral feeds, but only simple ammonium (nitrogen) salts for optimal growth and expression. See, e.g., U.S. Patent No. 5,324,639; Elliott et al., J. PROTEIN CHEM. (1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113, which are incorporated by reference In this article.

However, independent of the yeast host strain employed, the fermentation process may have certain common features. For example, a growth-limiting nutrient, usually carbon, can be added to the fermentor during the expansion phase to allow for maximum growth. In addition, fermentation methods generally employ fermentation media designed to contain sufficient amounts of carbon, nitrogen, basic salts, phosphorus, and other micronutrients (vitamins, trace minerals, salts, etc.). Examples of fermentation media suitable for use with Pichia are described in US Patent Nos. 5,324,639 and 5,231,178, which are incorporated herein by reference.

Baculovirus Infected Insect Cells The term "insect host" or "insect host cell" refers to an insect that can be used or has been used as a recipient of recombinant vectors or other transferred DNA. The term includes progeny of the original insect host cell that has been transfected. It is understood that the progeny of a single parental cell may not necessarily be identical in morphology or in genomic or total DNA that is complementary to the original parent, due to accidental or deliberate mutation. Progeny of a parent cell that closely resembles the parent to be characterized by a relevant property such as the presence of a nucleotide sequence encoding a GH (eg, hGH) polypeptide are included within the meaning of this definition.

The selection of suitable insect cells for expression of GH (eg, hGH) polypeptides is known to those of skill in the art. Several insect species are well described in the art and commercially available, including Aedes aegypti, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda and Trichoplusia ni. In selecting insect hosts for expression, suitable hosts may include those that exhibit, inter alia, good secretion capacity, low proteolytic activity, and overall robustness. Insects are generally available from a variety of sources including (but not limited to): Insect GeneticStock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and American Type Culture Collection ("ATCC") (Manassas , VA).

Typically, the components of a baculovirus-infected insect expression system include a transfer vector, usually a bacterial plasmid, containing a segment of the baculovirus genome and suitable restriction sites for insertion of a heterologous gene to be expressed; A wild-type baculovirus of sequences homologous to the baculovirus-specific fragment in the transfer vector (this allows homologous recombination of heterologous genes in the baculovirus genome); and appropriate insect host cells and growth media. Materials, methods and techniques for constructing vectors, transfecting cells, picking plaques, growing cells in culture, etc. are known in the art and manuals describing these techniques are available.

Following insertion of the heterologous gene into the transfer vector, the vector and wild-type viral genome are transfected into insect host cells where they recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for the baculovirus/insect cell expression system are commercially available as kits from, eg, Invitrogen Corp. (Carlsbad, CA). These techniques are generally known to those skilled in the art and are fully described in SUMMERS and SMITH, TEXAS AGRICULTURALEXPERIMENT STATION BULLETIN NO. 1555 (1987), which is incorporated herein by reference. See also, RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSION PROTOCOLS (1995); AUSUBEL et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY 16.9-16.11 (1994); K _ING and _POSSEE , THE BACULOVIRUS SYSTEM: A LABOR (AT) 'R _EILLY et al., BACULOVIRUSE EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins using baculovirus/insect cell expression systems is known to those skilled in the art. See, eg, U.S. Patent Nos. 6,368,825; 6,342,216; 6,338,846; 6,261,805; 6,245,528; 6,225,060;第5,965,393号；第5,939,285号；第5,891,676号；第5,871,986号；第5,861,279号；第5,858,368号；第5,843,733号；第5,762,939号；第5,753,220号；第5,605,827号；第5,583,023号；第5,571,709号；第5,516,657 No. 5,290,686; WO 02/06305; WO 01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO 99/51721; WO 99/45130; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO 89/01037; is incorporated herein by reference.

Vectors suitable for use in baculovirus/insect cell expression systems are known in the art and include, for example, the baculovirus derived from Autographa californica which is a helper-independent viral expression vector (Autographacalifornica) nuclear polyhedrosis virus (AcNPV) insect expression and transfer vector. Viral expression vectors derived from this system typically use the strong viral polyhedrin gene promoter to drive expression of the heterologous gene. See generally O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Before inserting the foreign gene into the baculovirus genome, the above-mentioned components including the promoter, leader sequence (if necessary), coding sequence of interest and transcription termination sequence are usually assembled into an intermediate dislocation construct (transfer vector ). Intermediate misplacement constructs are often maintained in replicons such as extrachromosomal elements (eg, plasmids) capable of stable maintenance in hosts such as bacteria. A replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and expansion. More specifically, the plasmid may contain the polyhedrin polyadenylation signal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and the prokaryotic ampicillin for selection and propagation in E. coli. ) resistance (amp) gene and origin of replication.

A commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors known to those of skill in the art have also been designed, including, for example, pVL985, which changes the polyhedrin start codon from ATG to ATT, and at 32 base pairs downstream of ATT A BamHI cloning site was introduced. See Luckow and Summers, VIROLOGY 170:31 (1989). Other commercially available vectors include, for example, PBlueBac4.5/V5-His, pBlueBacHis2, pMelBac, pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).

After insertion of the heterologous gene, the transfer vector and the wild-type baculovirus genome are co-transfected into insect cell hosts. Methods for introducing heterologous DNA into desired sites in baculovirus are known in the art. See, _SUMMERS and S _MITH , T _EXAS A _{GRICULTURAL EXPERIMENT STATION} B _ULLETIN No. 1555 (1987); Smith et al., MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989) 170:31. For example, insertion may be into a gene such as the polyhedrin gene by homologous double crossover recombination; insertion may also be into a restriction enzyme site designed into the desired baculovirus gene. See, Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection can be accomplished by electroporation. See, TROTTER and WOOD, 39 METHODS INMOLECULAR BIOLOGY (1995); Mann and King, J. GEN. VIROL. (1989) 70:3501. Alternatively, liposomes can be used to transfect insect cells with recombinant expression vectors and baculovirus. See, eg, Liebman et al., BIOTECHNIQUES (1999) 26(1):36; Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J.BIOL.CHEM. (1998) 273(22):13570 ; Schmidt et al., PROTEINE EXPRESSION AND PURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998) 18:45; TILKTNS et al., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154 (1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997) 190:139; Jakobsson et al., J.BIOL.CHEM.(1996) 271: 22203; Rowles et al., J.BIOL.CHEM.(1996) 271(37):22376; Reverey et al., JB _IOL.CHEM .(1996) 271(39):23607-10; Stanley et al., JB _IOL . CHEM. (1995) 270:4121; Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES (1993) 14(2):274. Commercially available liposomes include, for example, Cellfectin(R) and Lipofectin(R) (Invitrogen, Corp., Carlsbad, CA). Alternatively, calcium phosphate transfection can be used. See, TROTTER and WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990) 18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding baculovirus RNA polymerase and triggering the downstream (3') transcription of a coding sequence (eg, a structural gene) into mRNA. A promoter will have a transcription initiation region usually located closest to the 5' end of the coding sequence. The transcription initiation region generally includes an RNA polymerase binding site and a transcription initiation site. Baculovirus promoters can also have a second domain called an enhancer, which, if present, is usually located away from the structural gene. Furthermore, expression can be regulated or constitutive.

Structural genes that are well transcribed late in the infection cycle provide particularly useful promoter sequences. Examples include those derived from the gene encoding the viral polyhedron protein (FRIESEN et al., The Regulation of Baculovirus Gene Expression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476) and the gene encoding the p10 protein (Vlak et al. , J.GEN.VIROL. (1988) 69:765).

The newly formed baculovirus expression vectors are packaged into infectious recombinant baculoviruses, and grown plaques can then be purified by techniques known to those skilled in the art. See, Miller et al., BIOESSAYS (1989) 11(4):91; SUMMERS and SMITH, TEXAS AGRICULTURAL EXPERIMENTSTATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed for infection into some insect cells. For example, recombinants have been developed for Aedes aegypti (ATCC No. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster (ATCC No. 1963), Spodoptera frugiperda and Trichoplusia inter alia Baculovirus. See, Wright, _NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smith et al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., _IN VITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, cell lines for baculovirus expression vector systems typically include, but are not limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21 (Spodoptera frugiperda) (Invitrogen Corp., catalog No. 11497-013 (Carlsbad, CA)), Tri-368 (Trichoptera) and High-Five ^™ BTI-TN-5B1-4 (Trichoptera).

Cells and media for direct expression and fusion expression of heterologous polypeptides in baculovirus/expression are commercially available, and cell culture techniques are generally known to those of skill in the art.

E. coli, Pseudomonas species, and other prokaryotic bacterial expression techniques are known to those skilled in the art. A wide variety of vectors are available for use in bacterial hosts. Vectors can be single copy or low multicopy or high multicopy vectors. Vectors can be used for cloning and/or expression. In view of the extensive literature on vectors, the commercial availability of many vectors and also in view of the handbooks describing vectors and their restriction maps and characteristics, no extensive discussion is required here. As is well known, vectors generally involve selectable markers that may confer resistance to cytotoxic agents, prototrophy, or immunity. Often there are multiple markers that provide different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and triggering the downstream (3') transcription of a coding sequence (eg, a structural gene) into mRNA. A promoter will have a transcription initiation region usually located closest to the 5' end of the coding sequence. The transcription initiation region generally includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters can also have a second domain called an operator, which can overlap the adjacent RNA polymerase binding site where RNA synthesis begins. Operators allow negatively regulated (inducible) transcription because gene repressor proteins can bind to the operator and thereby repress the transcription of a particular gene. Constitutive expression can occur in the absence of negative regulatory elements such as operators. Additionally, positive regulation can be achieved by the gene activator protein binding sequence, which, if present, is usually closest (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli, E. coli) [Raibaud et al., ANNU.R _EV .G _ENET . (1984) 18:173]. Regulated expression can thus be positive or negative, thereby enhancing or decreasing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose (lac) [Chang et al., NATURE (1977) 198:1056] and maltose. Other examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al., Nuc.ACIDS RES. (1980) 8:4057; Yelverton et al., NUCL.ACIDS RES. (1981) 9 : 731; U.S. Patent No. 4,738,921; European Patent Publication Nos. 036 776 and 121 775, which are incorporated herein by reference]. β-galactosidase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes. "In Interferon 3 (I.Gresser edit)], bacteriophage lambda PL [people such as Shimatake, NATURE (1981) 292: 128] and T5 [US Patent No. 4,689,406, which is incorporated herein by reference] promoter systems also provide useful promoter sequences. Preferred methods of the invention utilize a strong promoter, such as the T7 promoter, to induce high levels of GH (eg, hGH) polypeptide. Examples of such vectors are known to those skilled in the art and include the pET29 series from Novagen and the pPOP vectors described in WO99/05297, incorporated herein by reference. The expression system produces high levels of GH (eg, hGH) polypeptide in the host without compromising host cell viability or growth parameters. pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, the transcriptional activation sequence of one bacterial or phage promoter can be joined to the operator sequence of another bacterial or phage promoter to produce a synthetic hybrid promoter [U.S. Patent No. 4,551,433, named incorporated herein by reference]. For example, the tac promoter is a hybrid trp-lac promoter consisting of a trp promoter and a lac operator sequence regulated by the lac repressor [Amann et al., GENE (1983) 25:167; de Boer et al., PROC .N _ATL .A _CAD .SCI.(1983) 80:21]. In addition, bacterial promoters can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Naturally occurring promoters of non-bacterial origin can also be coupled with compatible RNA polymerases to produce high-level expression of some genes in prokaryotes. The phage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al., J. MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985) 82 :1074]. Alternatively, a hybrid promoter can also consist of a phage promoter and an E. coli operon region (EP publication no. 267 851).

In addition to functional promoter sequences, efficient ribosome binding sites are also available for expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes the start codon (ATG) and is located 3-11 nucleosides upstream of the start codon A sequence of 3-9 nucleotides in length at the acid [Shine et al., _NATURE (1975) 254:34]. It is believed that the SD sequence promotes the binding of mRNA to the ribosome by base pairing between the SD sequence and the 3' end of the Escherichia coli 16S rRNA [Steitz et al. "Genetic signals and nucleotide sequences in messenger RNA", in Biological Regulation and Development: Gene Expression ( RF Goldberger ed., 1979)]. Eukaryotic and prokaryotic genes are expressed using weak ribosome binding sites [Sambrook et al., "Expression of cloned genes in Escherichia coli", Molecular Cloning: A Laboratory Manual, 1989].

The term "bacterial host" or "bacterial host cell" refers to a bacterium that can be used or has been used as a recipient of recombinant vectors or other transferred DNA. The term includes progeny of the original bacterial host cell that has been transfected. It is understood that the progeny of a single parental cell may not necessarily be identical in morphology or in genomic or total DNA that is complementary to the original parent, due to accidental or deliberate mutation. Progeny of a parental cell that closely resembles the parent to be characterized by a relevant property such as the presence of a nucleotide sequence encoding a GH (eg, hGH) polypeptide are included within the meaning of this definition.

The selection of suitable host bacteria for expression of a GH (eg, hGH) polypeptide is known to those of skill in the art. In selecting bacterial hosts for expression, suitable hosts may include those exhibiting, inter alia, good inclusion body forming ability, low proteolytic activity, and overall robustness. Bacterial hosts are generally available from a variety of sources including (but not limited to): Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and American TypeCulture Collection ("ATCC") (Manassas , VA). Industrial fermentation/pharmaceutical fermentation generally use bacteria derived from K strains (eg, W3110) or bacteria derived from B strains (eg, BL21). This is especially useful because the growth parameters of these strains are well known and stable. Additionally, these strains are non-pathogenic, which is commercially important for safety and environmental reasons. Other examples of suitable E. coli hosts include, but are not limited to, strains of BL21, DH10B, or derivatives thereof. In another embodiment of the methods of the invention, the E. coli host is a protease negative strain, including but not limited to OMP- and LON-. The host cell strain may be a Pseudomonas species including, but not limited to, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biotype 1 designated strain MB101 is known to be useful in recombinant production and in therapeutic protein production processes. Examples of Pseudomonas expression systems include those obtained from the Dow Chemical Company as host strains (Midland, MI, available at www.dow.com). US Patent Nos. 4,755,465 and 4,859,600, incorporated herein by reference, describe the use of Pseudomonas strains as host cells for GH (eg, hGH) production.

Once the recombinant host cell strain has been established (i.e., the expression construct has been introduced into the host cell and the host cell with the appropriate expression construct is isolated), it is cultured under conditions suitable for the production of a GH (e.g., hGH) polypeptide Recombinant host cell strains. As is readily understood by those of skill in the art, methods of culturing recombinant host cell strains will depend on the nature of the expression construct utilized and the properties of the host cell. Recombinant host strains are typically grown using methods known to those skilled in the art. Recombinant host cells are typically cultured in liquid media containing assimilable sources of carbon, nitrogen, and inorganic salts, and, if necessary, vitamins, amino acids, growth factors, and other protein culture supplements known to those of skill in the art. The liquid medium used for culturing the host cells may optionally contain antibiotics or antifungal agents to prevent the growth of undesired microorganisms and/or include, but not limited to, antibiotics (to select for host cells containing the expression vector). compound.

Recombinant host cells can be cultured in batch or in continuous format, with cell harvesting (in the case of intracellular accumulation of GH (eg, hGH) polypeptide) or collection of culture supernatant in batch or continuous format. Batch culture and cell harvesting are preferred for production in prokaryotic host cells.

The GH (eg, hGH) polypeptides of the invention are typically expressed in recombinant systems and then purified. GH (eg, hGH) polypeptides can be purified from host cells or culture medium by various methods known in the art. GH (eg, hGH) polypeptides produced in bacterial host cells may be poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment of the invention, amino acid substitutions can readily be made in a GH (e.g., hGH) polypeptide selected to increase recombinant production using the methods disclosed herein as well as those known in the art. the solubility of the protein. In the case of insoluble proteins, the protein can be collected from the host cell lysate by centrifugation and can be further followed by homogenization of the cells. In the case of poorly soluble proteins, compounds including, but not limited to, polyethyleneimine (PEI) can be added to induce precipitation of partially soluble proteins. The precipitated protein can then conveniently be collected by centrifugation. Recombinant host cells can be disrupted or homogenized to release inclusion bodies from within the cells using various methods known to those of skill in the art. The host cell disruption or homogenization process can be performed using well-known techniques including, but not limited to, enzymatic cell disruption, sonication, dounce homogenization, or high pressure release disruption. In one embodiment of the methods of the invention, E. coli host cells are disrupted using high pressure release techniques to release inclusion bodies of a GH (eg, hGH) polypeptide. When processing the inclusion bodies of GH (e.g., hGH) polypeptides, it may be advantageous to minimize repeated homogenization times to maximize the yield of inclusion bodies without due to factors such as solubilization, mechanical shear, or protein Losses caused by hydrolysis factors.

Insoluble or precipitated GH (eg, hGH) polypeptides can then be solubilized using any of a number of suitable solubilizing agents known in the art. GH (eg, hGH) polypeptides can be solubilized with urea or guanidine hydrochloride. The volume of dissolved GH (eg, hGH) polypeptide should be minimized so that large batches can be produced using manageable batch sizes. This factor may be important in large-scale commercial settings where recombinant hosts can be grown in batches with volumes of several thousand liters. In addition, when manufacturing GH (eg, hGH) polypeptides in large-scale commercial settings, especially for human pharmaceutical use, harsh chemicals that can damage machinery and containers or the protein product itself should be avoided if possible. It has been shown in the methods of the present invention that the milder denaturant urea can be used to solubilize GH (eg, hGH) polypeptide inclusion bodies in place of the harsher denaturant guanidine hydrochloride. The use of urea significantly reduces the risk of damage to stainless steel equipment utilized during the manufacture and purification of GH (eg, hGH) polypeptides, while efficiently solubilizing GH (eg, hGH) polypeptide inclusion bodies.

In the case of soluble hGH protein, hGH can be secreted into the periplasmic space or into the culture medium. In addition, soluble hGH can be present in the cytoplasm of the host cell. It may be necessary to concentrate the soluble hGH prior to performing the purification steps. Standard techniques known to those of skill in the art can be used to concentrate soluble hGH from, for example, cell lysates or culture media. Additionally, standard techniques known to those of skill in the art can be used to disrupt the host cell and release soluble hGH from the cytoplasmic or periplasmic space of the host cell.

When a GH (eg, hGH) polypeptide is produced as a fusion protein, the fusion sequence can be removed. Removal of fusion sequences can be achieved by enzymatic or chemical cleavage. Enzymatic removal of fusion sequences can be achieved using methods known to those skilled in the art. The choice of enzyme for removal of the fusion sequence will be determined by the nature of the fusion and, as will be readily understood by those skilled in the art, the reaction conditions will be determined by the choice of enzyme. Chemical cleavage can be accomplished using reagents known to those of skill in the art including, but not limited to, cyanogen bromide, TEV protease, and other reagents. A cleaved GH (eg, hGH) polypeptide can be purified from the cleaved fusion sequence by methods known to those of skill in the art. Such methods will be dictated by the identity and properties of the fusion sequence and the GH (eg, hGH) polypeptide, as will be readily apparent to those of skill in the art. Methods for purification may include, but are not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, or dialysis, or any combination thereof.

GH (eg, hGH) polypeptides can also be purified to remove DNA from protein solutions. DNA may be removed by any suitable method known in the art, such as precipitation or ion exchange chromatography, or by precipitation with a nucleic acid precipitating agent such as, but not limited to, protamine sulfate. GH (eg, hGH) polypeptides can be separated from precipitated DNA using standard, well-known methods including, but not limited to, centrifugation or filtration. Removal of host nucleic acid molecules is an important factor in the setting of GH (eg, hGH) polypeptides to be used to treat humans, and the methods of the invention reduce host cell DNA to pharmaceutically acceptable levels.

Methods of small-scale fermentation or large-scale fermentation can also be used for protein expression, including (but not limited to) fermentors, shake flasks, fluidized bed bioreactors, hollow fiber bioreactors, spinner bottle culture systems, and stirred tank bioreactors device system. Each of these processes can be performed as a batch process, a fed-batch process, or a continuous mode process.

Human GH polypeptides of the invention can generally be recovered using standard methods in the art. For example, culture medium or cell lysates can be centrifuged or filtered to remove cellular debris. The supernatant can be concentrated or diluted to the desired volume or diafiltered into an appropriate buffer to condition the preparation for further purification. Further purification of the GH (eg, hGH) polypeptides of the invention includes isolating deamidated and truncated forms of the GH (eg, hGH) polypeptide variants from the intact form.

GH (e.g., hGH) polypeptides of the invention can be purified using any of the following exemplary procedures: affinity chromatography; anion or cation exchange chromatography (using, but not limited to, DEAE SEPHAROSE); silica chromatography; Reverse phase HPLC; Gel filtration (using including but not limited to SEPHADEX G-75); Hydrophobic interaction chromatography; Size exclusion chromatography; Metal chelation chromatography; Ultrafiltration/diafiltration; Ethanol precipitation; Ammonium sulfate precipitation; chromatographic focusing; displacement chromatography; electrophoretic procedures (including, but not limited to, preparative isoelectric focusing), differential solubility methods (including, but not limited to, ammonium sulfate precipitation), SDS-PAGE, or extraction methods.

Proteins of the invention, including (but not limited to) proteins comprising unnatural amino acids, peptides comprising unnatural amino acids, antibodies to proteins comprising unnatural amino acids, binding partners of proteins comprising unnatural amino acids, etc. Partially or substantially purified to homogeneity using standard procedures known and used by those skilled in the art. Accordingly, the polypeptides of the present invention can be extracted by methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic Recovery and purification by any of a number of methods known to those skilled in the art such as sex interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis, and the like. Protein refolding steps can be used as desired in the production of properly folded mature proteins. High performance liquid chromatography (HPLC), affinity chromatography or other suitable methods may be employed in final purification steps requiring high purity. In one embodiment, antibodies raised against unnatural amino acids (or proteins or peptides comprising unnatural amino acids) are used as purification reagents, including but not limited to, for proteins comprising one or more unnatural amino acids or purification reagents for affinity-based purification of peptides. Once a polypeptide has been partially purified or purified to homogeneity as desired, it can be used in a variety of applications as desired, including, but not limited to, use as assay components, therapeutic agents, prophylactic agents, diagnostic agents, scientific research reagents and /or used as an immunogen for antibody production.

Various purification/protein folding methods are known to those skilled in the art, including (but not limited to) those described in the following references, in addition to the other references mentioned herein: R .Scopes, Protein Purification , Springer-Verlag, NY (1982); Deutscher, Methods in Enzymology, Vol. 182: Guide to Protein Purification , Academic Press, Inc. NY (1990); Sandana, (1997) Bioseparation of Proteins , Academic Press, Inc.; Bollag et al. (1996) Protein Methods , 2nd Edition, Wiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook Humana Press, NJ; Harris and Angal, (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes, (1993) Protein Purification: Principles and Practice, 3rd ed. , Springer Verlag, NY ; Janson and Ryden, (1998) Protein Purification: Principles, High Resolution Methods and Applications, 2nd Edition, Wiley-VCH, NY; and Walker (1998), Protein Protocols on CD-ROM Humana Press, NJ; and cited therein references.

One advantage of producing a protein or polypeptide of interest with an unnatural amino acid in a eukaryotic or non-eukaryotic host cell is that the protein or polypeptide will generally fold into its native conformation. However, in certain embodiments of the invention, those skilled in the art will recognize that, following synthesis, expression and/or purification, a protein or peptide may have a conformation that differs from the desired conformation of the related polypeptide. In one aspect of the invention, the expressed protein is optionally denatured and subsequently renatured. This is accomplished using methods known in the art including, but not limited to, by adding chaperonins to the protein or polypeptide of interest, by solubilizing the protein in a chaotropic agent such as guanidine hydrochloride , by using protein disulfide bond isomerase, etc. to achieve.

In general, it is sometimes desirable to denature and reduce the expressed polypeptide and then refold the polypeptide into a preferred conformation. For example, guanidine, urea, DTT, DTE and/or chaperones can be added to the translation product of interest. Methods for reducing, denaturing and renaturing proteins are known to those skilled in the art (see, references above and Debinski et al., (1993) J. Biol. Chem. , 268:14065-14070; Kreitman and Pastan (1993) Bioconiug. Chem ., 4:581-585; and Buchner et al., (1992) Anal. Biochem., 205:263-270). For example, Debinski et al. describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. Proteins can be refolded in redox buffers containing, but not limited to, oxidized glutathione and L-arginine. Refolding reagents can be flowed or otherwise moved into contact with one or more polypeptides or other expression products, or vice versa.

In the case of prokaryotic production of a GH (eg, hGH) polypeptide, the GH (eg, hGH) polypeptide so produced may be misfolded and thus lack or have reduced biological activity. The biological activity of proteins can be restored by "refolding". Generally, misfolded GH (e.g., hGH) polypeptides are detected by using, for example, one or more chaotropic agents (e.g., urea and/or guanidine) and reducing agents capable of reducing disulfide bonds (e.g., , dithiothreitol, DTT or 2-mercaptoethanol, 2-ME) solubilize the polypeptide chain (wherein the GH (eg, hGH) polypeptide is also insoluble), unfold and reduce to refold. An oxidizing agent (eg, oxygen, cystine, or cystamine) is then added at a moderate concentration of chaotropic agent, which reforms the disulfide bonds. GH (e.g., hGH) polypeptides can be refolded using standard methods known in the art, such as those described in U.S. Patent Nos. 4,511,502, 4,511,503, and 4,512,922, incorporated herein by reference method. GH (eg, hGH) polypeptides can also cofold with other proteins to form heterodimers or heteromultimers.

Following refolding or cofolding, the GH (eg, hGH) polypeptides can be further purified. Purification of GH (e.g., hGH) can be accomplished using various techniques known to those skilled in the art, including hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, reversed-phase high performance liquid Phase chromatography, affinity chromatography, etc. or any combination thereof. Additional purification may also include steps of drying or precipitating the purified protein.

After purification, GH (e.g., hGH) can be exchanged into a different buffer and/or concentrated by any of a variety of methods known in the art including, but not limited to, diafiltration and dialysis . GH (eg, hGH) provided as a single purified protein can be subject to aggregation and precipitation.

The purified GH (e.g., hGH) can be at least 90% pure (as measured by reverse phase high performance liquid chromatography RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE) or At least 95% pure, or at least 98% pure, or at least 99% or more pure. Regardless of the exact value of the purity of the GH (e.g. hGH), it is sufficient for use as a medicinal product or for further processing such as conjugation with a water-soluble polymer such as PEG pure.

Certain GH (e.g., hGH) molecules can be used as therapeutic agents in the absence of other active ingredients or proteins (other than excipients, carriers and stabilizers, serum albumin, etc.), or they can be combined with another protein or polymer composites.

General Purification Methods Any of the various isolation steps can be directed to a cell lysate, extract, culture medium, inclusion body, periplasmic space, cytoplasm of the host cell, or Other materials are performed, or performed on any mixture of GH (e.g., hGH) polypeptides resulting from any separation procedure including, but not limited to, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, coagulation Gel filtration chromatography, high performance liquid chromatography ("HPLC"), reverse phase HPLC ("RP-HPLC"), expanded bed adsorption, or any combination and/or repetition thereof, and may be performed in any suitable order.

Equipment and other necessary materials used in performing the techniques described herein are commercially available. Pumps, fraction collectors, monitors, recorders, and complete systems are available from, for example, Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA), and Amersham Biosciences, Inc. (Piscataway, NJ) obtained. Chromatographic materials including, but not limited to, exchange matrix materials, media, and buffers are also available from the company.

Equilibration and other steps in the column chromatography described herein, such as washing and elution, can be accomplished more rapidly using specialized equipment, such as pumps. Commercially available pumps include, but are not limited to, HILOAD( ^R ) pump P-50, peristaltic pump P-1, pump P-901, and pump P-903 (Amersham Biosciences, Piscataway, NJ).

Examples of fraction collectors include the RediFrac Fraction Collector, the FRAC-100 and FRAC-200 Fraction Collectors, and the SUPERFRAC(R) Fraction Collector (Amersham Biosciences, Piscataway, NJ). Mixers are also available to create pH and linear concentration gradients. Commercially available mixers include the Gradient Mixer GM-1 and the In-Line Mixer (Amersham Biosciences, Piscataway, NJ).

The chromatographic process can be monitored using any commercially available monitor. The monitors can be used to collect information such as UV, pH and conductivity. Examples of detectors include Monitor UV-1, UVICORD® S II, Monitor UV-MII, Monitor UV-900, Monitor UPC-900, Monitor pH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway , NJ). Indeed, complete systems including the various AKTA(R) systems from Amersham Biosciences (Piscataway, NJ) are commercially available.

In one embodiment of the invention, for example, a GH (e.g., hGH) polypeptide can be prepared by first denaturing the resulting purified GH (e.g., hGH) polypeptide in urea, followed by dilution to a reducing agent (such as, DTT) Reduction and denaturation in TRIS buffer with appropriate pH value. In another embodiment, a GH (eg, hGH) polypeptide is denatured in urea at a concentration ranging from about 2M to about 9M, followed by dilution into TRIS buffer having a pH ranging from about 5.0 to about 8.0. The refolding mixture of the examples can then be cultured. In one embodiment, the refolding mixture is incubated at room temperature for 4 hours to 24 hours. The reduced and denatured GH (eg, hGH) polypeptide mixture can then be further isolated or purified.

As described herein, the pH of the first GH (eg, hGH) polypeptide mixture can be adjusted prior to any subsequent isolation steps. Additionally, the first GH (eg, hGH) polypeptide mixture, or any subsequent mixture thereof, can be concentrated using techniques known in the art. In addition, the elution buffer comprising the first GH (eg, hGH) polypeptide mixture, or any subsequent mixture thereof, can be exchanged for a buffer suitable for the next isolation step using techniques known to those of skill in the art.

Ion Exchange Chromatography In one embodiment and as an optional, additional step, ion exchange chromatography can be performed on the first GH (eg, hGH) polypeptide mixture. See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21, Amersham Biosciences (Piscataway, NJ)). Commercially available ion exchange columns include HITRAP ^(R) , HIPREP ^(R) , and HILOAD ^(R) columns (Amersham Biosciences, Piscataway, NJ). The columns utilize strong anion exchangers such as Q SEPHAROSE ^(R) Fast Flow, Q SEPHAROSE ^(R) High Performance and Q SEPHAROSE(R) XL; strong cation exchangers such as SP SEPHAROSE ⁽ R) High Performance, SP SEPHAROSE ^(R) FastFlow and SP SEPHAROSE ^(R) XL Weak anion exchangers, such as DEAE SEPHAROSE ^(R) Fast Flow; and weak cation exchangers, such as CM SEPHAROSE ^(R) Fast Flow (Amersham Biosciences, Piscataway, NJ). Anion or cation exchange column chromatography can be performed on the GH (eg, hGH) polypeptide at any stage of the purification process to isolate a substantially purified GH (eg, hGH) polypeptide. The cation exchange chromatography step can be performed using any suitable cation exchange matrix. Useful cation exchange matrices include, but are not limited to, fibrous, porous, non-porous, particulate, beaded, or crosslinked cation exchange matrix materials. Such cation exchange matrix materials include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyethylene, polystyrene, silica, polyether, or composites of any of the foregoing.

The cation exchange matrix can be any suitable cation exchanger including strong cation exchangers and weak cation exchangers. A strong cation exchanger can remain ionized over a wide pH range and thus can be capable of binding GH (eg, hGH) over a wide pH range. However, weak cation exchangers can lose ionization with changes in pH. For example, weak cation exchangers may lose charge when the pH is lowered below about pH 4 or pH 5. Suitable strong cation exchangers include, but are not limited to, charged functional groups such as sulfopropyl (SP), methyl sulfonate (S) or sulfoethyl (SE). The cation exchange matrix can be a strong cation exchanger that preferably has a GH (eg, hGH) binding pH range of about 2.5 to about 6.0. Alternatively, the strong cation exchanger can have a GH (eg, hGH) binding pH range of about pH 2.5 to about pH 5.5. The cation exchange matrix can be a strong cation exchanger with a GH (eg, hGH) binding pH of about 3.0. Alternatively, the cation exchange matrix can be a strong cation exchanger preferably having a GH (eg, hGH) binding pH range of about 6.0 to about 8.0. The cation exchange matrix can be a strong cation exchanger that preferably has a GH (eg, hGH) binding pH range of about 8.0 to about 12.5. Alternatively, the strong cation exchanger can have a GH (eg, hGH) binding pH range of about pH 8.0 to about pH 12.0.

The cation exchange matrix can be equilibrated, for example, using several column volumes of diluted mild acid (eg, 4 column volumes of 20 mM acetic acid at pH 3) prior to loading with GH (eg, hGH). After equilibration, GH (eg, hGH) can be added and the column can be washed one to several times with a weak acid solution, such as a weak acetic or phosphoric acid solution, before substantially purified GH (eg, hGH) is eluted. For example, the column can be washed with approximately 2-4 column volumes of 20 mM acetic acid, pH 3. Additional washes with, for example, 2-4 column volumes of 0.05M sodium acetate at pH 5.5 or 0.05M sodium acetate at pH 5.5 mixed with 0.1M sodium chloride may also be used. Alternatively, the cation exchange matrix can be equilibrated using several column volumes of diluted weak base using methods known in the art.

Alternatively, substantially purified GH (eg, hGH) can be eluted by contacting the cation exchanger matrix with a buffer of sufficiently low pH or ionic strength to displace the GH (eg, hGH) from the matrix. The pH of the elution buffer can vary from a pH of about 2.5 to a pH of about 6.0. More specifically, the pH of the elution buffer can range from about pH 2.5 to about pH 5.5, from about pH 2.5 to about pH 5.0. The elution buffer may have a pH of about 3.0. Additionally, the amount of elution buffer can vary widely and will generally range from about 2 column volumes to about 10 column volumes.

After adsorption of the GH (e.g., hGH) polypeptide to a cation exchanger matrix, the substantially purified hGH polypeptide can be eluted by contacting the matrix with a buffer of sufficiently high pH or ionic strength to convert the GH (e.g., hGH) ) polypeptides are displaced from the matrix. Suitable buffers for high pH elution of substantially purified GH (e.g., hGH) polypeptides useful herein include, but are not limited to, citric acid at concentrations ranging from at least about 5 mM to at least about 100 mM Salt, Phosphate, Formate, Acetate, HEPES and MES buffers.

Reverse Phase Chromatography RP-HPLC can be performed to purify proteins following suitable protocols known to those skilled in the art. See, eg, Pearson et al., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J.CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM.(1986 ) 359:391-402. RP-HPLC can be performed on a GH (eg, hGH) polypeptide to isolate a substantially purified GH (eg, hGH) polypeptide. In this regard, various lengths (including, but not limited to, at least about C ₃ to at least about C ₃₀ , at least about C ₃ to at least about C ₂₀ , or at least about C ₃ to at least about C ₁₈ ) can be used. Alkyl functional silica derivatized resins. Alternatively, polymeric resins can be used. For example, TosoHaas Amberchrome CG1000sd resin, which is a styrene polymer resin, may be used. Cyano or polymeric resins with various alkyl chain lengths can also be used. In addition, the RP-HPLC column can be washed with a solvent such as ethanol. Source RP columns are another example of RP-HPLC columns.

A suitable elution buffer containing an ion pairing agent and an organic modifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile or ethanol can be used to elute the GH (eg, hGH) polypeptide from the RP-HPLC column. The most commonly used ion pairing agents include, but are not limited to, acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, triethylamine, tetramethylammonium, tetrabutylammonium, and triethylammonium acetate. Elution can be performed using one or more gradients or isocratic conditions, where gradient conditions are optimized to result in shorter separation times and reduced peak widths. Another approach involves the use of two gradients with different solvent concentration ranges. Examples of suitable elution buffers for use herein may include, but are not limited to, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification Techniques Hydrophobic interaction chromatography (HIC) can be performed on GH (eg, hGH) polypeptides. See generally, HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK: PRINCIPLES AND M _ETHODS (Cat. No. 18-1020-90), Amersham Biosciences (Piscataway, NJ), which is incorporated herein by reference. Suitable HIC matrices may include, but are not limited to, alkyl- or aryl-substituted matrices, such as butyl, hexyl, octyl, or phenyl-substituted matrices, including agarose, cross-linked agarose, Sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate) matrices; and mixed resins, including but not limited to polyvinylamine resins or butyl- or phenyl-substituted poly(methacrylate) matrix. Commercial sources for hydrophobic interaction column chromatography include, but are not limited to, HITRAP ^(R ), HIPREP ^(R) , and HILOAD( ^R) columns (Amersham Biosciences, Piscataway, NJ). Briefly, prior to loading, the HIC column can be equilibrated with standard buffers known to those skilled in the art, such as acetic acid/sodium chloride solution or HEPES with ammonium sulfate. Ammonium sulfate can be used as a buffer for loading HIC columns. Following loading of the GH (eg, hGH) polypeptide, the column can then be washed using standard buffers and conditions to remove unwanted material but retain the GH (eg, hGH) polypeptide on the HIC column. GH can be eluted with about 3 column volumes to about 10 column volumes of a standard buffer such as, inter alia, a HEPES buffer containing EDTA and a lower concentration of ammonium sulfate than the equilibration buffer, or an acetic acid/sodium chloride buffer (eg, hGH) polypeptides. Decreasing linear salt gradients using, for example, potassium phosphate gradients can also be used to elute GH (eg, hGH) molecules. The eluate can then be concentrated, for example, by filtration such as diafiltration or ultrafiltration. Salts used to elute the GH (eg, hGH) polypeptide can be removed using diafiltration.

Other purification techniques can be performed on the first GH (e.g. hGH) polypeptide mixture or any subsequent mixture thereof using, for example, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat. No. 18-1022-18, Amersham Biosciences, Piscataway , NJ), which is incorporated herein by reference), hydroxyapatite chromatography (suitable matrices include, but are not limited to, HA-Ultrogel, High Resolution (Calbiochem), CHT ceramic hydroxyapatite (BioRad ), Bio-Gel HTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration, lyophilization, etc. before another separation step to remove any excess salt and replace with an appropriate buffer buffer for the next separation step or even for formulation of the final drug product.

The yield of GH (eg, hGH) polypeptides, including substantially purified GH (eg, hGH) polypeptides, can be monitored at the various steps described herein using techniques known to those of skill in the art. The technique can also be used to assess the yield of substantially purified GH (eg, hGH) polypeptide following the final isolation step. For example, any of several reverse phase high pressure liquid chromatography (such as cyano RP-HPLC, C ₁₈ RP-HPLC) columns with various alkyl chain lengths can be used as well as cation exchange HPLC and gel filtration HPLC to monitor the yield of GH (eg, hGH) polypeptide.

In particular embodiments of the invention, the yield of GH (e.g., hGH) after each purification step can be at least about 30%, at least about 35%, of the GH (e.g., hGH) in the starting material of each purification step , at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% , at least about 99.9%, or at least about 99.99%.

Purity can be determined using standard techniques such as SDS-PAGE or by measuring GH (eg, hGH) polypeptide using Western blot and ELISA assays. For example, polyclonal antibodies can be raised against proteins isolated from negative control yeast fermentation and cation exchange recovery. Antibodies can also be used to detect the presence of contaminating host cell proteins.

The RP-HPLC material Vydac C4 (Vydac) consists of silica particles with C4-alkyl chains on their surface. Separation of GH (eg, hGH) polypeptides from proteinaceous impurities is based on differences in the strength of hydrophobic interactions. Elution was performed using an acetonitrile gradient in dilute trifluoroacetic acid. Preparative HPLC was performed using stainless steel columns packed with 2.8 to 3.2 liters of Vydac C4 silica gel. The hydroxyapatite Ultrogel eluate was acidified by addition of trifluoroacetic acid and loaded onto a Vydac C4 column. For washing and elution, a gradient of acetonitrile in dilute trifluoroacetic acid was used. Fractions were collected and immediately neutralized with phosphate buffered saline. GH (eg, hGH) polypeptide fractions within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl (DEAE) covalently bound to the surface of Sepharose beads. Binding of a GH (eg, hGH) polypeptide to the DEAE group is mediated by ionic interactions. Acetonitrile and trifluoroacetic acid pass through the column without retention. After these materials have been washed away, trace impurities are removed by washing the column with low pH acetate buffer. The column is then washed with neutral phosphate buffer and the GH (eg, hGH) polypeptide is eluted with a buffer of increasing ionic strength. The column was packed with DEAE Sepharose fast flow. The column volume is adjusted to ensure that the GH (eg, hGH) polypeptide loading is in the range of 3-10 mg GH (eg, hGH) polypeptide/ml gel. The column was washed with water and equilibration buffer (sodium/potassium phosphate). The pooled HPLC eluate fractions were loaded and the column was washed with equilibration buffer. Then, the column was washed with washing buffer (sodium acetate buffer), followed by washing with equilibration buffer. Subsequently, the GH (eg, hGH) polypeptide is eluted from the column with elution buffer (sodium chloride, sodium/potassium phosphate) and collected in a single fraction according to the master elution profile. Adjust the eluate of the DEAE Sepharose column to the specified conductivity. The resulting drug was sterile filtered into Teflon bottles and stored at -70°C.

Other methods that may be employed include, but are not limited to, steps to remove endotoxin. Endotoxins are lipopolysaccharides (LPS) located on the outer membrane of Gram-negative host cells such as Escherichia coli. Methods of reducing endotoxin levels are known to those skilled in the art and include, but are not limited to, the use of silica supports, glass powder or hydroxyapatite, reverse phase chromatography, affinity chromatography, size exclusion Purification techniques such as resistance chromatography, anion exchange chromatography, hydrophobic interaction chromatography, combinations of these methods, etc. Modifications or other methods may be required to remove contaminants such as co-migrating proteins from the polypeptide of interest. Methods for determining endotoxin levels are known to those of skill in the art and include, but are not limited to, the Limulus Amebocyte Lysate (LAL) assay.

A variety of methods and procedures are available to assess the yield and purity of GH (e.g., hGH) proteins comprising one or more non-naturally encoded amino acids, including, but not limited to, the Bradford Assays, SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry (including but not limited to MALDI-TOF) and other methods known to those skilled in the art for characterizing proteins .

Other methods include (but are not limited to): SDS-PAGE combined with protein staining methods, immunoblotting, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry methods, isoelectric focusing, analytical anion exchange, chromatofocusing and circular dichroism.

VIII. Expression in Alternative Systems

Several strategies have been employed to introduce unnatural amino acids into proteins in non-recombinant host cells, mutated host cells, or in cell-free systems. These systems are also suitable for use in the production of GH (eg, hGH) polypeptides of the invention. Derivatization of amino acids with reactive side chains such as Lys, Cys and Tyr results in the conversion of lysine to ^N2 -acetyl-lysine. Chemical synthesis also provides a straightforward method for incorporation of unnatural amino acids. With the recent development of enzymatic and native chemical ligation of peptide fragments, it is possible to make larger proteins. See, eg, PEDawson and SBHKent, Annu. Rev. Biochem , 69:923 (2000). Chemical peptide linkage and native chemical linkage are described in US Patent No. 6,184,344, US Patent Publication No. 2004/0138412, US Patent Publication No. 2003/0208046, WO 02/098902 and WO 03/042235, all The above references are incorporated herein by reference. A general in vitro biosynthetic approach that incorporates suppressor tRNAs chemically acylated with the desired unnatural amino acid into in vitro extracts capable of sustaining protein biosynthesis has been used to site-specifically target more than 100 unnatural amino acids. Incorporated into a wide variety of proteins of virtually any size. See, eg, VW Cornish, D. Mendel and PG Schultz, Angew. Chem. Int. Ed. Engl. , 1995, 34:621 (1995); CJ Noren, SJ Anthony-Cahill, MC Griffith, PG Schultz, A general method for site-specific Incorporation of unnatural amino acids into proteins, Science 244:182-188 (1989); and JDBain, CG Glabe, TADix, ARChamberlin, ESDiala, Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide, J.Am.Chem .Soc. 111:8013-8014 (1989). A wide range of functional groups have been introduced into proteins for the study of protein stability, protein folding, enzymatic mechanisms and signal transduction.

An in vivo approach called selective pressure incorporation was developed to exploit the promiscuity of wild-type synthetases. See, eg, N. Budisa, C. Minks, S. Alefelder, W. Wenger, FM Dong, L. Moroder, and R. Huber, FASEB J. , 13:41 (1999). An auxotrophic strain in which the relevant metabolic pathway supplying a cell-specific natural amino acid is cut off is grown in a minimal medium containing a limited concentration of natural amino acid while transcription of the target gene is repressed. At the onset of the quiescent growth phase, natural amino acids are depleted and replaced by unnatural amino acid analogs. Induction of expression of recombinant proteins results in the accumulation of proteins containing non-natural analogs. For example, using this strategy, o-, m-, and p-fluorophenylalanine have been incorporated into proteins and present two characteristic features that can be easily identified in the UV spectrum. Shoulder, see, e.g., C. Minks, R. Huber, L. Moroder, and N. Budisa, Anal. Biochem. , 284:29 (2000); trifluoromethionine has been used to replace bacteriophage T4 lytic Methionine in enzymes to study its interaction with oligochitosan ligands by ¹⁹ F NMR, see, for example, H.Duewel, E.Daub, V.Robinson and JFHonek, Biochemistry , 36:3404 (1997 ); and trifluoroleucine has been incorporated in place of leucine, resulting in increased thermal and chemical stability of leucine zipper proteins. See, eg, Y. Tang, G. Ghirlanda, WAPetka, T. Nakajima, WF DeGrado, and DATirrell, Angew Chem. Int. Ed . Engl., 40:1494 (2001). Furthermore, selenomethionine and telluromethionine were incorporated into various recombinant proteins to facilitate the dissolution of phases in X-ray crystallographic analysis. See, eg, WA Hendrickson, JR Horton, and DM Lemaster, EMBO J. , 9: 1665 (1990); JOBoles, K. Lewinski, M. Kunkle, JDOdom, B. Dunlap, L. Lebioda, and M. Hatada, Nat. Struct. Biol . , 1: 283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J. Kellermann and R. Huber, Eur. J. Biochem. , 230: 788 (1995); and N. Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind, L. Moroder, and R. Huber, J. Mol. Biol., 270:616 (1997). Methionine analogs with alkene or alkyne functionality have also been incorporated efficiently, allowing additional modification of proteins by chemical means. See, e.g., JC van Hest and DATirrell, FEBS Lett. , 428:68 (1998); JC van Hest, KL Kiick and DATirrell, J.Am.Chem.Soc ., 122:1282 (2000); and KL Kiick and DATirrell, Tetrahedron , 56:9487 (2000); US Patent No. 6,586,207; US Patent Publication 2002/0042097, which is incorporated herein by reference.

The success of this approach depends on the recognition of unnatural amino acid analogs by aminoacyl-tRNA synthetases, which generally require high selectivity to ensure the fidelity of protein translation. One way to expand the scope of this approach is to relax the substrate specificity of aminoacyl-tRNA synthetases, which has been achieved in limited circumstances. For example, replacement of Ala ²⁹⁴ with Gly in Escherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the size of the substrate-binding pocket and results in tRNAPhe being replaced by p-chlorophenylalanine (p- Cl-Phe) acylation. See, M. Ibba, P. Kast and H. Hennecke, Biochemistry , 33:7107 (1994). E. coli strains with this mutant PheRS allow the incorporation of p-chlorophenylalanine or p-bromophenylalanine in place of phenylalanine. See, eg, M. Ibba and H. Hennecke, FEBS Lett. , 364:272 (1995); and N. Sharma, R. Furter, P. Kast and DATirrell, FEBS Lett. , 467:37 (2000). Likewise, it was shown that the point mutation Phe130Ser near the amino acid binding site of Escherichia coli tyrosyl-tRNA synthetase allows more efficient incorporation of diazotyrosine than tyrosine. See, F. Hamano-Takaku, T. Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll and S. Nishimura, J. Biol. Chem. , 275:40324 (2000 ).

Another strategy for incorporating unnatural amino acids into proteins in vivo is to modify synthetases with proofreading mechanisms. These synthetases cannot discriminate between amino acids that are structurally similar to the cognate natural amino acids and therefore cannot activate said amino acids. This error is corrected at a separate site that deacylates the misloaded amino acid from the tRNA to maintain the fidelity of protein translation. If the proofreading activity of the synthetase is lost, misactivated structural analogs can escape the editing function and be incorporated. This approach has recently been demonstrated with valyl-tRNA synthetase (ValRS). See, V. Doring, HD Mootz, L. A. Nangle, TL Hendrickson, V. de Crecy-Lagard, P. Schimmel, and P. Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAVal with Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids are then hydrolyzed by the editing domain. After random mutations in the E. coli chromosome, a mutant E. coli strain with a mutation at the editing site of ValRS was selected. This editing-deficient ValRS improperly loads tRNAVal with Cys. Because Abu is sterically similar to Cys (the -SH group of Cys is replaced by _-CH3 in Abu), when this mutant E. coli strain is grown in the presence of Abu, the mutant ValRS also incorporates Abu protein. Mass spectrometry analysis showed that in the natural protein, about 24% of valines were replaced by Abu at each valine position.

Solid-phase synthetic and semi-synthetic methods also allow for the synthesis of large quantities of proteins containing novel amino acids. See, for example, the following publication and references cited therein, as follows: Crick, FHC, Barrett, L. Brenner, S. Watts-Tobin, R. General nature of the genetic code for proteins. Nature , 192:1227 -1232(1961); Hofmann, K., Bohn, H.Studies on polypeptides.XXXVI.The effect ofpyrazole-imidazole replacements on the S-protein activating potency of an S-peptide fragment, J.Am Chem , 88(24): 5914-5919(1966); Kaiser, ETSynthetic approaches tobiologically active peptides and proteins including enyzmes, Acc Chem Res , 22:47-54(1989); Nakatsuka, T., Sasaki, T., Kaiser, ETPeptide segment coupling catalystyzed by theseemis Enzyme thiosubtilisin, J Am Chem Soc , 109:3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease, Science , 256(5054): 221- 225 (1992); Chaiken, IMSemisynthetic peptides and proteins, CRC Crit Rev Biochem , 11(3):255-301 (1981); Offord, REProtein engineering by chemical means? Protein Eng. , 1(3):151-157 (1987); and Jackson, DY, Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, JAA Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues, Science , 266(5183):243(1994).

Chemical modification has been used to introduce various unnatural side chains including cofactors, spin labels and oligonucleotides into proteins in vitro. See, e.g., Corey, DR, Schultz, PG Generation of a hybrid sequence-specific single-stranded deoxyribonuclease, Science , 238(4832):1401-1403 (1987); Kaiser, ET, Lawrence DS, Rokita, SE The chemical modification of enzymatic specificity , Annu Rev Biochem , 54: 565-595 (1985); Kaiser, ET, Lawrence, DS Chemical mutation of enyzme active sites, Science , 226 (4674): 505-511 (1984); Neet, KE, Nanci A, Koshland, DE Properties of thiol-subtilisin, J Biol.Chem , 243(24):6392-6401(1968); Polgar, L. et MLBender.A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J.Am Chem Soc , 88 : 3153-3154 (1966); and Pollack, SJ, Nakayama, G. Schultz, PG Introduction of nucleophiles and spectroscopic probes into antibody combining sites, Science , 242(4881): 1038-1040 (1988).

Alternatively, biosynthetic methods employing chemically modified aminoacyl-tRNAs have been used to incorporate some biophysical probes into synthesized proteins in vitro. See the following publications and references cited therein: Brunner, J. New Photolabeling and crosslinking methods, Annu. Rev Biochem , 62:483-514 (1993); and Krieg, UC, Walter, P., Hohnson, AE Photocrosslinking of The signal sequence of nascent preprolactin of the 54-kilodalton polypeptide of the signal recognition particle, Proc. Natl. Acad. Sci , 83(22): 8604-8608 (1986).

It has previously been shown that unnatural amino acids can be site-specifically incorporated into proteins in vitro by adding chemically aminoacylated suppressor tRNAs to protein synthesis reactions programmed with genes containing desired amber nonsense mutations middle. Using these methods, one skilled in the art can substitute a number of the common 20 amino acids with closely related structural homologues (eg, fluorophenylalanine for phenylalanine) using specific amino acid auxotrophic strains. See, e.g., Noren, CJ, Anthony-Cahill, Griffith, MC, Schultz, PG General method for site-specific incorporation of unnatural amino acids into proteins, Science , 244:182-188 (1989): MW Nowak et al., Science 268: 439-42 (1995); Bain, JD, Glabe, CG, Dix, TA, Chamberlin, AR, Diala, ES Biosynthetic site-specific Incorporation of a non-natural aminoacid into a polypeptide, J. Am Chem Soc , 111:8013- 8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999); Ellman, JA, Mendel, D., Anthony-Cahill, S., Noren, CJ, Schultz, PGBiosynthetic method for introducing unnatural amino acids site-specifically into proteins. Methods in Enz. , Vol. 202, 301-336 (1992); and Mendel, D., Cornish, VW & Schultz, PGSite-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys. Biomol Struct . 24, 435-62 (1995).

For example, a suppressor tRNA that recognizes the stop codon UAG is prepared and chemically aminoacylated with an unnatural amino acid. Conventional site-directed mutagenesis can be used to introduce the stop codon TAG at the site of interest in the protein gene. See, eg, Sayers, JR, Schmidt, W. Eckstein, F. 5'-3' Exonucleases in phosphorothioate-based olignoucleotide-directed mutagensis, Nucleic Acids Res , 16(3):791-802 (1988). When the acylated suppressor tRNA and the mutant gene are combined in an in vitro transcription/translation system, the unnatural amino acid is incorporated in response to the UAG codon, resulting in a protein containing that amino acid at the indicated position. Experiments with [ ³ H]-Phe and experiments with α-hydroxy acids demonstrated that the desired amino acid was incorporated only at the position specified by the UAG codon, and that this amino acid was not incorporated anywhere else in the protein. See, eg, Noren et al., supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432; and Ellman, JA, Mendel, D., Schultz, PG Site-specific incorporation of novel backbone structures into proteins, Science , 255(5041): 197-200 (1992).

A tRNA can be aminoacylated with a desired amino acid by any method or technique including, but not limited to, chemical aminoacylation or enzymatic aminoacylation.

Aminoacylation can be accomplished by aminoacyl tRNA synthetases or by other enzymatic molecules including, but not limited to, ribozymes. The term "ribozyme" is interchangeable with "catalytic RNA". Cech and coworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987, Concepts Biochem. 64:221-226) demonstrated the presence of naturally occurring RNAs (ribozymes) that can act as catalysts. However, although these natural RNA catalysts have only been shown to act on ribonucleic acid substrates for cleavage and splicing, the recent development of artificial evolution of ribozymes has extended the repertoire of catalysis to various chemical reactions. Studies have identified RNA molecules that catalyze aminoacyl-RNA bonds at their own (2') 3'-ends (Illangakekare et al., 1995 Science 267:643-647) and that can transfer amino acids from one RNA molecule to another. An RNA molecule of an RNA molecule (Lohse et al., 1996, Nature 381:442-444).

US Patent Application Publication 2003/0228593, incorporated herein by reference, describes methods of constructing ribozymes and their use in aminoacylation of tRNAs with naturally encoded and non-naturally encoded amino acids. Substrate-immobilized forms of enzymatic molecules that can aminoacylate tRNAs, including but not limited to ribozymes, can enable efficient affinity purification of aminoacylated products. Examples of suitable substrates include agarose, sepharose and magnetic beads. The production and use of substrate-immobilized forms of ribozymes for aminoacylation are described in Chemistry and Biology 2003, 10: 1077-1084 and in U.S. Patent Application Publication 2003/0228593, which references are incorporated by reference incorporated into this article.

Chemical aminoacylation methods include, but are not limited to, those described by Hecht and colleagues (Hecht, S.M. Acc. Chem. Res. 1992, 25, 545; Heckler, T.G.; Roesser, J.R.; Xu, C; Chang, P.; Hecht , S.M.Biochemistry 1988, 27, 7254; Hecht, S.M.; Alford, B.L.; Kuroda, Y.; Kitano, S.J.Biol.Chem. 1978, 253, 4517) and by Schultz, Chamberlin, Dougherty and others (Cornish, V.W.; Mendel , D.; Schultz, P.G. Angew.Chem.Int.Ed.Engl.1995, 34, 621; Robertson, S.A.; Ellman, J.A.; Schultz, P.G.J.Am.Chem.Soc.1991, 113, 2722; Anthony-Cahill, S.J.; Griffith, M.C.; Schultz, P.G. Science 1989, 244, 182; Bain, J.D.; Glabe, C.G.; Dix, T.A.; et al., Nature 1992, 356, 537; Gallivan, J.P.; Lester, H.A.; Dougherty, D.A. Chem. Biol. 1997, 4, 740; Turcatti et al., J. Biol. Chem. 1996, 271, 19991; Nowak, M.W. et al., Science, 1995, 268, 439; Saks, M.E. et al., J.Biol.Chem.1996, 271, 23169; Hohsaka, T. et al., J.Am.Chem.Soc.1999, 121, 34) Those proposed to avoid the use of synthetases in aminoacylation, said references are incorporated herein by reference. This method or other chemical aminoacylation methods can be used to aminoacylate tRNA molecules.

The method for generating catalytic RNA may involve generating an independent collection of randomized ribozyme sequences, performing directed evolution against the collection, screening the collection for the desired aminoacylation activity and selecting those that exhibit the desired aminoacylation activity The sequence of the ribozyme.

Ribozymes may comprise motifs and/or regions that facilitate acylation activity, such as GGU motifs and U-rich regions. For example, it has been reported that U-rich regions can facilitate recognition of amino acid substrates, and that GGU motifs can form base pairs with the 3' end of tRNAs. When combined, the GGU motif and the U-rich region simultaneously facilitate simultaneous recognition of amino acids and tRNAs, and in turn facilitate aminoacylation of the 3' end of the tRNA.

Ribozymes can be generated by in vitro selection using partially randomized r24mini conjugated to tRNA ^Asn _CCCG , followed by systematic engineering of consensus sequences present in active clones. An exemplary ribozyme obtained by this method is called "Fx3 ribozyme" and is described in U.S. Published Application No. 2003/0228593, the contents of which are incorporated herein by reference, the Ribozymes serve as versatile catalysts for the synthesis of various aminoacyl-tRNAs loaded with cognate unnatural amino acids.

Immobilization on a substrate can be used to enable efficient affinity purification of aminoacylated tRNAs. Examples of suitable substrates include, but are not limited to, agarose, sepharose, and magnetic beads. Ribozyme can be immobilized on the resin by utilizing the chemical structure of RNA, such as the 3'-cis-diol on the ribose of RNA can be oxidized by periodate to produce the corresponding dialdehyde to facilitate the immobilization of RNA on the resin . Various types of resins can be used, including inexpensive hydrazide resins, where reductive amination makes the interaction between the resin and the ribozyme an irreversible bond. The synthesis of aminoacyl-tRNA can be greatly facilitated by this on-column aminoacylation technique. Kourouklis et al. (Methods 2005;36:239-4) describe a column-based aminoacylation system.

Isolation of aminoacylated tRNAs can be accomplished in various ways. A suitable method is to use a buffer (such as a solution of sodium acetate with 10 mM EDTA containing 50 mM N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid), 12.5 mM KCl (pH 7.0), 10 mM EDTA buffer or only EDTA buffered water (pH 7.0)) to elute the aminoacylated tRNA from the column.

Aminoacylated tRNAs can be added to a translation reaction to incorporate an amino acid with which the tRNA is aminoacylated at a selected position in the polypeptide produced by the translation reaction. Examples of translation systems that can use the aminoacylated tRNAs of the invention include, but are not limited to, cell lysates. Cell lysates provide the reaction components necessary for in vitro translation of polypeptides from input mRNA. Examples of such reaction components include, but are not limited to, ribosomal proteins, rRNA, amino acids, tRNA, GTP, ATP, translation initiation and elongation factors, and other factors associated with translation. In addition, the translation system may be batch translation or compartmentalized translation. Batch translation systems combine reaction components in a single compartment, while partitioned translation systems separate translation reaction components from reaction products that can inhibit translation efficiency. Such translation systems are commercially available.

In addition, coupled transcription/translation systems can be used. Coupled transcription/translation systems allow the transcription of input DNA into corresponding mRNA, which is in turn translated by the reaction components. One example of a commercially available coupled transcription/translation system is the Rapid Translation System (RTS, Roche Inc.). The system includes a mixture of E. coli lysates containing translational components such as ribosomes and translation factors. Additionally, RNA polymerase is included for transcription of input DNA into mRNA templates for translation. RTS can employ partitioned translation by separating reaction groups through membranes placed between reaction compartments, including supply/consumption compartments and transcription/translation compartments.

Aminoacylation of tRNA can be performed by other reagents including, but not limited to, transferases, polymerases, catalytic antibodies, multifunctional proteins, and the like.

Lu et al. in Mol Cell. 2001 Oct;8(4):759-69 describe a method for chemically linking proteins to synthetic peptides containing unnatural amino acids (expressed protein linking).

Unnatural amino acids have also been incorporated into proteins using microinjection techniques. See, e.g., MW Nowak, PC Kearney, JRSampson, MESaks, CG Labarca, SK Silverman, WG Zhong, J. Thorson, JNAbelson, N. Davidson, PG Schultz, DADougherty and HALester, Science , 268:439 (1995); and DADougherty, Curr . Opin. Chem. Biol., 4:645 (2000). Xenopus oocytes were co-injected with two RNA species produced in vitro: the mRNA encoding the target protein with a UAG stop codon at the amino acid position of interest, and the amino acid via the desired unnatural amino acid. Acylated succinates suppress tRNA. Next, the oocyte's translation machinery inserts the unnatural amino acid at the position specified by the UAG. This approach has enabled in vivo structure-function studies of integral membrane proteins that are not generally amenable to in vitro expression systems. Examples include the incorporation of fluorescent amino acids into the tachykinin neurokinin-2 receptor to measure distance by fluorescence resonance energy transfer, see, e.g., G. Turcatti, K. Nemeth, MD Edgerton, U. Meseth, F. Talabot, M. .Peitsch, J.Knowles, H.Vogel and A.Chollet, J.Biol.Chem ., 271:19991 (1996); Incorporation of biotinylated amino acids to identify surface-exposed residues in ion channels, see, e.g., JPGallivan, HALester, and DADougherty, Chem. Biol ., 4:739 (1997); Use of caged tyrosine analogs to monitor conformational changes in ion channels in real time, see, e.g., JCMiller, SK Silverman, PMEngland, DADougherty, and HALester, Neuron, 20:619 (1998); and the use of alpha hydroxyamino acids to alter the framework of ion channels for probing their gating mechanisms. See, e.g., PM England, Y. Zhang, DADougherty and HALester, Cell , 96:89 (1999); and T. Lu, AY Ting, J. Mainland, LY Jan, PG Schultz and J. Yang, Nat. Neurosci ., 4 : 239 (2001).

The ability to incorporate unnatural amino acids directly into proteins in vivo offers a variety of advantages including, but not limited to, high yields of mutant proteins, technical ease, studying mutations in cells or possibly in living organisms protein, and the use of these mutant proteins in therapeutic treatment and diagnostic applications. The ability to include unnatural amino acids of various sizes, acidity, nucleophilicity, hydrophobicity, and other properties into proteins can greatly expand our ability to rationally and systematically manipulate the structure of proteins to probe protein function and generate A new protein or organism with novel properties.

In an attempt to site-specifically incorporate p-fluorophenylalanine, yeast amber inhibits the tRNAPheCUA/phenylalanyl-tRNA synthetase pair for p-fluorophenylalanine resistance, phenylalanine auxotrophy type Escherichia coli strains. See, eg, R. Furter, Protein Sci., 7:419 (1998).

It is also possible to obtain expression of the GH (eg, hGH) polynucleotides of the invention using cell-free (in vitro) translation systems. The translation system can be a cellular system or a cell-free system, and can be a prokaryotic system or a eukaryotic system. Cellular translation systems include, but are not limited to, whole cell preparations such as permeabilized cells or cell cultures, in which a desired nucleic acid sequence can be transcribed into mRNA and translated. Cell-free translation systems are commercially available and many different types and systems are well known. Examples of cell-free systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysate, and prokaryotic lysates such as wheat germ extract, insect cell lysate, rabbit reticulocyte lysate, rabbit egg Eukaryotic lysates of mother cell lysates and human cell lysates. Eukaryotic extracts or lysates may be preferred when the resulting protein is glycosylated, phosphorylated, or otherwise modified, since many of these modifications are only possible in eukaryotic systems. Some of these extracts and lysates are commercially available (Promega; Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; Grand Island, N.Y.). Membrane extracts such as canine pancreas extracts containing microsomal membranes are also available, which can be used to translate secreted proteins. In these systems, which can include mRNA as a template (in vitro translation) or DNA as a template (combined in vitro transcription and translation), in vitro synthesis is directed by ribosomes. Considerable effort has been devoted to developing cell-free protein expression systems. See, e.g., Kim, D.M. and J.R. Swartz, Biotechnology and Bioengineering, 74:309-316 (2001); Kim, D.M. and J.R. Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D.M. and J.R. Swartz , Biotechnology Progress, 16, 385-390, (2000); Kim, D.M. and J.R.Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999); and Patnaik, R. and J.R.Swartz, Biotechniques 24, 862-868, ( 1998); U.S. Patent No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated herein by reference. Another method that can be applied to express a GH (eg, hGH) polypeptide comprising a non-naturally encoded amino acid includes mRNA-peptide fusion technology. See, eg, R. Roberts and J. Szostak, Proc. Natl Acad. Sci. (USA) 94: 12297-12302 (1997); A. Frankel et al., Chemistry & Biology 10: 1043-1050 (2003). In this method, an mRNA template linked to puromycin is translated into a peptide on the ribosome. Unnatural amino acids can also be incorporated into the peptide if one or more tRNA molecules have been modified. After the last mRNA codon has been read, puromycin captures the C-terminus of the peptide. If the resulting mRNA-peptide conjugates are found to have interesting properties in in vitro assays, their properties can be readily revealed from the mRNA sequence. Accordingly, one of skill in the art can screen libraries of GH (eg, hGH) polypeptides comprising one or more non-naturally encoded amino acids to identify polypeptides with desired properties. It has recently been reported that in vitro ribosomal translation using purified components allows the synthesis of peptides substituted with non-naturally encoded amino acids. See, eg, A. Forster et al., Proc. Natl Acad. Sci. (USA) 100:6353 (2003).

It is also possible to use the refactoring translation system. Mixtures of purified translation factors and purified translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 (alpha or beta), elongation factor T (EF-Tu), or termination factors Factor supplemented lysates or combinations of lysates have also been successfully used to translate mRNA into protein. The cell-free system can also be a coupled transcription/translation system, wherein DNA is introduced into the system, transcribed into mRNA, and translated as described in Current Protocols in Molecular Biology (eds. F.M. Ausubel et al., Wiley Interscience, 1993), which references It is hereby expressly incorporated by reference. RNA transcribed in eukaryotic transcription systems can be in the form of heterokaryotic RNA (hnRNA) or mature mRNA with a 5′-cap (7-methylguanosine) and a 3′-terminal poly A tail. This can be an advantage in some translation systems. For example, capped mRNAs are translated with high efficiency in the reticulocyte lysate system.

IX. Macromolecular polymers coupled to GH (eg, hGH) polypeptides

The various modifications to the non-natural amino acid polypeptides described herein can be achieved using the compositions, methods, techniques and strategies described herein. These modifications include the incorporation of another functional group on the non-natural amino acid component of the polypeptide including, but not limited to: labels; dyes; polymers; water-soluble polymers; derivatives of polyethylene glycol; coupling agent; radionuclide; cytotoxic compound; drug; affinity label; photoaffinity label; reactive compound; resin; second protein or polypeptide or polypeptide analog; antibody or antibody fragment; metal chelator; cofactor; Fatty acids; carbohydrates; polynucleotides; DNA; RNA; antisense polynucleotides; sugars; water-soluble dendrimers; cyclodextrins; inhibitory ribonucleic acids; biomaterials; nanoparticles; spin label; fluorophore; metal-containing moiety; radioactive moiety; novel functional group; group that interacts covalently or non-covalently with other molecules; photocage moiety); actinic radiation excitable moiety; photoisomerization moieties; biotin; derivatives of biotin; biotin analogs; moieties incorporating heavy atoms; chemically cleavable groups; photocleavable groups; elongated side chains; carbon-linked sugars; Redox-active agents; aminothioacids; toxic moieties; isotope-labeled moieties; biophysical probes; phosphorescent groups; chemiluminescent groups; electron-dense groups; magnetic groups; intercalating groups; emitting groups Chromophores; energy transfer agents; bioactive agents; detectable labels; small molecules; quantum dots; nanoconductors; radionucleotides; radioconductors; neutron capture agents; or any combination of the foregoing, or any other desired compound or substance. As an illustrative, non-limiting example of the compositions, methods, techniques and strategies described herein, the following description will focus on the addition of macromolecular polymers to non-natural amino acid polypeptides, provided that the compositions described therefor , methods, techniques and strategies are also applicable (where necessary, with appropriate modifications and for which those skilled in the art can use the disclosure herein to carry out) to add other functional substances, which include (but not limited to) Functional substances listed above.

A variety of macromolecular polymers and other molecules can be attached to the GH (e.g., hGH) polypeptides of the invention to modulate the biological properties of the GH (e.g., hGH) polypeptides, and/or to provide novel GH (e.g., hGH) molecules. biological properties. These macromolecular polymers can be combined with GH (e.g., hGH) polypeptide. The molecular weight of the polymers can be within a wide range, including, but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da , 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3, Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer can be between about 100 Da and 50,000 Da. In some embodiments, the molecular weight of the polymer may be between about 100 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer may be between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer may be between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of the polymer may be between about 10,000 Da and 40,000 Da.

The present invention provides substantially homogeneous formulations of polymer:protein conjugates. "Substantially homogeneous" as used herein means that the polymer:protein conjugate molecules are observed to be greater than half of the total protein. Polymer:protein conjugates are biologically active and "substantially homogeneous" PEGylated GH (e.g., hGH) polypeptide formulations of the invention provided herein are those that are sufficiently homogeneous to exhibit the advantages of homogeneous formulations , for example in clinical applications where batch-to-batch pharmacokinetic properties are readily predictable.

Those skilled in the art may also choose to prepare mixtures of polymer:protein conjugate molecules, and the advantage provided herein is that those skilled in the art may choose the ratio of single polymer:protein conjugates to include in the mixture. Thus, if necessary, one skilled in the art can prepare mixtures of various proteins with various numbers of polymer moieties attached (i.e., di-, tri-, tetra-, etc.) The compound is combined with the single polymer:protein conjugate prepared using the method of the present invention, and a mixture of single polymer:protein conjugate with a predetermined ratio is obtained.

The selected polymer may be water soluble so that the protein attached thereto does not precipitate in an aqueous environment, such as a physiological environment. This polymer can be branched or unbranched. This polymer will be pharmaceutically acceptable for therapeutic use in the final product formulation.

Examples of polymers include, but are not limited to, polyalkyl ethers and their alkoxy-terminated analogs (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol and their methoxy- or ethoxy-terminated analogs). substances, especially polyoxyethylene glycol, the latter also known as polyethylene glycol or PEG); polyvinylpyrrolidone; polyvinylalkyl ethers; polyoxazolines; polyalkyloxazolines and polyhydroxyalkanes oxazolines; polyacrylamides, polyalkylacrylamides and polyhydroxyalkylacrylamides (such as polyhydroxypropylmethacrylamide and its derivatives); polyhydroxyalkylacrylates; polysialic acids and the like substances; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, such as carboxymethyl dextran, dextran sulfate, aminodextran; cellulose and its derivatives , such as carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, such as chitosan, succinylated chitin, carboxymethyl chitin, carboxymethyl deacetylated Chitin; hyaluronic acid and its derivatives; starch; alginate; chondroitin sulfate; albumin; Lysine, polyaspartic acid, polyasparagine; maleic anhydride copolymers, such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; poly Vinyl alcohol; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives of the foregoing polymers.

The ratio of polyethylene glycol molecules to protein molecules will vary, as will its concentration in the reaction mixture. In general, the optimal ratio (in terms of the efficiency of the reaction in the presence of a minimal excess of unreacted protein or polymer) can be determined by the molecular weight of the polyethylene glycol selected and the available number of reactive groups available. When it comes to molecular weight, generally the higher the molecular weight of the polymer, the lower the number of polymer molecules that can be attached to the protein. Likewise, polymer branching should be considered when optimizing these parameters. In general, the higher the molecular weight (or the more branches), the higher the polymer:protein ratio.

As used herein, and when encompassing PEG:GH (eg, hGH) polypeptide conjugates, the term "therapeutically effective amount" refers to an amount that provides the desired benefit to a patient. This amount will vary from individual to individual and will depend on several factors including the general physical condition of the patient and the underlying cause of the condition being treated. The amount of GH (eg, hGH) polypeptide used for treatment provides an acceptable rate of change and maintains the desired response at a beneficial level. A therapeutically effective amount of a composition of the invention can be readily determined by one of ordinary skill in the art using publicly available materials and procedures.

The water soluble polymer can be in any structural form including, but not limited to, linear, forked or branched. Typically, the water soluble polymer is a poly(hydrocarbylene glycol), such as poly(ethylene glycol) (PEG), although other water soluble polymers may also be used. For example, PEG is used to describe certain embodiments of the invention.

PEG is a well-known water-soluble polymer that is commercially available or can be obtained according to methods known to those skilled in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pp. 138-161 ), prepared by ring-opening polymerization of ethylene glycol. The term "PEG" is used broadly to encompass any polyethylene glycol molecule, regardless of PEG size or terminal modification, and can be represented as linked to a GH (e.g., hGH) polypeptide by the following formula:

XO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y.

wherein n is 2 to 10,000 and X is H or a terminal modification including, but not limited to, C _1-4 alkyl, a protecting group, or a terminal functional group.

In some cases, PEGs used in the invention are terminated at one end with a hydroxyl or methoxy group, ie, X is H or _CH3 ("methoxy PEG"). Alternatively, PEG can be terminated with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups may include those commonly used to react with functional groups present in the 20 common amino acids (including but not limited to maleimide groups, activated carbonates including ( but not limited to) p-nitrophenyl esters), activated esters (including but not limited to N-hydroxysuccinimide, p-nitrophenyl esters) and aldehydes) and inert to 20 common amino acids Functional groups (including but not limited to, azido, alkynyl) that specifically react with complementary functional groups present in non-naturally encoded amino acids. It should be noted that the other end of the PEG represented by Y in the formula above will be directly or indirectly linked to a GH (eg, hGH) polypeptide via a naturally occurring amino acid or a non-naturally encoded amino acid. For example, Y can be an amide bond, a carbamate bond, or a urea bond attached to an amine group of the polypeptide, including but not limited to the epsilon amine or N-terminus of lysine. Alternatively, Y may be a maleimide bond attached to a thiol group, including but not limited to that of cysteine. Alternatively, Y may be a bond to a residue not normally accessible via the 20 common amino acids. For example, an azido group on a PEG can be reacted with an alkyne group on a GH (eg, hGH) polypeptide to form a Hoosier root [3+2] cycloaddition product. Alternatively, an alkynyl group on PEG can be reacted with an azido group present in a non-naturally encoded amino acid to form a similar product. In some embodiments, a strong nucleophile (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in a non-naturally encoded amino acid, as appropriate, to form hydrazones, oximes or semicarbazones, which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, strong nucleophiles can be incorporated into GH (eg, hGH) polypeptides via non-naturally encoded amino acids and used to preferentially react with ketone or aldehyde groups present in water-soluble polymers.

Any molecular mass of PEG can be used as practical, including, but not limited to, about 100 Daltons (Da) to 100,000 Da or more (sometimes including, but not limited to, 0.1 to 50 kDa or 10 to 40 kDa) as desired . The molecular weight of PEG can be within a wide range including, but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of PEG can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da , 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2, Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of PEG is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight of PEG is between about 100 Da and 40,000 Da. In some embodiments, the molecular weight of PEG is between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight of PEG is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of PEG is between about 10,000 Da and 40,000 Da. Branched chain PEGs may also be used, including but not limited to PEG molecules in which each chain has a molecular weight in the range of 1 to 100 kDa, including but not limited to 1 to 50 kDa or 5 to 20 kDa. The molecular weight of each chain of a branched PEG can be, including but not limited to, between about 1,000 Da and about 100,000 Da or more. The molecular weight of each chain of the branched PEG can be between about 1,000 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da , 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4, Da, 3,000Da, 2,000Da, and 1,000Da. In some embodiments, each chain of the branched PEG has a molecular weight of between about 1,000 Da and 50,000 Da. In some embodiments, each chain of the branched PEG has a molecular weight of between about 1,000 Da and 40,000 Da. In some embodiments, each chain of the branched PEG has a molecular weight of between about 5,000 Da and 40,000 Da. In some embodiments, each chain of the branched PEG has a molecular weight of between about 5,000 Da and 20,000 Da. A broad range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, Nektar Therapeutics catalog, which is incorporated herein by reference.

Generally, at least one end of the PEG molecule is available for reaction with a non-naturally encoded amino acid. For example, PEG derivatives having alkynyl and azido moieties suitable for reaction with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid contains an azide moiety, the PEG will generally contain an alkynyl moiety to form a [3+2] cycloaddition product, or be an activated PEG species (i.e., ester, carbonate) containing a sulfo group to form amide bond. Alternatively, if the non-naturally encoded amino acid contains an alkyne moiety, the PEG will generally contain an azide moiety to form a [3+2] Hoog radical cycloaddition product. If the non-naturally encoded amino acid contains a carbonyl group, the PEG will generally contain effective nucleophiles (including but not limited to, hydrazide, hydrazine, hydroxylamine, semicarbazide functional groups) to form the corresponding hydrazone linkages, oxime linkages, and semicarbazones, respectively key. In other alternative embodiments, reverse-directing groups of the reactive groups described above may be used, ie, reacting the azido moiety of the non-naturally encoded amino acid with an alkynyl-containing PEG derivative.

In some embodiments, a GH (eg, hGH) polypeptide variant having a PEG derivative contains a chemical functional group that is reactive with a chemical functional group present on the side chain of the non-naturally encoded amino acid.

The present invention provides, in some embodiments, azide-containing polymer derivatives and ethynyl-containing polymer derivatives comprising a water-soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer may be poly(ethylene glycol). However, it should be understood that a variety of water-soluble polymers, including but not limited to polyethylene glycol and other related polymers, including poly(dextran) and poly(propylene glycol), are also suitable for use in the practice of the present invention, and the term The use of PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, any form of poly(ethylene glycol), including bifunctional PEG, multiarmed PEG, derivatized PEG, bifurcated PEG, branched PEG, pendent PEG (i.e., having one or more side PEG with functional groups attached to the polymer backbone or related polymers) or PEG with degradable linkages therein.

PEG is generally clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is generally nontoxic. Poly(ethylene glycol) is considered biocompatible, which means that PEG can coexist with living tissue or organisms without causing damage. More specifically, PEG is substantially non-immunogenic, that is, PEG does not tend to generate an immune response in vivo. When attached to a molecule with some desired function in vivo, such as a biologically active agent, PEG tends to mask the agent and can reduce or eliminate any immune response so that the organism can tolerate the agent. PEG conjugates do not tend to produce a substantial immune response or cause clotting or other adverse effects. PEGs having the formula _-CH2CH2O- ( _CH2CH2O ) _n -- _{CH2CH2- ,} where n is from about 3 to about 4000, _typically from about 20 to about 2000, _are suitable for use in the present invention. PEG having a molecular weight of from about 800 Da to about 100,000 Da is particularly useful as the polymer backbone in some embodiments of the invention. The molecular weight of PEG can be within a wide range including, but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of PEG can be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da of Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of PEG is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight of PEG is between about 100 Da and 40,000 Da. In some embodiments, the molecular weight of PEG is between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight of PEG is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of PEG is between about 10,000 Da and 40,000 Da.

The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branched core portion and a plurality of linear polymer chains attached to the central branched core. PEG is generally used in branched form, which can be prepared by the addition of ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids such as lysine. Branched poly(ethylene glycol) can be represented by the general formula R(-PEG-OH) _m , where R is derived from a core moiety such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. No. 5,932,462; No. 5,643,575; No. 5,229,490; No. 4,289,872; U.S. Patent Application 2003/0143596; WO 96/21469; Multi-armed PEG molecules such as those described in middle) can also be used as polymer backbones.

Branched PEGs can also be in the form of forked PEGs represented by PEG(-- _YCHZ2 ) _n , where Y is the linking group and Z is an activated end group attached to CH through a chain of atoms of specified length.

Another branched form, pendant PEG, has reactive groups such as carboxyl groups along the PEG backbone rather than at the ends of the PEG chains.

In addition to these PEG forms, polymers can also be prepared with weak or degradable linkages in the backbone. For example, PEG can be prepared with readily hydrolyzable ester linkages in the polymer backbone. This hydrolysis results in the cleavage of the polymer into fragments of lower molecular weight as follows:

-PEG-CO ₂ -PEG-+H ₂ O→PEG-CO ₂ H+HO-PEG-

Those skilled in the art understand that the term poly(ethylene glycol) or PEG refers to or includes all forms known in the art, including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention. In some embodiments, water-soluble polymer backbones having 2 to about 300 termini are particularly suitable for use in the present invention. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols) such as poly(propylene glycol) (“PPG”), copolymers thereof (including but not limited to, copolymers of ethylene glycol and propylene glycol) , its trimer, its mixture, etc. While the molecular weight of the individual chains in the polymer backbone can vary, it typically ranges from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da. The molecular weight of each chain in the polymer backbone may be between about 100 Da and about 100,000 Da, including but not limited to 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of each chain in the polymer backbone is between about 100 Da and 50,000 Da. In some embodiments, the molecular weight of each chain in the polymer backbone is between about 100 Da and 40,000 Da. In some embodiments, the molecular weight of each chain in the polymer backbone is between about 1,000 Da and 40,000 Da. In some embodiments, the molecular weight of each chain in the polymer backbone is between about 5,000 Da and 40,000 Da. In some embodiments, the molecular weight of each chain in the polymer backbone is between about 10,000 Da and 40,000 Da.

Those skilled in the art will recognize that the foregoing list of substantially water-soluble backbones is by no means exhaustive, but illustrative only, and that all polymeric materials having the characteristics described above are considered suitable for use in the present invention.

In some embodiments of the invention, the polymer derivatives are "multifunctional," meaning that the polymer backbone has at least two, and possibly as many as about 300, functionalized or activated ends with functional groups. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, where each terminus is bonded to a functional group which may be the same or different.

In some embodiments, the polymer derivative has the following structure:

X-A-POLY-B-N=N=N,

in:

N=N=N is an azido moiety;

B is a linking moiety, which may or may not be present;

POLY is a water-soluble non-antigenic polymer;

A is a linking moiety, which may or may not be present, and which may be the same as or different from B; and

X is a second functional group.

Examples of linking moieties A and B include, but are not limited to, multiply functionalized alkyl groups containing up to 18 and possibly 1 to 10 carbon atoms. Heteroatoms such as nitrogen, oxygen or sulfur may be included in the alkyl chain. Alkyl chains may also be branched at heteroatoms. Other examples of linking moieties A and B include, but are not limited to, multifunctional aryl groups containing up to 10 and possibly 5 to 6 carbon atoms. This aryl group may be substituted by a further carbon atom, nitrogen atom, oxygen atom or sulfur atom. Other examples of suitable linking groups include those described in US Patent Nos. 5,932,462; 5,643,575; and US Patent Application Publication 2003/0143596, each of which is incorporated herein by reference. Those skilled in the art will recognize that the foregoing list of linking moieties is by no means exhaustive, but illustrative only, and that all polymeric materials having the properties described above are considered suitable for use in the present invention.

Examples of functional groups suitable for use as X include, but are not limited to, hydroxyl, protected hydroxyl, alkoxy, active esters (such as N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester), Activated carbonates (such as N-hydroxysuccinimidyl carbonate and 1-benzotriazolyl carbonate), acetals, aldehydes, hydrated aldehydes, alkenyls, acrylates, methacrylates, acrylamides , active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide , Vinylsulfone, Dithiopyridine, Vinylpyridine, Iodoacetamide, Epoxide, Glyoxal, Diketone, Mesylate, Tosylate, Trifluoroethanesulfonate, Alkene, Ketone and Azide compound functional groups. As will be appreciated by those skilled in the art, the moiety X is chosen to be compatible with the azido group so as not to react with the azido group. Azido-containing polymer derivatives are homobifunctional, which means that the second functional group (i.e., X) is also an azido moiety; or heterobifunctional, which means that the second functional group is a different functional group.

The term "protected" refers to the presence of a protecting group or moiety that prevents a chemically reactive functional group from reacting under certain reaction conditions. Protecting groups will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from the group of t-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be an ortho-pyridyl disulfide. If the chemically reactive group is a carboxylic acid (such as butyric acid or propionic acid) or a hydroxyl group, the protecting group may be benzyl or an alkyl group (such as methyl, ethyl or tert-butyl). Other protecting groups known in the art may also be used in the present invention.

Specific examples of terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see, e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amines (see, e.g., Buckmann et al. Makromol. Chem. 182: 1379 (1981), Zalipsky et al. Eur.Polym.J.19: 1177 (1983)), hydrazide (see for example Andresz et al. Makromol. Chem. 179: 301 (1978)), succinimidylpropionic acid Esters and succinimidyl butyrate (see e.g. Olson et al. in Poly(ethylene glycol) Chemistry & Biological Applications, pp. 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C., 1997; see also U.S. Patent No. 5,672,662), succinimidyl succinate (see, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) and Joppich et al. Makromol. Chem. 180:1381 (1979)), Succinimide esters (see, e.g., U.S. Patent No. 4,670,417), benzotriazole carbonates (see, e.g., U.S. Patent No. 5,650,234), glycidyl ethers (see, e.g., Pitha et al. Eur. J Biochem. 94:11 ( 1979), Elling et al, Biotech.Appl.Biochem.13:354 (1991)), oxycarbonylimidazole (see for example Beauchamp et al., Anal.Biochem.131:25 (1983), Tondelli et al. J.Controlled Release 1 : 251 (1985)), p-nitrophenyl carbonate (see, for example, Veronese et al., Appl. Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem. Biotech., 27: 45 (1991)), aldehydes (see for example Harris et al. J.Polym.Sci.Chem.Ed.22:341 (1984), U.S. Patent No. 5,824,784, U.S. Patent No. 5,252,714), maleimide ( See for example Goodson et al. Biotechnology (NY) 8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984) and Kogan, Synthetic Comm. 22:2417 (1992)), o-pyridyl disulfide Chem. 4:314 (1993)), propenyl alcohol (see, eg, Sawhney et al. Macromolecules, 26:581 (1993)), vinyl sulfone (see, eg, US Pat. No. 5,900,461). All of the above references and patents are incorporated herein by reference.

In a particular embodiment of the invention, the polymer derivatives of the invention comprise a polymer backbone having the following structure:

X- _CH2CH2O --( _CH2CH2O ) _{n -- CH2CH2 -} N ₌ N=N,

in:

X is a functional group as described above; and

n is from about 20 to about 4000.

In another embodiment, the polymer derivatives of the present invention comprise a polymer backbone having the following structure:

X- _CH2CH2O --( _CH2CH2O ) _n -- _{CH2CH2 -} O- _{( CH2} ) _{m -} WN=N=N,

in:

W is an aliphatic or aromatic linker moiety comprising 1 to 10 carbon atoms;

n is from about 20 to about 4000; and

X is a functional group as described above, and m is between 1 and 10.

The azide-containing PEG derivatives of the present invention can be prepared by a variety of methods known in the art and/or disclosed herein. In one method shown below, a water soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da, having a first end bonded to a first functional group and a second end bonded to a suitable leaving group The polymer backbone at both ends is reacted with an azide anion that can ion-pair with any of several suitable counterions including sodium, potassium, tert-butylammonium, etc. This leaving group undergoes nucleophilic displacement and is partially displaced by the azide group, yielding the desired azide-containing PEG polymer.

X-PEG-L+N ₃ ^- →X-PEG-N ₃

As shown therein, a suitable polymer backbone for use in the present invention has the formula X-PEG-L, where PEG is poly(ethylene glycol), and X is a functional group that does not react with azido groups, and L is a suitable leaving group. Examples of suitable functional groups include, but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl, amine, aminooxy, protected amine, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid , maleimide, dithiopyridine, and vinylpyridine, and ketone functional groups. Examples of suitable leaving groups include, but are not limited to, chloride, bromide, iodide, mesylate, triflate, and tosylate groups.

In another method of preparing the azide-containing polymer derivatives of the present invention, a linker having azide functionality is contacted with a water-soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da, wherein This linker has functional groups that will selectively react with chemical functional groups on the PEG polymer to form polymer derivative products containing azido groups separated from the polymer backbone by a linker group.

An exemplary reaction scheme is shown below:

X-PEG-M+N-Linker-N=N=N→PG-X-PEG-Linker-N=N=N,

in:

PEG is poly(ethylene glycol), and X is a capping group, such as an alkoxy group, or a functional group as described above; and

M is a functional group that does not react with the azido functional group but will efficiently and selectively react with the N functional group.

Examples of suitable functional groups include, but are not limited to: if N is an amine, then M is a carboxylic acid, carbonate or active ester; if N is a hydrazide or aminooxy moiety, then M is a ketone; if N is a nucleophile , then M is a leaving group.

Purification of crude products can be accomplished by known methods including, but not limited to, precipitation of the product followed, if necessary, by chromatography.

A more specific example is shown below in the case of PEG diamines, where one of these amines is protected with a protecting group moiety such as tert-butyl-Boc, and the resulting mono-protected PEG diamine is combined with an azide The linking part of the base functional group reacts:

BocHN-PEG-NH ₂ +HO ₂ C-(CH ₂ ) ₃ -N=N=N.

In this case, the amine group can be coupled to the carboxylic acid group using a variety of activators such as thionyl chloride or carbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazole , to form an amine bond between the monoamine PEG derivative and the linker moiety having an azide group. After successful amine bond formation, the resulting N-tert-butyl-Boc-protected azide-containing derivatives can be directly used to modify bioactive molecules, or they can be further refined to add other useful functional groups. For example, N-tert-butoxycarbonyl can be hydrolyzed by treatment with strong acid to generate ω-amino-PEG-azide. The resulting amines can be used as synthetic handles to add other useful functional groups such as maleimide groups, activated disulfides, activated esters, etc. to form valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when they are required to attach different molecules to each end of the polymer. For example, ω-N-amino-N-azido PEG will allow molecules with activated electrophilic groups (such as aldehydes, ketones, activated esters, activated carbonates, etc.) An ethynyl molecule is attached to the other end of PEG.

In another embodiment of the invention, the polymer has the following structure:

X-A-POLY-B-C≡C-R,

in:

R can be H or alkyl, alkenyl, alkoxy or aryl or substituted aryl;

B is a linking moiety, which may or may not be present;

POLY is a water-soluble non-antigenic polymer;

A is a linking moiety, which may or may not be present, and which may be the same as or different from B; and

X is a second functional group.

Examples of linking moieties A and B include, but are not limited to, multiply functionalized alkyl groups containing up to 18 and possibly 1 to 10 carbon atoms. Heteroatoms such as nitrogen, oxygen or sulfur may be included in the alkyl chain. Alkyl chains may also be branched at heteroatoms. Other examples of linking moieties A and B include, but are not limited to, multifunctional aryl groups containing up to 10 and possibly 5 to 6 carbon atoms. This aryl group may be substituted by a further carbon atom, nitrogen atom, oxygen atom or sulfur atom. Other examples of suitable linking groups include those described in US Patent Nos. 5,932,462; 5,643,575; and US Patent Application Publication 2003/0143596, each of which is incorporated herein by reference. Those skilled in the art will recognize that the foregoing list of linking moieties is by no means exhaustive, but is intended to be illustrative only, and that a variety of polymeric materials having the characteristics described above are considered suitable for use in the present invention.

Examples of functional groups suitable for use as X include, but are not limited to, hydroxyl, protected hydroxyl, alkoxy, active esters (such as N-hydroxysuccinimidyl ester and 1-benzotriazolyl ester), Activated carbonates (such as N-hydroxysuccinimidyl carbonate and 1-benzotriazolyl carbonate), acetals, aldehydes, hydrated aldehydes, alkenyls, acrylates, methacrylates, acrylamides , active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide , Vinylsulfone, Dithiopyridine, Vinylpyridine, Iodoacetamide, Epoxide, Glyoxal, Diketone, Mesylate, Tosylate, Trifluoroethanesulfonate, Alkene, Ketone and Ethynyl . As we understand, the selected X moiety should be compatible with the azide group so as not to react with the azide group. The ethynyl-containing polymer derivatives are homobifunctional, meaning that the second functional group (ie, X) is also an ethynyl moiety; or heterobifunctional, meaning that the second functional group is a different functional group.

In another embodiment of the invention, the polymer derivative comprises a polymer backbone having the following structure:

X- _{CH2CH2O --} ( _CH2CH2O ) _n -- _{CH2CH2 -} O-( _CH2 ) _m - _C≡CH ,

in:

X is a functional group as described above;

n is from about 20 to about 4000; and

m is between 1 and 10.

Specific examples of each heterobifunctional PEG polymer are shown below.

The ethynyl-containing PEG derivatives of the present invention can be prepared using methods known to those skilled in the art and/or disclosed herein. In one method, polymerizing a water soluble polymer backbone having an average molecular weight of from about 800 Da to about 100,000 Da, having a first end bonded to a first functional group and a second end bonded to a suitable nucleophilic group The polymer backbone is reacted with a compound having an ethynyl functional group and a leaving group suitable for reacting with a nucleophilic group on PEG. When a PEG polymer with a nucleophilic moiety is combined with a molecule with a leaving group, this leaving group undergoes nucleophilic displacement and is displaced by the nucleophilic moiety, resulting in the desired acetylene-containing polymer.

X-PEG-Nu+I-A-C→X-PEG-Nu-A-C≡CR’

As shown therein, a preferred polymer backbone for this reaction has the formula X-PEG-Nu, where PEG is poly(ethylene glycol), Nu is the nucleophilic moiety and X is non-reactive with Nu, L or ethynyl functional groups. functional groups.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy, mercapto, imino, carboxylate, hydrazide, aminooxy, which react primarily via an SN2-type mechanism. Other examples of Nu groups include those functional groups that will react primarily via nucleophilic addition reactions. Examples of L groups include chloride, bromide, iodide, mesylate, triflate, and tosylate groups and other groups expected to undergo nucleophilic displacement, such as ketones, aldehydes, sulfur Esters, alkenes, alpha-beta unsaturated carbonyls, carbonates and other electrophilic groups expected to undergo addition of nucleophiles.

In another embodiment of the present invention, A is an aliphatic linker of 1 to 10 carbon atoms or a substituted aromatic ring of 6 to 14 carbon atoms. X is a functional group that is not reactive with an azido group, and L is a suitable leaving group.

In another preparation method of the acetylenyl-containing polymer derivatives of the present invention, having an average molecular weight of about 800 Da to about 100,000 Da, having a protected functional group or capping agent on one end and a suitable The PEG polymer of the leaving group is contacted with the acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+-C≡CR’→X-PEG-C≡CR’

in:

PEG is poly(ethylene glycol) and X is a capping group, such as an alkoxy group or a functional group as described above; and

R' is H, alkyl, alkoxy, aryl, or aryloxy, or substituted alkyl, substituted alkoxy, substituted aryl, or substituted aryloxy.

In the above example, the leaving group L should be sufficiently reactive to undergo SN2-type displacement upon contact with a sufficient concentration of acetylene anion. The reaction conditions required to accomplish displacement of the leaving group by the acetylene anion SN2 are known to those skilled in the art.

Purification of crude products can generally be accomplished by methods known in the art including, but not limited to, precipitation of the product followed, if necessary, by chromatography.

A water soluble polymer can be attached to a GH (eg, hGH) polypeptide of the invention. The water soluble polymer can be incorporated into a non-naturally encoded amino acid in a GH (e.g., hGH) polypeptide, or any functional group or substituent of a non-naturally encoded or naturally encoded amino acid, or added to a non-naturally encoded or naturally encoded amino acid. Any functional group or substituent of the amino acid is connected. Alternatively, the water soluble polymer is a GH ( For example, hGH) polypeptides. In some instances, a GH (e.g., hGH) polypeptide of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Non-natural amino acids, wherein one or more non-naturally encoded amino acids are linked to a water-soluble polymer (including but not limited to, PEG and/or oligosaccharides). In some instances, a GH (e.g., hGH) polypeptide of the invention additionally comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Naturally encoded amino acid linked to a water soluble polymer. In some instances, a GH (eg, hGH) polypeptide of the invention comprises one or more non-naturally encoded amino acids linked to a water soluble polymer and one or more naturally occurring amino acids linked to a water soluble polymer. In some embodiments, water soluble polymers for use in the invention enhance the serum half-life of a GH (eg, hGH) polypeptide relative to the unconjugated form.

The amount of water-soluble polymer (i.e., degree of PEGylation or glycosylation) attached to a GH (e.g., hGH) polypeptide of the invention can be adjusted to provide altered (including but not limited to, increased or decreased) Pharmacological, pharmacokinetic or pharmacodynamic characteristics, such as in vivo half-life. In some embodiments, the half-life of a GH (e.g., hGH) polypeptide is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, relative to an unmodified polypeptide. 90%, 2 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times , 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, or at least about 100 times.

PEG derivatives containing strong nucleophilic groups (i.e., hydrazide, hydrazine, hydroxylamine, or semicarbazide)

In one embodiment of the invention, a GH (e.g., hGH) polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is transformed with PEG containing a terminal hydrazine, hydroxylamine, hydrazide, or semicarbazide moiety directly attached to the PEG backbone. Derivative modification.

In some embodiments, the hydroxylamine terminated PEG derivative will have the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -O-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000 (ie, an average molecular weight of 5 to 40 kDa).

In some embodiments, PEG derivatives containing a hydrazine or hydrazide moiety will have the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -X-NH-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000, and X is optionally a carbonyl group (C=O) which may or may not exist.

In some embodiments, a PEG derivative containing a semicarbazide moiety will have the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -NH-C(O)-NH-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000.

In another embodiment of the invention, GH (e.g., hGH) polypeptides comprising carbonyl-containing amino acids are derivatives of PEG containing terminal hydroxylamine, hydrazide, hydrazine, or semicarbazide moieties linked to the PEG backbone via amide bonds. grooming.

In some embodiments, the hydroxylamine-terminated PEG derivative has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _m -O-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000 (ie, an average molecular weight of 5 to 40 kDa).

In some embodiments, the PEG derivative containing a hydrazine or hydrazide moiety has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _m -X-NH-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10, n is 100 to 1,000, and X is a carbonyl group (C=O) which may or may not exist as needed.

In some embodiments, the PEG derivative containing a semicarbazide moiety has the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _m -NH-C(O)-NH-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000.

In another embodiment of the invention, a GH (eg, hGH) polypeptide comprising a carbonyl-containing amino acid is modified with a branched PEG derivative containing a terminal hydrazine, hydroxylamine, hydrazide, or semicarbazide moiety, wherein each of the branched PEG Chains have molecular weights in the range of 10 to 40 kDa and possibly 5 to 20 kDa.

In another embodiment of the invention, a GH (eg, hGH) polypeptide comprising a non-naturally encoded amino acid is modified with a PEG derivative having a branched structure. For example, in some embodiments, a hydrazine-terminated or hydrazide-terminated PEG derivative will have the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)] ₂ CH(CH ₂ ) _m -X-NH-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000, and X is optionally a carbonyl group (C=O) which may or may not exist.

In some embodiments, a PEG derivative containing a semicarbazide moiety will have the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -C(O)-NH-CH ₂ -CH ₂ ] ₂ CH-X-(CH ₂ ) _m -NH-C(O) -NH-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C(O) or absent, m is 2 to 10 and n is 100 to 1,000.

In some embodiments, PEG derivatives containing hydroxylamine groups will have the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -C(O)-NH-CH ₂ -CH ₂ ] ₂ CH-X-(CH ₂ ) _m -O-NH ₂ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C(O) or absent, m is 2 to 10 and n is 100 to 1,000.

The extent and site of attachment of the water soluble polymer to the hGH polypeptide can modulate the binding of the hGH polypeptide to the hGH polypeptide receptor at site 1 . In some embodiments, the present invention provides GH (eg, hGH) linked to at least one PEG via an oxime linkage, wherein the PEG used to form the oxime linkage in this reaction is a linear, 30 kDa monomethoxy-poly(ethyl) Diol)-2-aminooxyethylamine carbamate hydrochloride, as shown in FIG. 19 . Figure 20 presents an illustration of the synthesis of a linear, 30 kDa monomethoxy-poly(ethylene glycol)-2-aminooxyethylamine carbamate hydrochloride suitable for use in the synthesis of certain embodiments of the invention instance.

By way of example only and not limitation as to the type or class of PEG reagents that can be used with the compositions, methods, techniques and strategies described herein, Figure 21 presents additional illustrative examples of hydroxylamine-containing PEG reagents (these PEG reagents can be used with Carbonyl-Containing Non-Natural Amino Acid Polypeptides Reacted to Form Oxime-Containing Non-Natural Amino Acid Polypeptides Attached to a PEG Group) and Examples of Carbonyl-Containing PEG Reagents That Can React With Oxime-Containing Non-Natural Amino Acid Polypeptides Or Hydroxylamine-Containing Non-Natural Amino Acid Polypeptides to form a new oxime-containing non-natural amino acid polypeptide linked to a PEG group). Figure 22 presents four illustrative examples of synthetic methods for forming hydroxylamine-containing PEG reagents, or protected forms of hydroxylamine-containing PEG reagents, or masked forms of hydroxylamine-containing PEG reagents. Figure 23 presents an illustrative example of a synthetic method for forming an amide-linked hydroxylamine-containing PEG reagent, or a protected form of an amide-linked hydroxylamine-containing PEG reagent, or a masked form of an amide-linked hydroxylamine-containing PEG reagent. Figures 24 and 25 present the masking of carbamate-linked hydroxylamine-containing PEG reagents, or protected forms of carbamate-linked hydroxylamine-containing PEG reagents, or carbamate-linked hydroxylamine-containing PEG reagents. An illustrative example of the synthetic method of the form. Figure 26 presents an illustrative example of a synthetic method for forming a simple hydroxylamine-containing PEG reagent, or a protected form of a simple hydroxylamine-containing PEG reagent, or a simple masked form of a hydroxylamine-containing PEG reagent. In addition, Figure 27 presents an illustrative example of a hydroxylamine-containing reagent with multiple branches attached to PEG groups, and further shows that one such hydroxylamine-containing multi-PEG branched reagent is reacted with a carbonyl-containing non-natural amino acid polypeptide to form a Reaction of oxime-containing unnatural amino acid polypeptides with multiple PEG branch groups.

Other examples of water-soluble polymers (such as PEGs) suitable for use in the present invention (such as PEGs modified to form oxime linkages) can be found in U.S. Patent Application Nos. 60/638,418; 60/638,527 and filed in December 2004 No. 60/639,195, entitled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides," filed on the 22nd, the entirety of which is incorporated herein by reference. It is also in U.S. Patent Application No. 60/696,210; No. 60/696,302 and No. 60/696,302, entitled "Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides," filed July 1, 2005. 60/696,068, which reference is incorporated herein by reference in its entirety.

The extent and site of attachment of the water soluble polymer to the hGH polypeptide can modulate the binding of the GH (eg, hGH) polypeptide to the GH (eg, hGH) polypeptide receptor at site 1 . In some embodiments, the bond is configured such that the GH (e.g., hGH) polypeptide and the GH (e.g., hGH) polypeptide receptor at site 1 have a _Kd of about 400 nM or less, with a _Kd of 150 nM or less And in some cases bind with a _Kd of 100 nM or less ( _Kd is measured according to an equilibrium binding assay such as that described in Spencer et al., J. Biol. Chem., 263:7862-7867 (1988)).

Methods and chemistries for polymer activation and peptide conjugation are described in the literature and known in the art. Common methods used for polymer activation include, but are not limited to, the use of cyanogen bromide, periodate, glutaraldehyde, diepoxides, epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides , trichlorotriazine, etc. to activate functional groups (see R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S.S.Wong, (1992), CHEMISTRY OF PROTEINCONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, R.L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol.469, American D1. Chemical Chemical. Ashtony 9 ).

Several reviews and monographs on PEG functionalization and conjugation are available. See (eg) Harris, Macromol. Chem. Phys. C25: pp. 325-373 (1985); Scouten, Methods in Enzymology 135: pp. 30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: pp. 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: pp. 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: pp. 150-165 (1995).

Methods for polymer activation can also be found in WO 94/17039, U.S. Patent No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Patent No. 5,219,564, U.S. Patent No. 5,122,614, WO90/13540, U.S. Patent No. 5,281,698 and WO 93/15189, and methods can be found for conjugation between activated polymers and enzymes including, but not limited to, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027 ), oxygen-carrying molecules (US Patent No. 4,412,989), ribonucleases, and superoxide dismutases (Veronese et al., App. Biochem. Biotech. 11: pp. 141-52 (1985)). All literature and patents cited are incorporated herein by reference.

PEGylation (ie, addition of any water-soluble polymer) of a GH (eg, hGH) polypeptide containing a non-naturally encoded amino acid (such as p-azido-L-phenylalanine) is performed by any convenient method. For example, GH (eg, hGH) polypeptides are PEGylated with alkyne-terminated mPEG derivatives. Briefly, an excess of solid mPEG(5000)-O- _CH2 -C≡H was added to an aqueous solution of a p-azido-L-Phe-containing GH (eg, hGH) polypeptide with stirring at room temperature middle. The aqueous solution is typically buffered with a buffer having _a pK a close to the pH at which the reaction is performed (typically about pH 4 to 10). Examples of suitable buffers for PEGylation at pH 7.5 include, but are not limited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES, for example. The pH was continuously monitored and adjusted as necessary. Typically this reaction is allowed to continue for about 1 to 48 hours.

The reaction products are then subjected to hydrophobic interaction chromatography to allow the separation of PEGylated GH (e.g., hGH) polypeptide variants with free mPEG(5000)-O-CH ₂ -C≡CH and PEGylated GH (e.g., hGH) polypeptides Any high molecular weight complexes of any high molecular weight complexes that can form when unblocked PEG is activated on both ends of the molecule, thereby cross-linking the GH (eg, hGH) polypeptide variant molecules. Conditions during hydrophobic interaction chromatography are such that free mPEG(5000)-O- _CH2 -C≡CH flows through the column while any cross-linked PEGylated GH (e.g., hGH) polypeptide variant complexes The compound is eluted after the desired form containing one GH (eg, hGH) polypeptide variant molecule bound to one or more PEG groups. Suitable conditions will vary depending on the relative size of the cross-linked complex to the desired conjugate, and can be readily determined by one of ordinary skill in the art. The eluate containing the desired conjugate was concentrated by ultrafiltration and desalted by diafiltration.

If desired, PEGylated GH (e.g., hGH) polypeptides obtained by hydrophobic chromatography can be further purified by one or more of the procedures known to those skilled in the art, including but not limited to Affinity chromatography; anion exchange or cation exchange chromatography (use including but not limited to DEAE Sepharose); silica chromatography; reverse phase HPLC; gel filtration (use including but not limited to SEPHADEX G- 75); hydrophobic interaction chromatography; size-exclusion chromatography (size-exclusion chromatography); metal chelation chromatography; ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation; chromatographic focusing; displacement chromatography; electrophoresis procedures (including, but not limited to, preparative isoelectric focusing); differential solubility methods (including, but not limited to, ammonium sulfate precipitation); or extraction methods. Apparent molecular weights can be estimated by comparing GPC to globular protein standards (Preneta, AZ in PROTEINPURIFICATION METHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989, pp. 293-306). Purity of GH (eg, hGH)-PEG conjugates can be assessed by proteolytic degradation (including, but not limited to, trypsin cleavage) followed by mass spectrometric analysis. Pepinsky RB., et al., J. Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

PEGylation (ie, addition of any water-soluble polymer) of GH (eg, hGH) polypeptides containing a carbonyl-containing non-naturally encoded amino acid (such as p-acetyl-L-phenylalanine) is also performed by any convenient method. As a non-exclusive example, aminooxyethylamine carbamate mPEG derivatives (having a molecular weight of about 0.1 to 100 kDa, or about 1 to 100 kDa, or about 10 to 50 kDa, or about 20 to 40 kDa, or for example 30 kDa) are used. ) PEGylate a GH (hGH) polypeptide containing a carbonyl-containing non-naturally encoded amino acid (eg, p-acetyl-L-phenylalanine). Briefly, an excess of solid MPEG-hydroxylamine such as mPEG(30,000)-O-CO-NH-(CH ₂ ) ₂ -ONH ₃ ⁺ (linear 30 kDa monomethoxy- Poly(ethylene glycol)-2-aminooxyethylamine carbamate hydrochloride, 30K MPEG-hydroxylamine) was added to the GH (e.g., hGH) polypeptide containing p-acetyl-L-phenylalanine in aqueous solution. The molar ratio of PEG:GH (eg, hGH) can be about 2 to 15, or about 5 to 10, or about 5, 6, 7, 8, 9, or 10. The aqueous solution is typically buffered with a buffer having a pKa close to the pH at which the reaction is performed (typically about pH 2 to 8). Examples of suitable buffers for PEGylation at pH 4.0 include, but are not limited to, sodium acetate/glycine buffer adjusted to pH 4.0 by the addition of acetic acid, for example. Typically the reaction is allowed to continue for about 1 to 60 hours, or about 10 to 50 hours, or about 18 to 48 hours, or about 39 to 50 hours at room temperature with gentle shaking. PEGylation can be determined by SDS gel.

The reaction product is then subjected to any high molecular weight complexes from, e.g., free 30K MPEG-hydroxylamine and PEGylated GH (e.g., hGH) polypeptide (which may form when unblocked PEG is activated on both ends of the molecule) Purification, thereby cross-linking the GH (eg, hGH) polypeptide variant molecules. Any suitable method of purification may be used, eg column chromatography (such as SourceQ column chromatography using SourceQ Buffer A and SourceQ Buffer B). The reaction mixture was diluted with TRIS base and SourceQ Buffer A and MilliQ water before loading onto the column. The eluate containing the desired conjugate can be further concentrated by ultrafiltration and desalted by diafiltration.

If necessary, PEGylated GH (e.g., hGH) polypeptides obtained by chromatography may be further subjected to one or more of the procedures known to those of skill in the art and described herein (see e.g., above). And purified. The final PEGylated GH (eg, hGH) polypeptide may be obtained at a purity greater than 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 99.99%. Purity can be determined by methods known in the art. Exemplary, non-limiting methods of assessing purity include SDS-PAGE, measurement of GH (e.g., hGH) polypeptide using Western blot and ELISA assays, Bradford assay, mass spectrometry (including but not limited to, MALDI-TOF), HPLC methods ( Methods such as RP HPLC, cation exchange HPLC, and gel filtration HPLC) and other methods known to those skilled in the art for characterizing proteins.

The water soluble polymers linked to the amino acids in the GH (eg, hGH) polypeptides of the invention can be further derivatized or substituted without limitation.

Azido-containing PEG derivatives

In another embodiment of the invention, a GH (eg, hGH) polypeptide is modified with a PEG derivative containing an azido moiety that will react with an alkynyl moiety present on the side chain of a non-naturally encoded amino acid. Generally, these PEG derivatives will have an average molecular weight of 1 to 100 kDa, and in some embodiments 10 to 40 kDa.

In some embodiments, an azido-terminated PEG derivative will have the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -N ₃ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000 (ie, an average molecular weight of 5 to 40 kDa).

In another example, an azido-terminated PEG derivative would have the following structure:

RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) _m -NH-C(O)-(CH ₂ ) _p -N ₃ ,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10, p is 2 to 10 and n is 100 to 1,000 (ie, an average molecular weight of 5 to 40 kDa).

In another embodiment of the invention, GH is modified with a branched PEG derivative containing a terminal azido moiety wherein each chain of the PEG has a molecular weight in the range of 10 to 40 kDa and possibly 5 to 20 kDa (e.g. , hGH) polypeptide. For example, in some embodiments, an azido-terminated PEG derivative will have the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)] ₂ CH(CH ₂ ) _m -X-(CH ₂ ) _p N ₃

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10, p is 2 to 10 and n is 100 to 1,000, and X is optionally O, N, S or carbonyl (C =0), which may or may not be present in each case.

Alkyne-containing PEG derivatives

In another embodiment of the invention, a GH (eg, hGH) polypeptide is modified with a PEG derivative containing an alkynyl moiety that will react with an azido moiety present on the side chain of a non-naturally encoded amino acid.

In some embodiments, the alkynyl-terminated PEG derivative will have the following structure:

RO-( _CH2CH2O ) _n -O-( _CH2 ) _{m -} C≡CH,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10 and n is 100 to 1,000 (ie, an average molecular weight of 5 to 40 kDa).

In another embodiment of the invention, a GH containing an alkynyl-containing non-naturally encoded amino acid is modified with a PEG derivative containing a terminal azido moiety or a terminal alkynyl moiety attached to the PEG backbone via an amide bond. (eg, hGH) polypeptides.

In some embodiments, the alkynyl-terminated PEG derivative will have the following structure:

RO-( _{CH2CH2O ) n} -O-( _CH2 )m _- NH-C(O)-( _CH2 ) _p- C≡CH,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10, p is 2 to 10 and n is 100 to 1,000.

In another embodiment of the present invention, the azide-containing compound is modified with a branched PEG derivative containing a terminal alkynyl moiety wherein each chain of the PEG has a molecular weight in the range of 10 to 40 kDa and possibly 5 to 20 kDa. GH (e.g., hGH) polypeptides of amino acids. For example, in some embodiments, an alkynyl-terminated PEG derivative will have the following structure:

[RO-(CH ₂ CH ₂ O) _n -O-(CH ₂ ) ₂ -NH-C(O)] ₂ CH(CH ₂ ) _m -X-(CH ₂ ) _p C≡CH,

wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2 to 10, p is 2 to 10 and n is 100 to 1,000, and X is optionally O, N, S or carbonyl (C =0), or does not exist.

PEG Derivatives Containing Phosphine Moieties

In another embodiment of the present invention, a compound containing an activated functional group (including but not limited to, ester, carbonate) and further comprising an arylphosphine group (which will interact with the amino acid present on the side chain of the non-naturally encoded amino acid) is used. Azide moiety reacted) PEG derivatives modify GH (eg, hGH) polypeptides. Generally, these PEG derivatives will have an average molecular weight of 1 to 100 kDa, and in some embodiments 10 to 40 kDa.

In some embodiments, the PEG derivative will have the following structure:

wherein n is 1 to 10; X can be O, N, S or absent, Ph is phenyl, and W is a water-soluble polymer.

In some embodiments, the PEG derivative will have the following structure:

Where X can be O, N, S or absent, Ph is phenyl, W is a water soluble polymer, and R can be H, alkyl, aryl, substituted alkyl, and substituted aryl. Exemplary R groups include, but are not limited to, _-CH2 , -C( _CH3 ) ₃ , -OR', -NR'R", -SR', halo, -C(O)R', -CONR 'R', -S(O) _2R ', -S(O) _2NR'R ", -CN, and _-NO2 . R', R", R'', and R"" each independently refer to hydrogen , substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including but not limited to, aryl substituted with 1 to 3 halogen atoms), substituted or unsubstituted alkane radical, alkoxy or thioalkoxy or aralkyl. For example, when a compound of the invention includes more than one R group, each R group is independently selected; just as when more than one of R', R", R'', and R"" When present, the R', R", R'', and "" groups are each independently selected. When R' and R" are attached to the same nitrogen atom, they may combine with this nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" means including, but not limited to ) 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is meant to include groups comprising carbon atoms bonded to non-hydrogen groups, such as haloalkyl groups (including but not limited to) - CF ₃ and -CH ₂ CF ₃ ) and acyl groups (including but not limited to -C(O)CH ₃ , -C(O)CF ₃ , -C(O)CH ₂ OCH ₃ , etc.).

Other PEG derivatives and general PEGylation techniques

Other exemplary PEG molecules and methods of PEGylation that can be linked to a GH (e.g., hGH) polypeptide include those described in the following patents: U.S. Patent Publication No. 2004/0001838; No. 2002 No./0052009; No. 2003/0162949; No. 2004/0013637; No. 2003/0228274; No. 2003/0220447; No. 2003/0105224; No. 2003/0023023; No. 2002/0156047; No. 2002/0099133; 2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0021763;第5,219,564号；第5,629,384号；第5,736,625号；第4,902,502号；第5,281,698号；第5,122,614号；第5,473,034号；第5,516,673号；第5,382,657号；第6,552,167号；第6,610,281号；第6,515,100号；第6,461,603 No. 6,436,386; No. 6,214,966; No. 5,990,237; No. 5,900,461; No. 5,739,208; No. 5,672,662; No. 6,201,072; No. 6,451,346; No. 6,306,821; No. 5,559,213; No. 5,747,646; 5,834,594; 5,849,860; 5,980,948; No. 6,129,912; WO 97/32607, EP 229,29,129,32607,1299,307,1299,307,1299,299,307,1299,307,1299,299,307,12907,12907,12907,12907,12907,29,307,12907,29,32607,12907,29,32607,12907,29,32607,29,307,29,32607,299,3960 , WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP400 472, EP 183 503 and EP 154 316, which are incorporated herein by reference. Any of the PEG molecules described herein can be used in any form, including, but not limited to, single-chain, branched, multi-armed, monofunctional, multifunctional, or any combination thereof.

Enhanced affinity for serum albumin

Various molecules can also be fused to the GH (eg, hGH) polypeptides of the invention to modulate the half-life of the GH (eg, hGH) polypeptides in serum. In some embodiments, molecules are linked or fused to GH (eg, hGH) polypeptides of the invention to enhance affinity for the animal's endogenous serum albumin.

For example, in some cases, recombinant fusion of a GH (eg, hGH) polypeptide with an albumin binding sequence is performed. Exemplary albumin binding sequences include, but are not limited to, the albumin binding domain from streptococcal protein G (see, e.g., Makrides et al., J. Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al., J, Immunol. Methods 201: 115-123 (1997)), or albumin binding peptides (such as described in, eg, Dennis et al., J. Biol. Chem. 277: pp. 35035-35043 (2002)).

In other embodiments, GH (eg, hGH) polypeptides of the invention are acylated using fatty acids. In some embodiments, fatty acids promote binding to serum albumin. See, eg, Kurtzhals et al., Biochem. J. 312: pp. 725-731 (1995).

In other embodiments, the GH (eg, hGH) polypeptides of the invention are fused directly to serum albumin (including, but not limited to, human serum albumin). Those skilled in the art will recognize that a variety of other molecules can also be linked to the GH (eg, hGH) polypeptides of the invention to modulate binding to serum albumin or other serum components.

X. Glycosylation of GH (eg, hGH) Polypeptides

The invention includes GH (eg, hGH) polypeptides into which one or more non-naturally encoded amino acids having carbohydrate residues are incorporated. These carbohydrate residues may be natural (including, but not limited to, N-acetylglucosamine) or unnatural (including, but not limited to, 3-fluorogalactose). These carbohydrates can be linked via N-linked or O-linked glycosidic linkages (including but not limited to N-acetylgalactose-L-serine) or non-natural linkages (including but not limited to oxime or corresponding C-linked or S-linked glycosides) linked to non-naturally encoded amino acids.

Carbohydrate (including but not limited to, glycosyl) moieties can be added to a GH (eg, hGH) polypeptide in vivo or in vitro. In some embodiments of the invention, a GH (e.g., hGH) polypeptide comprising a carbonyl-containing non-naturally encoded amino acid is modified with an aminooxy-derivatized carbohydrate to produce a corresponding glycosylated polypeptide linked by an oxime linkage . Once linked to a non-naturally encoded amino acid, the carbohydrate can be further refined by treatment with glycosyltransferases and other enzymes to produce oligosaccharides that bind to the GH (eg, hGH) polypeptide. See eg H. Liu et al. J. Am. Chem. Soc. 125: pp. 1702-1703 (2003).

In some embodiments of the invention, GH (eg, hGH) polypeptides comprising carbonyl-containing non-naturally encoded amino acids are directly modified using glycans of defined structure prepared as aminooxy derivatives. Those skilled in the art will recognize that other functional groups, including azido, alkynyl, hydrazide, hydrazine, and semicarbazide, can be used to link carbohydrates to non-naturally encoded amino acids.

In some embodiments of the present invention, this can then be achieved by Hu's radical [3+2] cycloaddition of (including but not limited to) to (including but not limited to) alkynyl or azido derivatives, respectively reactions to modify GH (eg, hGH) polypeptides comprising azido- or alkynyl-containing non-naturally encoded amino acids. This method allows proteins to be modified with very high selectivity.

Dimers and multimers of XI.GH supergene family members

The invention also provides combinations of members of the GH supergene family (including but not limited to, GH, such as hGH and hGH analogs), such as homodimers, heterodimers, homomultimers or heteromultimers (i.e., trimers or tetramers, etc.), wherein a GH supergene family member polypeptide (such as GH, e.g. hGH) containing one or more non-naturally encoded amino acids is associated with another GH supergene family member or variant thereof or any other polypeptide that is not a member of the GH supergene family or a variant thereof, and is connected to the polypeptide backbone directly or through a linker. Due to their higher molecular weights compared to monomers, dimer or multimeric conjugates of GH supergene family members (such as GH, e.g. hGH) can exhibit new or desirable properties, including but not limited to ) different pharmacological properties, pharmacokinetic properties, pharmacodynamic properties, modulated half-life or modulated plasma half-life relative to monomeric GH supergene family members. In some embodiments, dimers of GH supergene family members (such as GH, eg, hGH) of the invention will modulate dimerization of GH supergene family member receptors. In other embodiments, the GH supergene family member dimers or multimers of the invention will act as GH supergene family member receptor antagonists, agonists or iambic agents.

In some embodiments, one or more GH (e.g., hGH) molecules present in a dimer- or multimer-containing GH (e.g., hGH) comprise a link to a water-soluble polymer present within the site II binding region non-naturally encoded amino acids. Thus, each molecule of the dimer or multimer readily binds to a GH (eg, hGH) polypeptide receptor through a site I interface, but cannot bind to a second GH (eg, hGH) polypeptide receptor through a site II interface. Thus, a GH (e.g., hGH) polypeptide dimer or multimer can bind to the Site I binding site of each of two different GH (e.g., hGH) polypeptide receptors, but since the GH (e.g., hGH) polypeptide The molecule has a water soluble polymer linked to a non-genetically encoded amino acid present in the site II region such that the GH (e.g., hGH) polypeptide receptor is unable to bind to the site II region of the GH (e.g., hGH) polypeptide ligand, and The dimer or multimer acts as a GH (eg, hGH) polypeptide antagonist. In some embodiments, one or more GH (e.g., hGH) molecules present in a dimer- or multimer-containing GH (e.g., hGH) comprise a link to a water-soluble polymer present within the Site I binding region non-naturally encoded amino acid to allow binding to the site II region. Alternatively, in some embodiments, one or more molecules of GH (e.g., hGH) present in a dimer- or multimer-containing GH (e.g., hGH) comprise a combination of GH (e.g., hGH) molecules that are not present within the site I or site II binding region A non-naturally encoded amino acid linked to a water-soluble polymer to allow binding of both domains. In some embodiments, a combination of GH (eg, hGH) molecules with available Site I, available Site II, or both is used. Combinations of GH (eg, hGH) molecules in which at least one molecule has Site I available for binding and at least one molecule has Site II available for binding can provide molecules with desired activities or properties. In addition, combinations of GH (eg, hGH) molecules having both Site I and Site II available for binding can result in super-potent GH (eg, hGH) molecules.

In some embodiments, GH supergene family member polypeptides, including but not limited to, are directly linked via an Asn-Lys amine bond or a Cys-Cys disulfide. In some embodiments, linked GH supergene family member polypeptides and/or linked non-GH supergene family members will comprise different non-naturally encoded amino acids to facilitate dimerization, including but not limited to An alkynyl group in one non-naturally encoded amino acid of a first GH (e.g., hGH) polypeptide and an azido group in a second non-naturally encoded amino acid of a second GH supergene family member polypeptide will pass through the Hood root [ 3+2] cycloaddition binding linkage. Alternatively, a first GH supergene family member and/or a linked non-GH supergene family member, a polypeptide comprising a keto-containing non-naturally encoded amino acid can be conjugatively linked to a second GH supergene family comprising a hydroxylamine-containing non-naturally encoded amino acid. Gene family member polypeptides that react by forming the corresponding oximes.

Alternatively, two GH supergene family member polypeptides and/or linked non-GH supergene family members are linked by a linker. Any heterobifunctional or homobifunctional linker may be used to link two GH supergene family members and/or linked non-GH supergene family members, polypeptides (which may have the same or different primary sequences). In some embodiments, the linker used to link together GH supergene family members and/or linked non-GH supergene family members, polypeptides may be a bifunctional PEG reagent. The linker can have a wide range of molecular weights or molecular lengths. Linkers of larger or smaller molecular weight can be used between a GH supergene family member and a linking entity, or between a GH supergene family member and a GH supergene family member receptor, or between a linking entity and a GH supergene family member The desired spatial relationship or configuration is provided between the receptors. Linkers with longer molecular lengths or shorter molecular lengths can also be used between a GH supergene family member and a linking entity, or between a GH supergene family member and its receptor, or between a linking entity and a GH supergene family member recipient. Provide the required space and flexibility between the bodies. Similarly, linkers having a particular shape or configuration can be used to impart a particular shape or configuration to a GH supergene family member or linking entity either before or after the GH supergene family member reaches its target. This optimization of the spatial relationship between GH supergene family members and linking entities can provide molecules with new, indirectly modulated or desired properties.

In some embodiments, the present invention provides a water-soluble bifunctional linker having a dumbbell structure comprising: a) an azido, alkynyl, hydrazine, acyl group on at least a first end of the polymer backbone; hydrazine, hydroxylamine, or a carbonyl-containing moiety; and b) at least one second functional group on the second end of the polymer backbone. This second functional group can be the same as or different from the first functional group. In some embodiments, the second functional group does not react with the first functional group. In some embodiments, the present invention provides water-soluble compounds comprising at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one or more members of the GH supergene family, such as GH, e.g. hGH, formed by reaction with a water soluble activated polymer having the following structure:

R-( _CH2CH2O ) _n -O-( _CH2 ) _m -X _,

Where n is 5 to 3,000, m is 2 to 10, X can be azido, alkynyl, hydrazine, hydrazide, aminooxy, hydroxylamine, acetyl or a carbonyl-containing moiety, and R is a capping group, a functional group or a leaving group, which may be the same as or different from X. R can be, for example, a functional group selected from the group consisting of hydroxyl, protected hydroxyl, alkoxy, N-hydroxysuccinimide ester, 1-benzotriazolyl ester, N -Hydroxysuccinimide carbonate, 1-benzotriazolyl carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, activated sulfone, amine, amino Oxygen, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine , vinylpyridine, iodoacetamide, epoxides, glyoxal, diketones, mesylate, tosylate, trifluoroethanesulfonate, alkenes and ketones.

XII. Measurement of hGH polypeptide activity and affinity of hGH polypeptide for hGH polypeptide receptor

[586] hGH receptors can be prepared as described in McFarland et al., Science, 245: pp. 494-499 (1989) and Leung, D. et al., Nature, 330: pp. 537-543 (1987). hGH polypeptide activity can be determined using standard or known in vivo or in vitro assays. For example, cell lines that proliferate in the presence of hGH (eg, cell lines expressing hGH receptor or prolactin receptor) can be used to monitor hGH receptor binding. See eg Clark, R. et al., J. Biol. Chem. 271(36): p. 21969 (1996); Wada et al., Mol. Endocrinol. 12: pp. 146-156 (1998); Gout, PW et al. Human Cancer Res. 40, pp. 2433-2436 (1980); WO 99/03887. For non-PEGylated or PEGylated hGH polypeptides comprising unnatural amino acids, the affinity of the hormone for its receptors can be measured using a BIAcore ^™ biosensor (Pharmacia). See, eg, US Patent No. 5,849,535, Spencer, SA et al., J. Biol. Chem., 263: pp. 7862-7867 (1988). In vivo animal models for testing hGH activity include those described, for example, in Clark et al., J. Biol. Chem. 271(36): pp. 21969-21977 (1996). It can be carried out as described in Cunningham, B. et al., Science, 254: pp. 821-825 (1991) and Fuh, G. et al., Science, 256: pp. 1677-1680 (1992). Assay of the dimerization ability of hGH polypeptides of the above non-naturally encoded amino acids. All cited literature and patents are incorporated herein by reference.

The literature compilation on assay methods is not exhaustive, and those skilled in the art will recognize other assays suitable for testing the desired end result.

XIII. Measurements for Potency, Functional In Vivo Half-Life, and Pharmacokinetic Parameters

An important aspect of the invention is the extended biological half-life achieved by the construction of the hGH polypeptide with or without association of the polypeptide with a water-soluble polymer moiety. The rapid decrease in serum concentrations of hGH polypeptides has made it important to assess the biological response to therapy with binding or non-binding hGH polypeptides and variants thereof. Following subcutaneous or intravenous administration, binding or non-binding hGH polypeptides of the invention and variants thereof may also have a persistent serum half-life, enabling measurement by, for example, ELISA methods or by primary screening assays. ELISA or RIA kits from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX) can be used. In vivo biological half-life measurements were performed as described herein.

The potency and functional in vivo half-life of hGH polypeptides comprising non-naturally encoded amino acids can be determined according to the methods described in Clark, R. et al., J. Biol. Chem. 271(36): pp. 21969-21977 (1996). Determination.

Pharmacokinetic parameters of hGH polypeptides comprising non-naturally encoded amino acids can be assessed in normal Sprague-Dawley male rats (N=5 animals/treatment group). Animals will receive a single dose of 25 μg/rat IV or 50 μg/rat sc, according to a predetermined time course (generally encompassing GH containing non-naturally encoded amino acids, not conjugated to a water-soluble polymer (e.g., hGH ) polypeptides, and approximately 5 to 7 blood samples are collected for approximately 6 hours for GH (eg, hGH) polypeptides comprising non-naturally encoded amino acids and bound to water-soluble polymers, and approximately 4 days). Pharmacokinetic data for GH (eg, hGH) polypeptides is well-studied in several substances and can be compared directly with data obtained for GH (eg, hGH) polypeptides comprising non-naturally encoded amino acids. For studies related to GH (eg, hGH) polypeptides, see Mordenti J. et al., Pharm. Res. 8(11): pp. 1351-59 (1991).

Pharmacokinetic parameters can also be assessed in primates such as rhesus monkeys. Typically, a single injection is administered subcutaneously or intravenously, and serum GH (eg, hGH) levels are monitored over time. For further description see eg "Examples".

In some embodiments, the present invention provides at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 when administered subcutaneously to a mammal , GH (eg hGH) with a mean serum half-life of 16 hours or more. In some embodiments, the present invention provides at least about 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 when administered subcutaneously to a mammal Or a GH (eg hGH) with a mean serum half-life of more than 12 hours. An "average" serum half-life is the average of at least three animals, or at least four animals, or at least five animals, or more than five animals. In some embodiments, the mammal is a rat; in some embodiments, the mammal is a primate, such as a macaque, or such as a human. In some embodiments, the invention provides PEGylated GH (eg, PEGylated hGH) that when administered subcutaneously has a mean serum half-life that is at least about 2-fold, 3-fold comparable to non-PEGylated forms of GH (eg, hGH). , 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more than 50-fold mean serum half-life. In some embodiments, the invention provides a PEGylated GH (eg, PEGylated hGH) that when administered intravenously has a mean serum half-life that is at least about 2-fold, 3-fold equivalent to that of a non-PEGylated form of GH (eg, hGH). Times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more than 50-fold mean serum half-life. An "average" serum half-life is the average of at least three animals, or at least four animals, or at least five animals, or more than five animals. In some embodiments, the mammal is a rat; in some embodiments, the mammal is a primate, such as a macaque, or such as a human. In some embodiments, the GH is GH (eg, hGH). In some embodiments, the growth hormone is linked to at least one water soluble polymer via a covalent bond, wherein the covalent bond is an oxime bond. GH can be GH (eg hGH). In some embodiments, the GH (eg, hGH) includes a non-naturally encoded amino acid, such as a carbonyl-containing non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid is a keto-containing amino acid, such as p-acetylphenylalanine. In some embodiments, the GH (eg, hGH) contains a non-naturally encoded amino acid, eg, acetylphenylalanine substituted at a position in the GH (eg, hGH) corresponding to amino acid 35 in SEQ ID NO: 2. The water soluble polymer can be PEG. Suitable PEGs include linear PEGs and branched PEGs; any PEG described herein can be used. In particular embodiments, the PEG is a linear PEG of about 0.1 to 100 kDa, or about 1 to 100 kDa, or about 10 to 50 kDa, or about 20 to 40 kDa, or about 30 kDa. In some embodiments, the pharmaceutical composition comprises a GH (e.g., hGH) linked to a 30 kDa PEG by an oxime linkage in the para-acetyl group at a position corresponding to amino acid 35 in SEQ ID NO:2 between phenylalanine and PEG.

The specific activity of a GH (eg, hGH) polypeptide according to the invention can be determined by a variety of assays known in the art. The biological activity of a GH (eg, hGH) polypeptide mutein or fragment thereof obtained and purified according to the present invention can be tested by methods described or referenced herein, or methods known to those skilled in the art.

XIV. Administration and Pharmaceutical Compositions

Polypeptides or proteins of the present invention (including but not limited to GH (such as hGH), synthetases, proteins comprising one or more amino acids, etc.) are used in combination with suitable pharmaceutical carriers as needed (including but not limited to) therapeutic use. For example, such compositions comprise a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. Make the formulation suitable for administration. In general, methods of administering proteins are known to those of skill in the art and can be applied to administer polypeptides of the invention.

In some embodiments, the present invention provides a pharmaceutical composition comprising a hormone composition comprising growth hormone linked by a covalent bond to at least one water-soluble polymer, wherein the covalent bond is an oxime bond; and a pharmaceutical Pharmaceutically acceptable excipients. GH can be hGH. In some embodiments, the GH (eg, hGH) includes a non-naturally encoded amino acid, such as a carbonyl-containing non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid is a keto-containing amino acid, such as p-acetylphenylalanine. In some embodiments, the GH (eg, hGH) contains a non-naturally encoded amino acid, eg, acetylphenylalanine substituted at a position in the GH (eg, hGH) corresponding to amino acid 35 in SEQ ID NO: 2. The water soluble polymer can be PEG. Suitable PEGs include linear PEGs and branched PEGs; any PEG described herein can be used. In particular embodiments, the PEG is a linear PEG of about 0.1 to 100 kDa, or about 1 to 100 kDa, or about 10 to 50 kDa, or about 20 to 40 kDa, or about 30 kDa. In some embodiments, the pharmaceutical composition comprises a GH (e.g., hGH) linked to a 30 kDa PEG by an oxime linkage in the para-acetyl group at a position corresponding to amino acid 35 in SEQ ID NO:2 between phenylalanine and PEG.

Therapeutic compositions comprising one or more polypeptides of the present invention are optionally tested in one or more appropriate in vivo and/or in vitro animal disease models to confirm efficacy, tissue Metabolism and dose estimation. Specifically, unnatural amino acids can be compared to natural amino acid homologues (including but not limited to, GH (e.g., hGH) polypeptides modified to include one or more unnatural amino acids herein and natural amino acid GH (e.g., , comparison of hGH) polypeptides), that is, activity, stability or other suitable measures in relevant assays to preliminarily determine the dosage.

Administration is by any of the routes commonly used to introduce molecules into ultimate contact with blood or tissue cells. The non-natural amino acid polypeptide of the present invention is administered in any suitable manner, optionally using one or more pharmaceutically acceptable carriers. Suitable methods of administering the polypeptides are available in the context of the present invention, and although more than one route may be used to administer a particular composition, a particular route will often provide a more immediate and potent effect than other routes or react.

The pharmaceutically acceptable carrier will be determined in part by the particular composition being administered and the method used to administer the composition. Accordingly, there are various suitable formulations of the pharmaceutical composition of the present invention.

The hGH polypeptides of the invention (including but not limited to PEGylated hGH) can be administered by any conventional route suitable for proteins and polypeptides, including but not limited to parenteral means such as injection or infusion (including (but not limited to) subcutaneous or intravenous or any other form of injection or infusion. Polypeptide compositions can be administered by several routes including but not limited to oral, intravenous, intraperitoneal, intramuscular, via Skin, subcutaneous, topical, sublingual, or rectal. Compositions (modified or unmodified) comprising non-natural amino acid polypeptides can also be administered via liposomes. The route of administration and appropriate formulations are known to those skilled in the art It is generally known that hGH polypeptides comprising non-naturally encoded amino acids, including but not limited to PEGylated hGH, can be used alone or in combination with other suitable components, such as pharmaceutical carriers.

GH (eg, hGH) polypeptides comprising unnatural amino acids can also be formulated in aerosol formulations (ie, they can be "nebulized"), alone or in combination with other suitable components, for administration via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration (such as by intra-articular, intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffering agents , bacteriostatic agents and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations of hGH may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.

Parenteral and intravenous administration are preferred methods of administration. In particular, natural amino acid homologs that have been used in therapeutics (including but not limited to those used in EPO, GH, G-CSF, GM-CSF, IFN, interleukins, antibodies and/or any other medicinal The route of administration of the protein delivered by means), together with the formulation currently used, provides the preferred route of administration and the preferred formulation of the polypeptide of the invention.

Depending on the application, the dosage administered to a patient in the context of the present invention is sufficient to have a beneficial therapeutic response or other suitable activity in the patient over time. The dosage will be determined by the potency of the particular carrier or formulation, the activity, stability or serum half-life of the non-natural amino acid polypeptide employed, the condition of the patient, and the body weight and surface area of the patient to be treated. The size of the dose will also be determined by the existence, nature and extent of any adverse side effects accompanying the administration of a particular carrier, formulation, etc. in a particular patient.

In determining an effective amount of a vector or formulation to be administered in the treatment or prevention of a disease, including but not limited to, cancer, genetic disease, diabetes, AIDS, or the like, the physician assesses circulating plasma levels, formulation toxicity, The course of the disease and/or where the anti-non-natural amino acid polypeptide antibodies arise.

Doses administered to a patient of eg 70 kg are generally in the range equivalent to doses of therapeutic proteins currently used that are modulated for altered activity or serum half-life of the corresponding composition. The vector or formulation of the present invention can be treated by any known conventional treatment (including administration of antibodies, administration of vaccines, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide homologues, biological response modifiers, etc.) The situation is supplemented.

For administration, follow the LD-50 or ED-50 of the corresponding formulation and/or for (including but not limited to) any unnatural amino acid at various concentrations when applied as corresponding to the quality and overall health of the patient. The formulations of the invention are administered at rates determined by observation of side effects. Administration can be accomplished in single or divided doses.

If a patient receiving formula infusion develops fever, chills, or muscle aches, he or she may take appropriate doses of aspirin, ibuprofen, acetaminophen, or other antipyretic and pain relievers medicine. Patients who have a reaction to the infusion (such as fever, muscle aches, or chills) are prodromal before continuing the infusion of aspirin, acetaminophen, or (including but not limited to) diphenhydramine Use the medicine for 30 minutes. Meperidine is used for more severe colds and muscle pains that do not respond quickly to antipyretics and antihistamines. Slow or stop cell infusion based on severity of reaction.

The human GH polypeptides of the present invention can be administered directly to mammalian subjects. Administration is by any route commonly used to introduce hGH polypeptide into a subject. hGH polypeptide compositions according to embodiments of the invention include those suitable for oral, rectal, topical, inhalation (including but not limited to, via aerosol), buccal (including but not limited to, sublingual), vaginal, parenteral Enteral (including, but not limited to, subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, intracerebral, intraarterial, or intravenous), topical (ie, skin and mucosal surfaces, including tracheal surfaces), and transdermal The composition of administration, however, the most suitable route in any given case will depend on the nature and severity of the condition being treated. Administration can be local or systemic. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. The GH (eg, hGH) polypeptides of the invention are prepared in admixture with a pharmaceutically acceptable carrier in unit dosage injectable forms, including but not limited to solutions, suspensions, or emulsions. GH (eg, hGH) polypeptides of the invention can also be administered by continuous infusion (using, including but not limited to, small vacuum pumps such as osmotic pumps), individual boluses, or sustained release depot-type formulations.

Formulations suitable for administration include aqueous and nonaqueous solutions, isotonic sterile solutions which may contain antioxidants, buffers, bacteriostats and solutes to render the formulation isotonic; and aqueous and nonaqueous sterile suspensions, It may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. Solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

Freeze-drying is a technique commonly used to express proteins to remove water from the protein preparation. Freeze-drying or lyophilization is a method in which the material to be dried is first frozen and then the ice or freezing solvent is removed by sublimation in a vacuum environment. Excipients may be included in the pre-lyophilized formulation to enhance stability during the lyophilization process and/or to improve the stability of the lyophilized product upon storage. Pikal, M. Biopharm. 3(9) pp. 26-30 (1990) and Arakawa et al. Pharm. Res. 8(3): pp. 285-291 (1991).

Spray drying of pharmaceuticals is also known to those skilled in the art. See, for example, "The Spray Drying of Pharmaceuticals," by Broadhead, J. et al. in Drug Dev. hid. Pharm, 18 (11 & 12), pp. 1169-1206 (1992). In addition to small molecule drugs, a variety of biological materials have been spray dried and these include: enzymes, serum, plasma, microorganisms, and yeast. Spray drying is a suitable technique because it converts liquid drug preparations into fine, dust-free or agglomerated powders in a one-step process. The basic technique involves the following four steps: a) atomization of the feed solution into a spray; b) spray-air contact; c) spray drying; d) separation of the dried product from the drying air. US Patent Nos. 6,235,710 and 6,001,800, which are incorporated herein by reference, describe the preparation of recombinant erythropoietin by spray drying.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier, excipient or stabilizer. A pharmaceutically acceptable carrier is determined in part by the particular composition being administered and the particular method used to administer the composition. Accordingly, the pharmaceutical compositions of the present invention exist in a variety of suitable formulations (including optionally pharmaceutically acceptable carriers, excipients or stabilizers) (see e.g. Remington's Pharmaceutical Sciences, 17th Edition, 1985)) .

Suitable carriers include buffers, which contain succinic acid, phosphoric acid, boric acid, HEPES, citric acid, histidine or histidine derivatives, imidazole, acetic acid, bicarbonic acid, and other organic acids; antioxidants, which include (but Without limitation) ascorbic acid; low molecular weight polypeptides including but not limited to those less than about 10 residues; proteins including but not limited to serum albumin, gelatin or immunoglobulin; hydrophilic polymers substances including but not limited to polyvinylpyrrolidone; amino acids including but not limited to glycine, glutamine, asparagine, arginine, histidine or histidine derivatives, methionine , glutamic acid or lysine; monosaccharides, disaccharides and other carbohydrates including but not limited to trehalose, sucrose, glucose, mannose or dextrin; chelating agents including but not limited to EDTA ; divalent metal ions including (but not limited to) zinc, cobalt or copper ions; sugar alcohols including (but not limited to) mannitol or sorbitol; salt-forming counterions including (but not limited to) Tween ^™ (including but not limited to) Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), Pluronics ^™ and other polyether acids including but not limited to polyether acid F68 (polyether acid poloxamer 188) or PEG. Suitable surfactants include, for example but not limited to, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (ie (PEO-PPO-PEO)) based or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (i.e. (PPO-PEO ^- PPO)) or a combination thereof. , R-Pluronics ^™ , Tetronics ^™ , and R-Tetronics ^™ (BASF Wyandotte Corp., Wyandotte, Mich.), and are further described in US Patent No. 4,820,352 (herein incorporated by reference in its entirety). Other ethylene/polyethylene block polymers may be suitable surfactants. Surfactants or combinations of surfactants may be used to target one or more pressures (which include, but are not limited to, pressures induced by agitation) Stabilizes PEGylated hGH. Some of the above agents may be referred to as "bulking agents." Some may also be referred to as "tonicity modifiers."

GH (eg, hGH) polypeptides of the invention, including those linked to water-soluble polymers such as PEG, can also be administered by or as part of a sustained-release system. Sustained release compositions include, but are not limited to, semipermeable polymer matrices in the form of shaped articles, which include, but are not limited to, films or microcapsules. Sustained-release matrices include biocompatible materials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: pp. 267-277 (1981); Langer, Chem. .Tech., 12: pp. 98-105 (1982)), ethyl vinyl acetate (Langer et al., supra) or poly-D-(-)-3-hydroxybutyrate (EP 133,988), polypropylene Lactide (polylactic acid) (US Patent No. 3,773,919; EP 58,481), polyglycolide (polymer of glycolic acid), polylactide-glycolide copolymer (copolymer of lactic acid and glycolic acid) polyanhydride, Copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, pp. 547-556 (1983)), poly(ortho)esters, polypeptides, hyaluronic acid Acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipid, polysaccharide, nucleic acid, polyamino acid, amino acid (such as phenylalanine, tyrosine, isoleucine), polynucleotide, polyethylene propylene, Polyvinylpyrrolidone and silicone. Sustained release compositions also include liposome-encapsulated compounds. Liposomes containing this compound are prepared by the following methods known per se: DE 3,218,121; Eppstein et al., Proc. Natl. Acad. Sci. U.S.A., 77: pp. 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Patent No. 4,619,794; EP 143,949; U.S. Patent No. 5,021,234; US Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. All cited literature and patents are incorporated herein by reference.

Liposome-encapsulated GH (e.g., hGH) polypeptides can be prepared, for example, by methods described in the following literature and patents: DE 3,218,121; Eppstein et al., Proc.Natl.Acad.Sci.USA, 82: pp. 3688- 3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; No.; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. The composition and size of liposomes are well known or can be readily determined experimentally by those skilled in the art. Some examples of liposomes are described in, for example, the following literature and patents: Park JW et al., Proc. OF LIPOSOMES (1998); Drummond DC et al. Liposomal drug delivery systems forcancer therapy in Teicher B(ed): C _ANCER D _RUG D _{ISCOVERY AND} D _EVELOPMENT (2002); Park JW et al., Clin. Cancer Res. 8: pp. 1172-1181 (2002); Nielsen UB et al., Biochim. Biophys. Acta 1591(1-3): pp. 109-118 (2002); Mamot C et al., Cancer Res. 63: pp. 3154-3161 (2003). All cited literature and patents are incorporated herein by reference.

The dosage administered to a patient in the context of the present invention should be sufficient to elicit a beneficial response in the patient over time. Generally, the pharmaceutically effective total amount of parenterally administered GH (e.g., hGH) polypeptides of the present invention per dose ranges from about 0.01 μg/kg/day to about 100 μg/kg, or about 0.05 μg/kg based on patient body weight. mg/kg to about 1 mg/kg, however this should depend on the therapy. Dosing frequency will also depend on the therapy and may be more or less frequent than commercially available GH (eg, hGH) polypeptide products approved for use in humans. In general, PEGylated GH (eg, hGH) polypeptides of the invention can be administered by any of the routes of administration described above. In some embodiments, the invention provides a composition comprising any of the GH (eg, hGH) polypeptides described herein in a pharmaceutical composition described herein that is sufficiently stable for storage and administration regimens. Methods of testing stability are known in the art.

XV. Therapeutic Uses of GH (e.g., hGH) Polypeptides of the Invention

The GH (eg, hGH) polypeptides of the invention are useful in the treatment of a variety of disorders.

GH (eg, hGH) agonist polypeptides of the invention are useful, for example, in the treatment of growth defects, immune disorders, and in promoting cardiac function. Individuals with growth defects include, for example, individuals with Turner's Syndrome, GH-deficient individuals (including children), whose normal growth curve develops growth approximately 2 to 3 years before growth plate closure Slow or delayed children (sometimes referred to as "short normal children") and adult patients in whom the response of IGF-I to GH has been naturally reduced chemically (i.e., by glucocorticoid treatment) or by natural conditions (such as Middle) Individuals with blocked insulin-like growth factor (IGF-I) response to GH. The hGH polypeptides of the invention may be useful in the treatment of individuals suffering from pediatric growth hormone deficiency, idiopathic short stature, childhood-onset adult growth hormone deficiency, adult-onset adult growth hormone deficiency, or Secondary Growth Hormone Deficiency. Adults diagnosed with adult-onset growth hormone deficiency may have pituitary tumors or radiation. Conditions including, but not limited to, metabolic syndrome, head injury, obesity, osteoporosis, or depression may cause growth hormone deficiency-like symptoms in adults.

Agonist GH (e.g., hGH) variants can act to stimulate the immune system of a mammal by increasing its immune function, whether this increase is due to antibody modulation or cellular modulation, and whether the immune system responds to GH (e.g., hGH) Whether the host for polypeptide therapy is endogenous or transplanted from a donor to a host recipient receiving the GH (eg, hGH) polypeptide (eg, in a bone marrow transplant). "Immune dysregulation" includes any condition in which an individual's immune system has less than a normal immune system's antibody or cellular response to an antigen, including, but not limited to, those in which small spleens are immunoreduced due to drug (e.g., chemotherapy) treatment individual. Examples of individuals with immune disorders include, for example, elderly patients, individuals undergoing chemotherapy or radiation therapy, individuals recovering from serious illness or individuals about to undergo surgery, individuals with AIDS, individuals with congenital and acquired Patients with B cell deficiencies such as hypogammaglobulinemia, common variant agammaglobulinemia, and selective immunoglobulin deficiency (eg, IgA deficiency), infected with viruses such as rabies virus (which has a comparative patients with a shorter latency to the patient's immune response); and patients with a genetic disorder such as diGeorge syndrome.

The GH (e.g., hGH) antagonist polypeptides of the invention are useful in the treatment of gigantism and acromegaly, diabetes and complications resulting from diabetes (diabetic retinopathy, diabetic neuropathy), vascular eye diseases (e.g. including hyperplasia angiogenesis), kidney disease, and GH-responsive malignancies.

Vascular eye diseases include, for example, retinopathy (caused, for example, by anemia of prematurity or sickle cell anemia) and macular degeneration.

GH-responsive malignancies include, for example, Wilm's tumor, sarcomas (eg, bone-derived sarcomas), breast, colon, prostate, and thyroid cancers, and GH receptor mRNA-expressing Cancers of tissue (ie, placenta, thymus, brain, salivary glands, prostate, bone marrow, skeletal muscle, trachea, spinal cord, retina, lymph nodes and from Burkitt's lymphoma, colorectal cancer, lung cancer , lymphoid leukemia and melanoma).

For example, the GH (e.g., hGH) agonist polypeptides of the invention are useful in the treatment of chronic renal failure, growth impairment associated with chronic renal insufficiency (CRI), growth disorders associated with Turner's Syndrome short stature, pediatric Prader-Willi Syndrome (PWS), HIV patients with wasting or cachexia, children born small for gestational age (SGA), obesity, and osteoporosis.

The average amount of GH (eg, hGH) can vary and should be based inter alia on the recommendation and prescription of a qualified physician. The exact amount of GH (eg, hGH) is a matter of preference governed by the exact type of condition being treated, the condition of the patient being treated, and the other ingredients in the composition. The invention also provides for the administration of a therapeutically effective amount of another active agent. The amount to be administered can be readily determined by one of skill in the art based on the treatment with hGH.

The pharmaceutical compositions of the present invention can be manufactured in a conventional manner.

In some embodiments, the present invention provides a method of treatment comprising administering to an individual in need of treatment an effective amount of a hormonal composition comprising a compound covalently linked to at least one water-soluble polymer. Growth hormone (GH), wherein the covalent bond is an oxime bond. In some embodiments, the methods comprise administering GH (eg, hGH) to a subject (eg, a human). In some embodiments, the GH (eg, hGH) includes a non-naturally encoded amino acid, such as a carbonyl-containing non-naturally encoded amino acid. In some embodiments, the non-naturally encoded amino acid is a keto-containing amino acid, such as p-acetylphenylalanine. In some embodiments, the GH (eg, hGH) contains a non-naturally encoded amino acid, eg, acetylphenylalanine substituted at a position in the GH (eg, hGH) corresponding to amino acid 35 in SEQ ID NO: 2. The water soluble polymer can be PEG. Suitable PEGs include linear PEGs and branched PEGs; any PEG described herein can be used. In particular embodiments, the PEG is a linear PEG of about 0.1 to 100 kDa, or about 1 to 100 kDa, or about 10 to 50 kDa, or about 20 to 40 kDa, or about 30 kDa. In some embodiments, the pharmaceutical composition comprises a GH (e.g., hGH) linked to a 30 kDa PEG by an oxime linkage in the para-acetyl group at a position corresponding to amino acid 35 in SEQ ID NO:2 between phenylalanine and PEG. In some embodiments, the individual to be treated has pediatric growth hormone deficiency, idiopathic short stature, childhood-onset adult growth hormone deficiency, adult-onset adult growth hormone deficiency, or secondary growth hormone deficiency Hormone deficiency.

GH (eg, hGH) can be administered to an individual in any suitable form, route, dosage, frequency and duration, as described herein and as known in the art. In some embodiments, the present invention provides a method of treatment comprising administering to an individual in need of treatment an effective amount of a hormonal composition comprising a compound covalently linked to at least one water-soluble polymer. Growth hormone (GH), wherein the water soluble polymer is a linear polymer, and wherein the hormone composition is administered only about once every other day, or once every 3, 4, 5 or 6 days, and every Once a week, every 8, 9, 10, 11, 12, or 13 days, every two weeks, every 15, 16, 17, 18, 19, or 20 days, every three days Once a week, once every 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, monthly or less frequently than about monthly. It is understood that the frequency of dosing may vary according to the judgment of the individual or, more usually, the treating professional, and that any combination of frequencies may be used. In some embodiments, the GH composition is administered at a frequency of only about once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, the GH composition is administered at a frequency of only about once a week, once every two weeks, or once a month. In some embodiments, the GH composition is administered at a frequency of only about once per week. In some embodiments, the GH composition is administered at a frequency of only about once every two weeks. In some embodiments, the GH composition is administered at a frequency of only about once per month.

The invention also provides for the administration of a therapeutically effective amount of another active agent together with hGH of the invention. The amount to be administered can be readily determined by one of skill in the art based on the treatment with hGH.

The pharmaceutical compositions of the present invention can be manufactured in a conventional manner.

example

The following examples are offered to illustrate, but not limit, the claimed invention.

Example 1

This example describes one of many potential criteria sets for selecting preferred sites for incorporation of non-naturally encoded amino acids into hGH.

This example shows how to select preferred locations in hGH polypeptides for the introduction of non-naturally encoded amino acids. The crystal structure 3HHR consisting of hGH in complex with two molecules of the extracellular domain of the receptor (hGHbp) was used to determine preferred locations where one or more non-naturally encoded amino acids may be introduced. Potential changes in primary and secondary structure elements between crystal structure datasets were examined using other hGH structures (eg, 1AXI). Coordinates for these structures are available from the Protein Data Bank (PDB) (Bernstein et al. J. Mol. Biol. 1997, 112, p. 535) or via The Research Collaboratory for Structural Bioinformatics PDB at www.rcsb.org . Structural model 3HHR contains the complete mature 22 kDa hGH sequence except for residues 148 to 153 and the C-terminal F191 residue (which were ignored due to irregularities in the crystal structure). There are two disulfide bridges formed by C53 and C165 and C182 and C185. The sequence numbering used in this example corresponds to the amino acid sequence of mature hGH (22 kDa variant) shown in SEQ ID NO:2.

The following criteria were used to estimate individual hGH positions for the introduction of non-naturally encoded amino acids: Residue (a) should not interfere with the crystallographic structure based on 3HHR, 1AXI and 1HWG (hGH bound to hGHbp monomer or dimer). hGHbp binding by structural analysis, (b) should not be affected by alanine or homolog scanning mutations (Cunningham et al. Science (1989) 244:1081-1085 and Cunningham et al. Science (1989) 243:1330-1336), (c) should be surface exposed and exhibit minimal van der Waals or hydrogen bonding interactions with surrounding residues, (d) should be absent or variable in hGH variants (e.g. Tyr35, Lys38, Phe92, Lys140), (e) will result in a conservative change after substitution with a non-naturally encoded amino acid, (f) can be found in highly flexible regions (including, but not limited to, the CD loop) or structure In regions that are upwardly rigid (including, but not limited to, helix B). In addition, additional calculations were performed on the hGH molecule using the Cx program (Pintar et al. (2002) Bioinformatics, 18, p. 980) to estimate the degree of protrusion of each protein atom. Accordingly, in some embodiments, one or more non-naturally encoded amino acids are incorporated in hGH at one or more of, but not limited to, the following positions: before position 1 (i.e., at the N-terminus), Position 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98 ,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131 ,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156 . : 1 or the corresponding amino acid in SEQ ID NO: 3).

In some embodiments, at one or more of the following positions: positions 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186 and 187 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3), with one or more unnatural Coded amino acid substitutions.

In some embodiments, at one or more of the following positions: positions 29, 33, 35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 187 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3), substituted with one or more non-naturally encoded amino acids.

In some embodiments, at one or more of the following positions: positions 35, 88, 91, 92, 94, 95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145 and 155 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3), substituted with one or more non-naturally encoded amino acids.

In some embodiments, at one or more of the following positions: position 30, 74, 103 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3), with one or more Non-naturally encoded amino acid substitutions. In some embodiments, at one or more of the following positions: position 35, 92, 143, 145 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3), with one or More than one non-naturally encoded amino acid substitution.

In some embodiments, the non-naturally occurring amino acid is attached to the water soluble polymer at one or more of these positions including, but not limited to: Before position 1 (i.e., at the N-terminus) , position 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 2, or SEQ ID NO: the corresponding amino acid of 1 or 3). In some embodiments, a non-naturally occurring amino acid is attached to a water soluble polymer at one or more of these positions including, but not limited to: positions 29, 30, 33, 34, 35 ,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126 , 129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (SEQ ID NO: 2 , or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3).

In some embodiments, a non-naturally occurring amino acid is attached to a water soluble polymer at one or more of these positions including, but not limited to: positions 29, 33, 35, 37, 39 ,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136 , 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 186 and 187 (SEQ ID NO: 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3).

In some embodiments, a non-naturally occurring amino acid is attached to a water soluble polymer at one or more of these positions including, but not limited to: positions 35, 88, 91, 92, 94 , 95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145 and 155 (SEQ ID NO: 2, or SEQ ID NO: 1 or SEQ ID NO: 3 corresponding amino acids).

In some embodiments, a non-naturally occurring amino acid at one or more of these positions including, but not limited to, the following positions is attached to the water soluble polymer: positions 30, 74, 103 (SEQ ID NO : 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3). In some embodiments, the non-naturally occurring amino acid at one or more of said positions is linked to a water soluble polymer: positions 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO : 2, or the corresponding amino acid of SEQ ID NO: 1 or SEQ ID NO: 3). In some embodiments, the non-naturally occurring amino acid at one or more of said positions is linked to a water soluble polymer: positions 35, 92, 143, 145 (SEQ ID NO: 2, or SEQ ID NO: 1 or the corresponding amino acid of SEQ ID NO: 3).

Some of the sites used to generate hGH antagonists include: positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, 119, 120, 123, 127 or addition before position 1, or any combination thereof (SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 1, SEQ ID NO: 3 or any other GH sequence). These sites were selected using criteria (c)-(e) for agonist design. Antagonist design may also include site-directed modification of site I residues to increase binding affinity for hGHbp.

Example 2

This example details the cloning and expression of hGH polypeptides, including non-naturally encoded amino acids in E. coli. This example also describes a method for assessing the biological activity of modified hGH polypeptides.

Methods for cloning hGH and fragments thereof are described in detail in U.S. Patent Nos. 4,601,980; 4,604,359; 4,634,677; 4,658,021; 4,898,830; way incorporated into this article. The cDNAs encoding full-length hGH or the mature form of hGH lacking the N-terminal signal sequence are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

hGH containing non-naturally encoded amino acids was expressed using an introduced translation system comprising an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). O-RSs preferentially aminoacylate O-tRNAs with non-naturally encoded amino acids. The translation system, in turn, inserts the non-naturally encoded amino acid into hGH in response to the encoded selector codon.

Table 2: O-RS and O-tRNA sequences.

SEQ ID NO: 4 Tyr Methanococcus jannaschii (M.jannaschii) mtRNACUA tRNA SEQ ID NO: 5 HLAD03; optimized amber suppressor tRNA tRNA SEQ ID NO: 6 HL325A; optimized AGGA frameshift suppressor tRNA tRNA SEQ ID NO: 7 Aminoacyl tRNA synthetase p-Az-PheRS for incorporation of p-azido-L-phenylalanine (6) RS SEQ ID NO: 8 Aminoacyl tRNA synthetase p-BpaRS for incorporation of p-benzoyl-L-phenylalanine (1) RS SEQ ID NO: 9 Aminoacyl tRNA synthetase propargyl-PheRS for incorporation of propargyl-phenylalanine RS SEQ ID NO: 10 Aminoacyl tRNA synthetase propargyl-PheRS for incorporation of propargyl-phenylalanine RS SEQ ID NO: 11 Aminoacyl tRNA synthetase propargyl-PheRS for incorporation of propargyl-phenylalanine RS SEQ ID NO: 12 Aminoacyl tRNA synthetase p-Az-PheRS for incorporation of p-azido-phenylalanine (1) RS SEQ ID NO: 13 Aminoacyl tRNA synthetase p-Az-PheRS for incorporation of p-azido-phenylalanine (3) RS SEQ ID NO: 14 Aminoacyl tRNA synthetase p-Az-PheRS for incorporation of p-azidophenylalanine (4) RS SEQ ID NO: 15 Aminoacyl tRNA synthetase p-Az-PheRS for incorporation of p-azido-phenylalanine (2) RS

SEQ ID NO: 16 Aminoacyl tRNA synthetase (LW1) for incorporation of p-acetyl-phenylalanine RS SEQ ID NO: 17 Aminoacyl tRNA synthetase (LW5) for incorporation of p-acetyl-phenylalanine RS SEQ ID NO: 18 Aminoacyl tRNA synthetase (LW6) for incorporation of p-acetyl-phenylalanine RS SEQ ID NO: 19 Aminoacyl tRNA synthetase for incorporation of p-azido-phenylalanine (AzPheRS-5) RS SEQ ID NO: 20 Aminoacyl tRNA synthetase for incorporation of p-azido-phenylalanine (AzPheRS-6) RS

Transformation of Escherichia coli (E. coli) with a plasmid containing a modified hGH gene and an orthogonal aminoacyl tRNA synthetase/tRNA pair specific for the desired non-naturally encoded amino acid to allow site-specification of the non-naturally encoded amino acid Sexually incorporated into hGH polypeptide. Transformed E. coli (grown at 37°C in media containing 0.01 to 100 mM of the specific non-naturally encoded amino acid) expressed the modified hGH with high fidelity and efficiency. His-tagged hGH containing non-naturally encoded amino acids is produced by E. coli host cells in the form of inclusion bodies or aggregates. The aggregates were solubilized and affinity purified in 6M guanidine HCl under denaturing conditions. Refolding was performed by overnight dialysis at 4°C in 50 mM TRIS-HCl, pH 8.0, 40 μM CuSO ₄ and 2% (w/v) Sarkosyl. The material was then dialyzed against 20 mM TRIS-HCl, pH 8.0, 100 mM NaCl, 2 mM CaCl ₂ , followed by removal of the His tag. See Boissel et al., (1993) J. Bio. Chem. 268:15983-93. Methods for hGH purification are known to those skilled in the art and are confirmed by SDS-PAGE, Western blot analysis or electrospray ion trap mass spectrometry, among others.

Figure 6 is an SDS-PAGE of purified hGH polypeptide. His-tagged mutant hGH proteins were purified using ProBond nickel chelating resin (Invitrogen, Carlsbad, CA) via standard His-tagged protein purification procedures provided by the manufacturer, followed by anion exchange columns, and loaded onto gels. Lane 1 shows molecular weight markers and lane 2 represents N-His hGH without incorporation of unnatural amino acids. Lanes 3 to 10 contain N-His hGH mutants that contain the unnatural amino acid p-acetylphenylalanine at each of positions Y35, F92, Y111, G131, R134, K140, Y143, and K145, respectively.

To further assess the biological activity of modified hGH polypeptides, assays that measure downstream markers of the interaction of hGH with its receptors are used. Interaction of hGH with its endogenously produced receptor leads to tyrosine phosphorylation of the signal transducer and activator of transcription family member (STAT5) in the human IM-9 lymphoid cell line. Two forms of STAT5, STAT5A and STAT5B, were identified from the IM-9 cDNA library. See, eg, Silva et al., Mol. Endocrinol. (1996) 10(5): pp. 508-518. The human growth hormone receptor on IM-9 cells is selective for human growth hormone since neither rat growth hormone nor human prolactin resulted in detectable STAT5 phosphorylation. Importantly, rat GHR (L43R) extracellular domain and G120R with hGH efficiently competed for hGH-stimulated pSTAT5 phosphorylation.

IM-9 cells are stimulated with hGH polypeptides of the present invention. Human IM-9 lymphocytes were purchased from ATCC (Manassas, VA) and incubated with sodium pyruvate, penicillin, streptomycin (Invitrogen, Carlsbad, San Diego) and 10% heat-inactivated fetal calf serum (Hyclone , Logan, UT) grown in RPMI 1640. IM-9 cells were starved overnight in assay medium (RPMI without phenol red, 10 mM Hepes, 1% heat-inactivated charcoal/dextran-treated FBS, sodium pyruvate, penicillin, and streptomycin), This was followed by stimulation with a 12-point dose range of hGH polypeptide for 10 minutes at 37°C. Stimulated cells were fixed with 1% formaldehyde and then permeabilized with 90% ice-cold methanol for 1 hour on ice. STAT5 phosphorylation levels were detected by intracellular staining with a primary phospho-STAT5 antibody (Cell Signaling Technology, Beverly, MA) for 30 min at room temperature, followed by staining with a PE-conjugated secondary antibody. Samples were collected using FACSArray, and the collected data were analyzed with Flowjo software (Tree Star Inc., Ashland, OR). EC50 values were obtained from dose response curves plotted as mean fluorescence intensity (MFI) versus protein concentration using SigmaPlot.

Table 3 below summarizes the IM-9 data generated for the mutant hGH polypeptides. The human IM-9 cells were used to test various hGH polypeptides with unnatural amino acid substitutions at different positions. Specifically, Figure 7, panel A shows IM-9 data for a His-tagged hGH polypeptide, and Figure 7, panel B shows the IM-9 data for a His-tagged hGH comprising the unnatural amino acid p-acetylphenylalanine substituted for Y143. IM-9 data. The same assay was used to assess the biological activity of PEGylated hGH polypeptides comprising unnatural amino acids.

Table 3LE3 GH _EC50 (nM) GH _EC50 (nM) WHO WT 0.4±0.1(n=8) G120R >200,000 N-6His WT 0.6±0.3(n=3) G120pAF >200,000 Rat GH WT >200,000 G131pAF 0.8±0.5(n=3) wxya 0.7±0.2(n=4) P133pAF 1.0 wxya 0.9 R134pAF 0.9±0.3(n=4) wxya 2.0±0.6(n=2) T135pAF 0.9 wxya 0.8±0.4(n=9) G136pAF 1.4 wxya 0.7 F139pAF 3.3 wxya 16.7±1.0(n=2) K140pAF 2.7±0.9(n=2) N99pAF 8.5 Y143pAF 0.8±0.3(n=3) wxya 130,000 K145pAF 0.6±0.2(n=3)

wxya 1.0 A155pAF 1.3

Example 3

This example details the introduction of a carbonyl-containing amino acid and subsequent reaction with an aminooxy-containing PEG.

This example demonstrates a method for producing hGH polypeptides incorporating a keto-containing non-naturally encoded amino acid, which is subsequently reacted with an aminooxy-containing PEG of approximately 5,000 molecular weight. Residues 35, 88, 91, 92, 94, 95, 99, 101, 103, 111, 120, 131, 133, 134, 135, 136, 139, 140, 143 determined according to the criteria of Example 1 (hGH) Each of , 145 and 155 is independently substituted with a non-naturally encoded amino acid having the following structure:

The sequence for site-specifically incorporating p-acetylphenylalanine into hGH is SEQ ID NO: 2 (hGH)

Tyr and SEQ ID NO: 4 (muttRNA, M. jannaschii mtRNACUA) and 16, 17 or 18 (TyrRS LW1, 5 or 6) (described in Example 2 above).

Once modified, hGH polypeptide variants comprising carbonyl-containing amino acids are reacted with aminooxy-containing PEG derivatives of the form:

R-PEG(N)-O-(CH ₂ ) _n -O-NH ₂

wherein R is methyl, n is 3 and N is a molecular weight of about 5,000. Dissolve in 25mM MES (Sigma Chemical, St.Louis, MO) pH 6.0, 25mM Hepes (Sigma Chemical, St.Louis, MO) pH 7.0 or 10mM sodium acetate (Sigma Chemical, St.Louis, MO) with 10mg/mL Purified hGH containing p-acetylphenylalanine in MO) pH 4.5 was reacted with 10 to 100-fold excess of aminooxy-containing PEG, followed by stirring at room temperature for 10 to 16 hours (Jencks, W.J.Am.Chem .Soc.1959, 81, p. 475). PEG-hGH is then diluted into a suitable buffer for immediate purification and analysis.

Example 4

Conjugation to PEG comprising a hydroxylamine group attached to PEG via an amide bond.

A PEG reagent having the following structure was coupled to a keto-containing non-naturally encoded amino acid using the procedure described in Example 3:

R-PEG(N)-O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _n -O-NH ₂ .

where R = methyl, n = 4 and N is a molecular weight of approximately 20,000. Reaction conditions, purification conditions and analysis conditions were as described in Example 3.

Example 5

This example details the incorporation of two different non-naturally encoded amino acids into hGH polypeptides.

This example illustrates a method for producing hGH polypeptides incorporating non-naturally encoded amino acids comprising keto functionality at two positions in the following residues: E30, E74, Y103, K38, K41 , K140 and K145. hGH polypeptides were prepared as described in Examples 1 and 2, except that a selector codon was introduced at two different locations within the nucleic acid.

Example 6

This example details conjugation of hGH polypeptide to hydrazide-containing PEG and subsequent in situ reduction.

hGH polypeptides incorporating carbonyl-containing amino acids were prepared according to the procedures described in Examples 2 and 3. Once modified, a hydrazide-containing PEG with the following structure binds to the hGH polypeptide:

R-PEG(N)-O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _n -X-NH-NH ₂ ,

where R=methyl, n=2 and N=molecular weight of 10,000, and X is carbonyl (C=O). Purified hGH containing p-acetylphenylalanine was dissolved in 25 mM MES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical, St. Louis, MO) pH 7.0 or 10 mM sodium acetate (Sigma Chemical, St. Louis, MO) pH 4.5, react it with a 1 to 100-fold excess of hydrazide-containing PEG, and add the stock solution dissolved in H ₂ O 1M _NaCNBH3 (Sigma Chemical, St. Louis, MO) to final concentrations of 10 to 50 mM reduced the corresponding hydrazones in situ. React in the dark at 4°C to room temperature for 18 to 24 hours. Reactions were stopped by the addition of 1M Tris (Sigma Chemical, St. Louis, MO) (pH ~7.6) to a final Tris concentration of 50 mM, or the reaction was diluted into an appropriate buffer for immediate purification.

Example 7

This example details the introduction of an alkyne-containing amino acid into a hGH polypeptide and derivatization with mPEG-azide.

The following residues: 35, 88, 91, 92, 94, 95, 99, 101, 131, 133, 134, 135, 136, 140, 143, 145, and 155 are each the following non-naturally encoded amino acid (hGH; SEQ ID NO: 2) replace:

The sequence for site-specifically incorporating p-propargyltyrosine into hGH is SEQ ID NO: 2 (hGH),

TyrSEQ ID NO: 4 (muttRNA, M. jannaschii mtRNACUA) and 9, 10 or 11 (described in Example 2 above). Propargyltyrosine-containing hGH polypeptides were expressed in E. coli and purified using the conditions described in Example 3.

The purified hGH containing propargyl tyrosine was dissolved in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8) at 0.1 to 10 mg/mL, and a 10 to 1000-fold excess of azide-containing The base PEG was added to the reaction mixture. Catalytic amounts of CuSO ₄ and Cu filaments were then added to the reaction mixture. After incubating the mixture (including, but not limited to, about 4 hours at room temperature or 37°C, or overnight at 4°C), _H2O was added and the mixture was filtered through a dialysis membrane. This sample can be analyzed for addition by, including but not limited to, the procedures described in Example 3.

In this instance, the PEG will have the following structure:

R-PEG(N)-O-(CH ₂ ) ₂ -NH-C(O)(CH ₂ ) _n -N ₃ ,

wherein R is methyl, n is 4 and N is a molecular weight of 10,000.

Example 8

This example details the substitution of large hydrophobic amino acids with propargyl tyrosine in hGH polypeptides.

Phe, Trp or Tyr residues present in one of the following regions of hGH: 1-5 (N-terminal), 6-33 (helix A), 34-74 (region between helix A and helix B, A-B loop), 75-96 (helix B), 97-105 (region between helix B and helix C, B-C loop), 106-129 (helix C), 130-153 (region between helix C and helix D , C-D loop), 154-183 (helix D), 184-191 (C-terminus) (SEQ ID NO: 2), substituted as described in Example 7 with the following non-naturally encoded amino acids:

Once modified, PEG is linked to a hGH polypeptide variant comprising an alkynyl-containing amino acid. The PEG will have the following structure:

Me-PEG(N)-O-(CH ₂ ) ₂ -N ₃ ,

And the coupling procedure will follow the procedure in Example 7. This will result in hGH polypeptide variants comprising a non-naturally encoded amino acid that is approximately isosteric to one of the naturally occurring large hydrophobic amino acids and that is modified with a PEG derivative at various locations within the polypeptide.

Example 9

This example details the generation of hGH polypeptide homodimers, heterodimers, homomultimers or heteromultimers separated by one or more PEG linkers.

The alkyne-containing hGH polypeptide variants produced in Example 7 were reacted with bifunctional PEG derivatives of the form:

N ₃ -(CH ₂ ) _n -C(O)-NH-(CH ₂ ) ₂ -O-PEG(N)-O-(CH ₂ ) ₂ -NH-C(O)-(CH ₂ ) _n - N ₃ ,

wherein n is 4 and PEG has an average molecular weight of about 5,000 to produce a corresponding hGH polypeptide homodimer in which two hGH molecules are physically separated by PEG. In a similar manner, hGH polypeptides can be coupled with one or more other polypeptides to form heterodimers, homomultimers or heteromultimers. Coupling, purification and analysis will be performed as in Examples 7 and 3.

Example 10

This example details the coupling of carbohydrate moieties to hGH polypeptides.

One of the following residues: 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95 ,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154 , 155, 156, 159, 183, 186, and 187 (hGH, SEQ ID NO: 2), substituted as described in Example 3 with the following non-naturally encoded amino acids:

Once modified, the hGH polypeptide variant comprising a carbonyl-containing amino acid is reacted with a beta-linked aminooxy analog of N-acetylglucosamine (GlcNAc). hGH polypeptide variants (10 mg/mL) and aminooxysaccharides (21 mM) were mixed in aqueous 100 mM sodium acetate buffer (pH 5.5) and incubated at 37°C for 7 to 26 hours. By adding carbohydrate-conjugated hGH polypeptide (5 mg/mL) with UDP-galactose (16 mM) and β-1,4-galactosyltransferase (galacytosyltransferase) (0.4 units/mL) in 150 mM HEPES buffer at ambient temperature solution (pH 7.4) for 48 hours to enzymatically couple the second carbohydrate to the first carbohydrate (Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11

This example details the generation of PEGylated hGH polypeptide antagonists.

One of the following residues: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, 119, 120 , 123, or 127 (hGH, SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 1 or 3), substituted as described in Example 3 with the following non-naturally encoded amino acids.

Once modified, hGH polypeptide variants comprising carbonyl-containing amino acids will be reacted with aminooxy-containing PEG derivatives of the form:

R-PEG(N)-O-(CH ₂ ) _n -O-NH ₂ ,

wherein R is methyl, n is 4 and N is a molecular weight of 20,000 to generate hGH polypeptide antagonists comprising non-naturally encoded amino acids modified with PEG derivatives at a single site within the polypeptide. Coupling, purification and analysis were performed as in Example 3.

Example 12

Production of homodimers, heterodimers, homomultimers or heteromultimers wherein the hGH molecule is a directly linked hGH polypeptide

A hGH polypeptide variant comprising an alkynyl-containing amino acid can be directly coupled to another hGH polypeptide variant comprising an azide-containing amino acid, each comprising a non-naturally occurring amino acid at a position described in Example 10, including but not limited to. Coded amino acid substitutions. This will result in a corresponding hGH polypeptide homodimer in which the two hGH polypeptide variants are physically linked at the Site II binding interface. In a similar manner, hGH polypeptides can be coupled to one or more other polypeptides to form heterodimers, homomultimers, or heteromultimers. Coupling, purification and analysis were performed as in Examples 3, 6 and 7.

Example 13

PEG-OH+Br-(CH ₂ ) _n -C≡CR'→PEG-O-(CH ₂ ) _n -C≡CR'

A B

Polyalkylene glycol (P-OH) is reacted with alkyl halide (A) to form ether (B). In said compound, n is an integer from 1 to 9 and R' can be linear or branched, saturated or unsaturated C ₁ to C ₂₀ alkyl or heteroalkyl. R' can also be C _{to C} saturated or unsaturated cycloalkyl or cyclic heteroalkyl, substituted or unsubstituted aryl or heteroaryl, or substituted or unsubstituted alkaryl ( The alkyl group is a _C1 to _C20 saturated or unsaturated alkyl group) or a heteroalkaryl group. Typically, PEG-OH is polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) with a molecular weight of 800 to 40,000 Daltons (Da).

Example 14

mPEG-OH+Br-CH ₂ -C≡CH→mPEG-O-CH ₂ -C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g; 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). An 80 wt% solution of propargyl bromide in xylene (0.56 mL, 5 mmol, 50 equiv, Aldrich) and a catalytic amount of KI were then added to the solution, and the resulting mixture was heated to reflux for 2 hours. Water (1 mL) was then added and the solvent was removed under vacuum. To the residue was added CH ₂ Cl ₂ (25 mL) and the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and the volume was reduced to about 2 mL. This _{CH2Cl2 solution} was added dropwise to diethyl ether (150 mL). The resulting precipitate was collected, washed with several portions of cold diethyl ether and dried to give propargyl-O-PEG.

Example 15

mPEG-OH+Br-(CH ₂ ) ₃ -C≡CH→mPEG-O-(CH ₂ ) ₃ -C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g; 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Then 50 equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and a catalytic amount of KI were added to the mixture. The resulting mixture was heated to reflux for 16 hours. Water (1 mL) was then added and the solvent was removed under vacuum. To the residue was added CH ₂ Cl ₂ (25 mL) and the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and the volume was reduced to about 2 mL. This _{CH2Cl2 solution} was added dropwise to diethyl ether (150 mL). The resulting precipitate was collected, washed with several portions of cold diethyl ether and dried to give the corresponding alkyne. 5-Chloro-1-pentyne can be used in a similar reaction.

Example 16

(1) m-HOCH ₂ C ₆ H ₄ OH+NaOH+Br-CH ₂ -C≡CH→m-HOCH ₂ C ₆ H ₄ O-CH ₂ -C≡CH

(2) m-HOCH ₂ C ₆ H ₄ O-CH ₂ -C≡CH+MsCl+N(Et) ₃ →m-MsOCH ₂ C ₆ H ₄ O-CH ₂ -C≡CH

(3) m-MsOCH ₂ C ₆ H ₄ O-CH ₂ -C≡CH+LiBr→m-Br-CH ₂ C ₆ H ₄ O-CH ₂ -C≡CH

(4) mPEG-OH+m-Br-CH ₂ C ₆ H ₄ O-CH ₂ -C≡CH→mPEG-O-CH ₂ -C ₆ H ₄ O-CH ₂ -C=CH

To a solution of 3-hydroxybenzyl alcohol (2.4 g, 20 mmol) in THF (50 mL) and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g, 37.5 mmol), followed by propargyl bromide dissolved in 80 wt% solution in xylene (3.36 mL, 30 mmol). The reaction mixture was heated at reflux for 6 hours. To the mixture was added 10% citric acid (2.5 mL) and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3 x 15 mL), and the combined organic layers were washed with saturated NaCl solution (10 mL), dried over MgSO ₄ , and concentrated to give 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL, 20 mmol) were added to a solution of compound ₃ (2.0 g, 11.0 mmol) in _CH2Cl2 at 0 °C, and the reaction was placed in 16 hours in the refrigerator. Usual work-up gave the mesylate as a pale yellow oil. The oil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL), and LiBr (2.0 g, 23.0 mmol) was added. The reaction mixture was heated to reflux for 1 hour, then cooled to room temperature. Water (2.5 mL) was added to the mixture and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3 x 15 mL), and the combined organic layers were washed with saturated NaCl solution (10 mL), dried over anhydrous Na ₂ SO ₄ and concentrated to give the desired bromide.

mPEG-OH 20kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL), and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) was added over a period of several minutes with vigorous stirring, followed by the bromide obtained above (2.55 g, 11.4 mmol) and a catalytic amount of KI. The cooling bath was removed, and the resulting mixture was heated to reflux for 12 hours. Water (1.0 mL) was added to the mixture and the solvent was removed under vacuum. To the residue was added CH ₂ Cl ₂ (25 mL) and the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and the volume was reduced to about 2 mL. Addition to ether solution (150 mL) dropwise gave a white precipitate which was collected to give the PEG derivative.

Example 17

mPEG-NH ₂ +XC(O)-(CH ₂ ) _n -C≡CR'→mPEG-NH-C(O)-(CH ₂ ) _n -C≡CR'

Poly(ethylene glycol) polymers containing terminal alkyne groups can also be obtained by coupling poly(ethylene glycol) polymers containing terminal functional groups to reactive molecules containing alkynyl functional groups as indicated above. n is between 1 and 10. R' can be H or a small _C1 to _C4 alkyl group.

Example 18

(1) HO ₂ C-(CH ₂ ) ₂ -C≡CH+NHS+DCC→NHSO-C(O)-(CH ₂ ) ₂ -C≡CH

(2) mPEG-NH ₂ +NHSO-C(O)-(CH ₂ ) ₂ -C≡CH→mPEG-NH-C(O)-(CH ₂ ) ₂ -C=CH

4-Pentynoic acid (2.943 g, 3.0 mmol) was dissolved in _CH2Cl2 (25 _mL ). N-Hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) were added, and the solution was stirred overnight at room temperature. The resulting crude NHS ester 7 was used in the next reaction without further purification.

mPEG- _NH2 having a molecular weight of 5,000 Da (mPEG-NH2, 1 g, Sunbio) was dissolved in THF (50 mL), and the mixture was cooled to 4°C. NHS ester 7 (400 mg, 0.4 mmol) was added portionwise with vigorous stirring. The mixture was allowed to stir for 3 hours while warming to room temperature. Water (2 mL) was then added and the solvent was removed under vacuum. To the residue was added CH ₂ Cl ₂ (50 mL) and the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and the volume was reduced to about 2 mL. This _{CH2Cl2 solution} was added dropwise to ether (150 mL). The resulting precipitate was collected and dried under vacuum.

Example 19

This example presents the preparation of poly(ethylene glycol) methanesulfonyl ester, which may also be referred to as poly(ethylene glycol) methanesulfonate or methanesulfonic acid ester. The corresponding tosylate and halides can be prepared by analogous procedures.

mPEG-OH _{+ CH3SO2Cl +} N(Et) ₃ →mPEG-O- _SO2CH3 →mPEG- _N3

150 mL of mPEG-OH (MW = 3,400, 25 g, 10 mmol) in toluene was azeotropically distilled under nitrogen for 2 hours, and the solution was cooled to room temperature. 40 mL of anhydrous _CH2Cl2 and _2.1 mL of anhydrous triethylamine (15 mmol) were added to the solution. The solution was cooled in an ice bath, and 1.2 mL of distilled methanesulfonyl chloride (15 mmol) was added dropwise. The solution was stirred overnight at room temperature under a nitrogen atmosphere, and the reaction was quenched by adding 2 mL of absolute ethanol. The mixture was evaporated under vacuum to remove solvents (mainly solvents other than toluene), filtered, concentrated under vacuum again, and precipitated into 100 mL of diethyl ether. The filtrate was washed with several portions of cold diethyl ether and dried in vacuo to give the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml THF, and the solution was cooled to 4°C. Sodium azide (1.56 g, 24 mmol) was added to the cooled solution. The reaction was heated to reflux under nitrogen for 2 hours. Then the solvent was evaporated and the residue was diluted with _{CH2Cl2 (} 50 mL). The organic fraction was washed with NaCl solution and dried over anhydrous MgSO ₄ . The volume was reduced to 20 mL and the product was precipitated by adding to 150 mL of cold anhydrous ether.

Example 20

(1) N ₃ -C ₆ H ₄ -CO ₂ H→N ₃ -C ₆ H ₄ CH ₂ OH

(2) N ₃ -C ₆ H ₄ CH ₂ OH→Br-CH ₂ -C ₆ H ₄ -N ₃

(3) mPEG-OH+Br-CH ₂ -C ₆ H ₄ -N ₃ →mPEG-O-CH ₂ -C ₆ H ₄ -N ₃

4-Azidobenzyl alcohol can be made using the methods described in US Patent No. 5,998,595, which is incorporated herein by reference. Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL, 20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0 mmol) in _CH2Cl2 at ₀ °C , and the reaction was placed in the refrigerator for 16 hours. Usual work-up afforded the mesylate as a pale yellow oil. The oil (9.2 mmol) was dissolved in THF (20 mL), and LiBr (2.0 g, 23.0 mmol) was added. The reaction mixture was heated to reflux for 1 hour, then cooled to room temperature. Water (2.5 mL) was added to the mixture and the solvent was removed under vacuum. The residue was extracted with ethyl acetate (3 x 15 mL), and the combined organic layers were washed with saturated NaCl solution (10 mL), dried over anhydrous Na ₂ SO ₄ and concentrated to give the desired bromide.

mPEG-OH 20kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL), and bromide (3.32 g, 15 mmol) was added to the mixture along with a catalytic amount of KI. The resulting mixture was heated to reflux for 12 hours. Water (1.0 mL) was added to the mixture and the solvent was removed under vacuum. To the residue was added CH ₂ Cl ₂ (50 mL) and the organic layer was separated, dried over anhydrous Na ₂ SO ₄ , and the volume was reduced to about 2 mL. Addition dropwise to ether solution (150 mL) gave a precipitate which was collected to give mPEG- _O - _CH2 - _C6H4 - _N3 .

Example 21

NH ₂ -PEG-O-CH ₂ CH ₂ CO ₂ H+N ₃ -CH ₂ CH ₂ CO ₂ -NHS→N ₃ -CH ₂ CH ₂ -C(O)NH-PEG-O-CH ₂ CH ₂ CO ₂ h

_NH2 -PEG-O- _CH2CH2CO2H (molecular weight 3,400 Da, 2.0 g) _was dissolved in saturated aqueous _NaHCO3 (10 mL), and _the solution was cooled to 0 °C. 3-Azido-1-N-hydroxysuccinimidylpropionate (5 eq.) was added with vigorous stirring. After 3 hours, 20 ml _H2O were added and the mixture was stirred for a further 45 minutes at room temperature. The pH was adjusted to 3 with 0.5N _H2SO4 , and NaCl was added up to _a concentration of approximately 15% by weight. The reaction mixture was extracted with CH ₂ Cl ₂ (100 mL×3), dried over Na ₂ SO ₄ and concentrated. After precipitation with cold diethyl ether, the product was collected by filtration and dried under vacuum to give the ω-carboxy-azidated PEG derivative.

Example 22

mPEG-OMs+HC≡CLi→mPEG-O-CH ₂ -CH ₂ -C≡CH

To a solution of lithium acetylide in THF (4 eq) (prepared as known in the art and cooled to -78 °C) was added dropwise the solution of mPEG-OM dissolved in THF with vigorous stirring. After 3 hours, the reaction was allowed to warm to room temperature and quenched by adding 1 mL of butanol. Then 20 mL of _H2O was added, and the mixture was stirred for another 45 minutes at room temperature. The pH was adjusted to 3 with 0.5N _H2SO4 , and NaCl was added up to _a concentration of approximately 15% by weight. The reaction mixture was extracted with CH ₂ Cl ₂ (100 mL×3), dried over Na ₂ SO ₄ and concentrated. After precipitation with cold diethyl ether, the product was collected by filtration and dried under vacuum to give 1-(but-3-ynyloxy)-methoxypolyethylene glycol (mPEG).

Example 23

Azido- and ethynyl-containing amino acids were site-selectively incorporated into proteins using methods described in: L. Wang et al., (2001), Science 292:498-500; JW Chin et al. , Science 301: 964-7 (2003)); JW Chin et al., (2002), Journal of the American Chemical Society 124: 9026-9027; JW Chin, & P. G. Schultz, (2002), Chem Bio Chem 3 (11): 1135-1137; JW Chin et al., (2002), PNAS United States of America 99:11020-11024; and L. Wang, & PGSchultz, (2002), Chem.Comm., 1:1-11. Once the amino acid is incorporated, it is carried out with 0.01 mM protein in the presence of 2 mM PEG derivative, 1 mM CuSO ₄ and about 1 mg Cu silk in phosphate buffer (PB) at pH 8 at 37 °C. The cycloaddition reaction took 4 hours.

Example 24

This example describes the synthesis of p-acetyl-D,L-phenylalanine (pAF) and m-PEG-hydroxylamine derivatives.

Racemic was synthesized using the procedure previously described in Zhang, Z., Smith, B.A.C, Wang, L., Brock, A., Cho, C. & Schultz, P.G., Biochemistry, (2003) 42, 6735-6746. pAF.

The following procedure was performed to synthesize m-PEG-hydroxylamine derivatives. To (N-tert-butoxycarbonyl-aminooxy)acetic acid (0.382g, 2.0mmol) and 1,3-diisopropylcarbodiimide (0.16mL, 1.0mmol) in dichloromethane (DCM, 70mL) (which was stirred at room temperature for 1 hour) were added methoxy-polyethylene glycol amine (m-PEG-NH ₂ , 7.5 g, 0.25 mmol, Mt.30 K, purchased from BioVectra) and diiso Propylethylamine (0.1 mL, 0.5 mmol). The reaction product was stirred at room temperature for 48 hours, then concentrated to about 100 mL. The mixture was added dropwise to cold ether (800 mL). The tert-butoxycarbonyl protected product precipitated and was collected by filtration, washing with ether (3 x 100 mL). It was further purified by redissolving in DCM (100 mL) and precipitating twice in ether (800 mL). Drying in vacuo yielded 7.2 g (96%) of product, confirmed by NMR and Nihydrin tests.

Des-tert-butoxycarbonylation of the protected product obtained above (7.0 g) was carried out in 50% TFA/DCM (40 mL) at 0° C. for 1 hour followed by 1.5 hours at room temperature. After removing most of the TFA in vacuo, the TFA salt of the hydroxylamine derivative was converted to the HCl salt by adding 4N HCl in dioxane (1 mL) to the residue. The precipitate was dissolved in DCM (50 mL) and reprecipitated in ether (800 mL). The final product (6.8 g, 97%) was collected by filtration, washed with ether (3 x 100 mL), dried in vacuo and stored under nitrogen. Other PEG(5K, 20K) hydroxylamine derivatives were synthesized using the same procedure.

Example 25

This example describes expression and purification methods for hGH polypeptides comprising unnatural amino acids. Host cells have been transformed with orthogonal tRNA, orthogonal aminoacyl tRNA synthetase and hGH constructs.

A few stab cultures (stab) from frozen glycerol stocks of transformed DH10B(fis3) cells were first incubated at 37°C in 2 ml defined medium (supplemented with leucine, Glucose minimal medium containing isoleucine, trace metals and vitamins). When the _OD600 reached 2 to 5, 60 ul was transferred to 60 ml fresh defined medium with 100 μg/ml ampicillin and grown again at 37C to an _OD600 of 2 to 5. 50 ml of the culture was transferred to 2 liters of defined medium with 100 μg/ml ampicillin in a 5 liter fermenter (Sartorius BBI). The fermenter pH was controlled at pH 6.9 with potassium carbonate, the temperature was controlled at 37° C., the air flow rate was 51 pm, and the polyalkylene defoamer KFO F1 19 (Lubrizol) was used for foam control. The speed of the stirrer was automatically adjusted to maintain dissolved oxygen levels greater than 30%, and if the speed of the stirrer was at its maximum, the air sparge was supplemented with pure oxygen. After 8 h at 37 °C, cultures were fed with 50-fold concentration of defined medium at exponentially increasing rates to maintain a specific growth rate of 0.15 h ⁻¹ . When the _OD600 reached approximately 100, the racemic mixture of p-acetylphenylalanine was added to a final concentration of 3.3 mM and the temperature was lowered to 28°C. After 0.75 hours, isopropyl-bD-thiogalactopyranoside was added to a final concentration of 0.25 mM. Cells were grown for an additional 8 hours at 28°C, pelleted, and frozen at -80°C until further processing.

His-tagged mutant hGH proteins were purified using ProBond nickel chelating resin (Invitrogen, Carlsbad, CA) via standard His-tagged protein purification procedures provided by Invitrogen's instruction manual followed by anion exchange columns.

Purified hGH was concentrated to 8 mg/ml, and the buffer was changed to reaction buffer (20 mM sodium acetate, 150 mM NaCl, 1 mM EDTA, pH 4.0). MPEG-hydroxylamine powder was added to the hGH solution at a PEG:hGH molar ratio of 20:1. The reaction was performed at 28° C. with gentle shaking for 2 days. PEG-hGH was purified from unreacted PEG and hGH via anion exchange column.

The quality of each PEGylated mutant hGH was assessed by three assays, and then proceeded to animal experiments. The purity of PEG-hGH was checked by 4% to 12% acrylamide NuPAGE Bis-Tris gel electrophoresis with MES SDS running buffer under non-reducing conditions (Invitrogen). The gel was stained with Coomassie blue. The PEG-hGH band was greater than 95% pure according to densitometry scans. Each PEG-hGH was tested for endotoxin levels by kinetic LAL assay using a KTA ² kit from Charles River Laboratories (Wilmington, MA) and was less than 5 EU/dose. The biological activity of PEG-hGH was assessed using the IM-9 pSTAT5 bioassay (mentioned in Example 2) and had an _EC50 value of less than 15 nM.

Example 26

This example describes methods for assessing the purification and homogeneity of hGH polypeptides comprising unnatural amino acids.

Figure 8 is an SDS-PAGE of a hGH polypeptide comprising an unnatural amino acid at position 92. Lanes 3, 4 and 5 of the gel show hGH containing p-acetylphenylalanine at position 92 covalently linked to a 5 kDa, 20 kDa or 30 kDa PEG molecule. Other PEGylated hGH polypeptides comprising unnatural amino acids are shown in FIG. 11 . 5 μg of each PEG-hGH protein was loaded on each SDS-PAGE. Figure 11, panel A: color band 1, molecular weight marker; color band 2, WHO rhGH reference standard (2μg); color band 3 and 7, 30KPEG-F92pAF; color band 4, 30KPEG-Y35pAF; color band 5, 30KPEG- R134pAF; color band 6, 20KPEG-R134pAF; color band 8, WHO rhGH reference standard (20 μg). Figure 11, panel B: color band 9, molecular weight marker; color band 10, WHO rhGH reference standard (2 μg); color band 11, 30KPEG-F92pAF; color band 12, 30KPEG-K145pAF; color band 13, 30KPEG-Y143pAF; Color band 14, 30KPEG-G131pAF; color band 15, 30KPEG-F92pAF/G120R; color band 16, WHO rhGH reference standard (20μg). Figure 9 shows the biological activity of PEGylated hGH polypeptides (5kDa, 20kDa or 30kDa) in IM-9 cells; the method was performed as described in Example 2.

The purity of hGH-PEG conjugates can be assessed by proteolytic degradation (including, but not limited to, trypsin cleavage) followed by mass spectrometry analysis (Pepinsky RB., et al., J. Pharmcol. & Exp. Ther. 297(3) : 1059-66 (2001)). Methods for performing trypsinization are also described in European Pharmacopoeia (2002) 4th edition, p. 1938. Modifications were made to the method. Samples were dialyzed overnight against 50 mM TRIS-HCl (pH 7.5). The rhGH polypeptide was incubated with insulin (TPCK-treated trypsin, Worthington) at a mass ratio of 66:1 in a water bath at 37° C. for 4 hours. Samples were incubated on ice for several minutes to stop the digestion reaction and then maintained at 6°C during HPLC analysis. The digested sample (approximately 200 μg) was loaded onto a 25 x 0.46 cm Vydac C-8 column (5 μm particle size, 100 A pore size) in 0.1% trifluoroacetic acid and incubated at 30°C with a gradient of 0 to 80% Acetonitrile was eluted at a flow rate of 1 ml/min over 70 minutes. Elution of tryptic peptides was monitored by absorbance at 214 nm.

Figure 10, Panel A illustrates the primary structure of hGH with the trypsin cleavage site indicated and the arrow illustrating the unnatural amino acid substitution F92pAF (modified from Becker et al. Biotechnol Appl Biochem. (1988) 10(4):326-337 resulting figure). Panel B shows peptides produced from PEGylated hGH polypeptides containing non-naturally encoded amino acids (30K PEG His ₆ -F92pAF rhGH, labeled A), peptides produced from hGH polypeptides containing non-naturally encoded amino acids (His ₆ -F92pAF Overlaid tryptic maps of rhGH, labeled B) and the peptide produced by wild-type hGH (WHO rhGH, labeled C). Comparison of the tryptic maps of WHO rhGH and His ₆ -F92pAF rhGH showed only two peak shifts (peptide peak 1 and peptide peak 9), while the rest of the peaks were identical. These differences are caused by the addition of His ₆ to the N-terminus of expressed His ₆ -F92pAF rhGH, which results in a shift of peak 1; caused by acid substitution. Panel C shows a magnification of peak 9 from panel B. Comparison of the His ₆ -F92pAF and 30K PEG His ₆ -F92pAF rhGH tryptic maps showed that peak 9 disappeared after PEGylation of His ₆ -F92pAF rhGH, thus confirming that the modification was specific for peptide 9.

Example 27

This example describes a homodimer formed by two hGH polypeptides each comprising an unnatural amino acid.

Figure 12 joins together a His-tagged hGH polypeptide comprising a p-acetylphenylalanine substitution at position 92 with a bifunctional linker having the functionality and reactivity for PEGylation of hGH as described in Example 25 The IM-9 assay results of homodimers of this modified polypeptide were compared.

Example 28

This example describes monomeric and dimeric hGH polypeptides that act as hGH antagonists.

The hGH mutant protein in which the G120R substitution was introduced into site II was able to bind a single hGH receptor but was unable to dimerize both receptors. The mutant protein may act as an hGH antagonist in vitro by occupying the receptor site without activating intracellular signaling pathways (Fuh, G. et al. Science 256:1677-1680 (1992)). Figure 13, panel A shows data from an IM-9 assay measuring phosphorylation of pSTAT5 by hGH with a G120R substitution. As shown in Figure 13, panel B, hGH polypeptides with unnatural amino acids incorporated at the same position (G120) result in molecules that can also act as hGH antagonists. Dimers of hGH antagonists shown in Figure 13, Panel B were constructed linked together with bifunctional linkers having the functional groups and reactivity described in Example 25 for PEGylation of hGH. Figure 14 shows that this dimer also lacks biological activity in the IM-9 assay.

Additional assays were performed to compare dimers of hGH polypeptides comprising G120pAF substitutions to G120pAF modified hGH polypeptides linked by PEG linkers. Monomers in a dose-responsive range and dimers linked by pEG linkers compete for WHO hGH-induced STAT5 phosphorylation. Surface receptor competition studies were also performed to show that monomers and dimers compete with GH for cell surface receptor binding on IM-9 and rat GHR(L43R)/BAF3 cells. The dimers act as more potent antagonists than the monomers.

Table 4 shows the data from the study.

Table 4 cell line IM-9 IM-9 Rat GHR(L43R)/BAF3 test Inhibition of pSTAT5 surface receptor competition surface receptor competition IC ₅₀ (nM) IC ₅₀ (nM) IC ₅₀ (nM) G120pAF monomer 3.3 8.4 3.1 (G120pAF) dimer, PEG linker 0.7 2.7 1.4

Example 29

This example details the measurement of hGH activity and the affinity of hGH polypeptides for the hGH receptor.

Cloning and purification of the rat GH receptor The extracellular domain of the rat GH receptor (GHR ECD, amino acids S29-T238) was cloned into pET20b vector (Novagen) between the Nde I and Hind III sites in frame with the C-terminal 6His tag between. A mutation of L43 to R was introduced to provide further access to the human GH receptor binding site (Souza et al., ProcNatl Acad Sci USA. (1995) 92(4):959-63). Recombinant proteins were produced in BL21(DE3) E. coli cells (Novagen) by induction with 0.4 mM IPTG for 4 to 5 hours at 30°C. After the cells were lysed, they were washed four times by resuspending in a dounce with 30 mL of 50 mM Tris (pH 7.6), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, and washed with Wash twice with the same buffer (without Triton X-100). Herein, inclusion bodies consisted of more than 95% GHR ECD, and were dissolved in 0.1M Tris (pH 11.5), 2M urea. By passing an aliquot of the inclusion body solution through a S100 (Sigma) gel filtration column (equilibrated with 50 mM Tris (pH 7.8), 1 M L-arginine, 3.7 mM cystamine, 6.5 mM cysteamine), Thus, refolding is completed. Fractions containing soluble protein were combined and dialyzed against 50 mM Tris, pH 7.6, 200 mM NaCl, 10% glycerol. Samples were centrifuged briefly to remove any precipitate and incubated with aliquots of Talon resin (Clontech) according to the manufacturer's instructions. After washing with 20 volumes of dialysis buffer supplemented with 5 mM imidazole, proteins were eluted with 120 mM imidazole in dialysis buffer. Finally, samples were dialyzed overnight against 50 mM Tris (pH 7.6), 30 mM NaCl, 1 mM EDTA, 10% glycerol, centrifuged briefly to remove any precipitate, adjusted to a final glycerol concentration of 20%, etc. at -80°C points and storage. Protein concentration was measured by OD(280) using the calculated extinction coefficient ε = 65,700 M ⁻¹ * μm ⁻¹ .

Bio core ^TM analysis of binding of GH to GHR

Approximately 600 to 800 RU of soluble GHR ECD were immobilized on a Biacore ^™ CM5 chip using standard amine coupling procedures as recommended by the manufacturer. Even if most of the receptors are inactivated by this technique, it was experimentally found that this degree of immobilization is sufficient to generate a maximally specific GH binding response of approximately 100 to 150 RU without significant changes in binding kinetics. See eg Cunningham et al. J Mol Biol. (1993) 234(3):554-63 and Wells JA. Proc Natl Acad Sci USA (1996) 93(1):1-6).

Various concentrations of wild-type mutant GH (0.1 to 300 nM) in HBS-EP buffer (Biacore ^™ , Pharmacia) were injected on the GHR surface at a flow rate of 40 μl/min for 4 to 5 minutes, and after injection Split monitoring for 15 min. The surface was regenerated by 15 s pulses of 4.5M _MgCl2 . Only minimal loss of binding affinity (1% to 5%) was observed after at least 100 regeneration cycles. Reference cells with unfixed receptors were used to subtract out any buffer volume effects and non-specific binding.

Kinetic binding data obtained from GH titration experiments were processed using BiaEvaluation 4.1 software (BIACORE ^™ ). The "bivalent analyte" combination model provides a satisfactory fit (chi ² values are generally below 3), consistent with the proposed sequential 1:2 (GH:GHR) dimerization (Wells JA. Proc Natl Acad Sci US A (1996) 93(1): 1-6). Equilibrium splitting constants (Kd) were calculated as individual ratio constants (k _off /k _on ).

Table 5 shows binding parameters from Biacore ^™ using rat GHR ECD (L43R) immobilized on a CM5 chip.

table 5 GH k _on ，× 10 ^-5 1/M*s k _off , × 10 ⁴ , 1/s _Kd , nM WHO WT 6.4 3.8 0.6 N-6His WT 9 5.6 0.6

Rat GH WT 0.33 83 250 N12pF 12.5 4.6 0.4 wxya 6.8 4.8 0.7 wxya 7.8 5.3 0.7 wxya 6.8 5.4 0.8 wxya 6.6 4.9 0.7 wxya 8.6 5.0 0.6 wxya 5.6 6.0 1.1 wxya 0.7 3.1 4.3 N99pAF 2.2 3.8 1.7 wxya about 0.06 about 6 >100 wxya 8.4 4.8 0.6 G120R 2.2 twenty two 10 G120pAF 1.1 twenty three 20 G131pAF 6.0 5.3 0.9 P133pAF 6.4 4.9 0.8 R134pAF 8.4 5.8 0.7 T135pAF 7.2 4.5 0.6 G136pAF 6.2 4.3 0.7 F139pAF 6.8 4.4 0.7 K140pAF 7.2 3.7 0.5 Y143pAF 7.8 6.7 0.9 K145pAF 6.4 5.0 0.8 A155pAF 5.8 4.4 0.8 F92pAF-5KD PEG 6.2 2.3 0.4 F92pAF-20KD PEG 1.7 1.8 1.1 F92pAF-30KD PEG 1.3 0.9 0.7 R134pAF-5KD PEG 6.8 2.7 0.4 R134pAF-30KD PEG 0.7 1.7 2.4 Y35pAF-30KD PEG 0.9 0.7 0.7 (G120pAF) dimer 0.4 1.5 3.4 (F92pAF) dimer 3.6 1.8 0.5

GHR stable cell line

IL-3-dependent mouse cell line (BAF3) was prepared in a conventional manner in RPMI 1640, sodium pyruvate, penicillin, streptomycin, 10% heat-inactivated fetal bovine serum, 50 μM 2-mercaptoethanol and as IL-3 source Passage of 10% WEHI-3 cell line conditioned medium. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% _CO2 .

The rat GHR(L43R) stable cell clone, 2E2-2B12-F4, was established using the BAF3 cell line. Briefly, 1 x ¹⁰ moderately confluent BAF3 cells were polarized with 15 μg of linearized pcDNA3.1 plasmid containing full-length rat GHR(L43R) cDNA. Transfected cells were allowed to recover for 48 hours before cloning by limiting dilution in media containing 800 μg/ml G418 and 5 nM WHO hGH. GHR expressing transfectants were identified by surface staining with an anti-human GHR antibody (R & D Systems, Minneapolis, MN) and analyzed on a FACS Array (BD Biosciences, San Diego, CA). Transfectants expressing good GHR grades were then screened for proliferative activity against WHO hGH in the BrdU proliferation assay (described below). Stably transfected rat GHR (L43R) cell clones were established from two additional rounds of repeated subcloning of the desired transfectants in the presence of 1.2 mg/ml G418 and 5 nM hGH with a constant profile of surface receptor expression and proliferative capacity. The cell clone thus established (2E2-2B12-F4) was maintained in a conventional manner in BAF3 medium (plus 1.2 mg/ml G418 in the absence of hGH). Proliferation via BrdU labeling

Serum-starved rat GHR (L43R) expressing BAF3 cell line (2E2-2B 12-F4) was plated in a 96-well plate at a density of 5×10 ⁴ cells/well. Cells were activated with a 12-point dose range of hGH protein and simultaneously labeled with 50 μM BrdU (Sigma, St. Louis, MO). After 48 hours of incubation, cells were fixed/permeabilized with 100 [mu]l of BD cytofix/cytoperm solution (BD Biosciences) for 30 minutes at room temperature. To expose the BrdU epitope, fixed/permeabilized cells were treated with 30 μg/well DNase (Sigma) for 1 hour at 37°C. Sample analysis on the FACS Array was achieved using fluorescent immunoassay staining with APC-conjugated anti-BrdU antibody (BD Biosciences).

Table 6 shows the biological activity of the PEG hGH mutants as outlined in the pSTAT5(IM-9) and BrdU proliferation assays. For comparison between assays, WHO hGH expression was consistent.

Table 6 hGH pSTAT5 EC ₅₀ (nM) Proliferation EC ₅₀ (nM) WHO WT 1.0 1.0 wxya 1.3 1.6±0.8(n=3) Y35pAF-30KPEG 10 5.4±2.8(n=4) Y35pAF-40KPEG 53.3 24.0+11.0(n=3) wxya 2.2±0.4(n=9) 1.4±0.7(n=4) F92pAF-5KPEG 5.1+0.4(n=3) ND F92n AF-20KPEG 10.5+0.8(n=3) ND F92pAF-30KPEG 8.8±1.2(n=8) 4.1±0.9(n=3) F92pAF/G120R >200,000 >200,000 F92pAF/G120R-30KPEG >200,000 >200,000 G131pAF 2.3±1.8(n=2) 2.1±1.1(n=3) G131pAF-30KPEG 23.8±1.7(n=2) 4.6±2.4(n=3) R134pAF 1.1±0.2(n=2) 1.7±0.3(n=3) R134pAF-20KPEG 5.3 ND R134pAF-30KPEG 11.3±1.1(n=2) 2.5±0.7(n=4) Y143pAF 1.6±0.1(n=2) 1.8±0.6(n=2) Y143pAF-30KPEG 12.3±0.9(n=2) 6.6±2.7(n=3) K145pAF 2.3±0.5(n=2) 3.0±1.4(n=2) K145pAF-30KPEG 20.6±9.8(n=2) 5.3±3.5(n=3)

Example 30

This example describes the method used to measure the in vitro and in vivo activity of PEGylated hGH.

cell binding assay

In the absence or presence of various concentrations (volume: 10 μl) of unlabeled GH, hGH or GM-CSF, and in the presence of ¹²⁵ I-GH (approximately 100,000 cpm or 1 ng), the two Aliquots of cells (3×10 ⁶ ) were incubated in PBS/1% BSA (100 μl) for 90 minutes (total volume: 120 μl). Cells were then resuspended and layered through 200 μl of ice-cold FCS in 350 μl plastic centrifuge tubes and centrifuged (1000 g; 1 min). The pellets were collected by cutting off the end of the tube, and the pellets and supernatant were counted independently in a gamma counter (Packard).

Specific binding (cpm) was determined as total binding in the absence of competitor (average of replicates) minus binding in the presence of 100-fold excess of unlabeled GH (non-specific binding) (cpm). Non-specific binding was measured for each cell type used. Experiments were performed on different days using the same preparation of ¹²⁵ I-GH and should show internal consistency. ^125I -GH demonstrates binding to cells producing GH receptors. The binding is limited in a dose-dependent manner by unlabeled native GH or hGH, but not by GM-CSF or other negative controls. The ability of hGH to compete for binding of native ¹²⁵ I-GH (similar to native GH) suggests that both forms are equally well recognized by the receptor.

In vivo studies of PEGylated hGH

PEG-hGH, unmodified hGH and buffer solution are administered to mice or rats. The results will show the superior activity and prolonged half-life of the PEGylated hGH of the present invention compared to unmodified hGH exhibited by significant body weight gain.

In vivo half-life measurements of bound and unbound hGH and its variants .

All animal experiments were performed in AAALAC-accredited facilities and under protocols approved by the Institutional Animal Care and Use Committee of St. Louis University. Rats were housed individually in cages in rooms with a 12-hour light/dark cycle. Animals were provided with certified Purina chow 5001 and unlimited water. For hypophysectomized rats, the drinking water additionally contained 5% glucose.

Pharmacokinetic Studies

The quality of the PEGylated mutant hGH was assessed by three assays, followed by animal experiments. The purity of PEG-hGH was checked by 4% to 12% acrylamide NuPAGE Bis-Tris gel electrophoresis with MES SDS running buffer under non-reducing conditions (Invitrogen, Carlsbad, CA). The gel was stained with Coomassie blue. The PEG-hGH band was greater than 95% pure according to densitometry scans. Each PEG-hGH was tested for endotoxin levels by kinetic LAL assay using a KTA ² kit from Charles River Laboratories (Wilmington, MA) and was less than 5 EU/dose. The biological activity of PEG-hGH was assessed using the IM-9 pSTAT5 bioassay (described in Example 2), and _EC50 values were confirmed to be less than 15 nM.

The pharmacokinetics of PEG-modified somatotropin compounds were compared to each other and to non-PEGylated somatotropin in male Sprague-Dawley rats (261 to 425 g) obtained from Charles River Laboratories. A catheter is surgically installed in the carotid artery for blood collection. Following successful catheterization, animals were assigned to treatment groups (3 to 6 per group) followed by dosing. Animals were dosed subcutaneously at 1 mg/kg of compound (0.41-0.55 ml/kg in dose volume). Blood samples were collected at various time points via an indwelling catheter and into EDTA-coated microcentrifuge tubes. Plasma was collected after centrifugation and stored at -80°C until analysis. Compound concentrations were measured using antibody sandwich growth hormone ELISA kits purchased from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX). Concentrations are calculated using standards corresponding to the analogs administered. Pharmacokinetic parameters were estimated using the modeling program WinNonlin (Pharsight, version 4.1). Interval-free analysis with linear-up/log-down trapezoidal integration was used and the concentration data were weighted uniformly.

Figure 15 shows the mean (+/-S.D.) plasma concentrations following a single subcutaneous dose in rats. Rats (n=3 to 4 per group) were provided with 1 mg/kg hGH wild-type protein (WHO hGH), His-tagged hGH polypeptide (his-hGH) or a non-native protein containing a covalently linked to 30kDa PEG at position 92 A single bolus dose of the His-tagged hGH polypeptide (30KPEG-pAF92(his)hGH) of the amino acid p-acetylphenylalanine. Plasma samples were collected at the indicated time intervals and assayed for the injected compound. 30KPEG-pAF92(his)hGH had a significantly extended cycle compared to control hGH.

Figure 16 shows the mean (+/-SD) plasma concentrations following a single subcutaneous dose in rats. Rats (n=3 to 6 per group) were given a single bolus dose of 1 mg/kg protein. A hGH polypeptide comprising the unnatural amino acid p-acetylphenylalanine covalently linked to a 30 kDa PEG at each of 6 different positions was compared to WHO hGH and (his)-hGH. Plasma samples were collected at the indicated time intervals and assayed for the injected compound. Table 7 shows the values of pharmacokinetic parameters for single-dose administration of the hGH polypeptides shown in FIG. 16 . Concentration versus time curves were estimated by intervalless analysis (Pharsight, version 4.1). Values shown are mean values (+/- standard deviation). Cmax: maximum concentration; terminal _t1/2 : terminal half-life; AUC _0->inf : area under the concentration-time curve extrapolated to infinity; MRT: mean residence time; Cl/f: apparent total plasma clearance; Vz /f: Apparent volume distributed during the final period.

Table 7 : Values of pharmacokinetic parameters for subcutaneous administration of a single 1 mg/kg bolus to normal male Sprague-Dawley rats.

Compound (n) parameter Cmax(ng/ml) end t _1/2 (h) AUC _0->inf (ngXhr/ml) MRT(h) Cl/f(ml/hr/kg) Vz/f(ml/kg)

WHO hGH(3) 529(±127) 0.53(±0.07) 759(±178) 1.29(±0.05) 1,368(±327) 1051(±279) (his)hGH(4) 680(±167) 0.61(±0.05) 1,033(±92) 1.30(±0.17) 974(±84) 853(±91) 30KPEG-pAF35(his)hGH(4) 1,885 (±1,011) 4.85(+0.80) 39,918 (±22,683) 19.16(±4.00) 35(±27) 268(±236) 30KPEG-pAF92(his)hGH(6) 663(±277) 4.51(+0.90) 10,539 (±6,639) 15.05(±2.07) 135(±90) 959(±833) 30KPEG-pAF131(his)hGH(5) 497(±187) 4.41(±0.27) 6,978 (±2,573) 14.28(±0.92) 161(±61) 1,039 (±449) 30KPEG-pAF134(his)hGH(3) 566(±204) 4.36(±0.33) 7,304 (±2,494) 12.15(±1.03) 151(±63) 931(±310) 30KPEG-pAF143(his)hGH(5) 803(±149) 6.02(±1-43) 17,494 (±3,654) 18.83(±1.59) 59(±11) 526(±213) 30KPEG-pAF145(his)hGH(5) 634(+256) 5.87(+0.09) 13,162 (±6,726) 17.82 (+0.56) 88(±29) 743(+252)

Drug efficacy research

Hypophysectomized male Sprague-Dawley rats were obtained from Charles River Laboratories. The pituitary gland is surgically removed at 3 to 4 weeks of age. Animals were allowed to acclimatize for a period of 3 weeks, during which time body weight was monitored. Animals with a body weight gain of 0 to 8 g over the 7 day period prior to study initiation were included and randomized to treatment groups. Rats were administered subcutaneously as bolus doses or daily doses. Rats were weighed, paralyzed, bled and dosed (when appropriate) sequentially daily throughout the study period. Blood was drawn from the orbital sinus using heparinized capillaries and placed in EDTA-coated microcentrifuge tubes. Plasma was isolated by centrifugation and stored at -80°C until analysis.

Figure 17 shows the mean (+/-S.D.) plasma concentrations following a single subcutaneous dose in hypophysectomized rats. Rats (n=5 to 7 per group) were given a single bolus dose of 2.1 mg/kg protein. Results are shown for hGH polypeptides comprising the unnatural amino acid p-acetylphenylalanine covalently linked to 30 kDa PEG at each of two different positions (positions 35, 92). Plasma samples were collected at the indicated time intervals and assayed for the injected compound.

The peptide IGF-1 is a member of the somatomodulin or insulin-like growth factor family. IGF-1 mediates multiple growth-promoting effects of growth hormone. IGF-1 concentrations were measured using a competition binding enzyme immunoassay kit against the provided rat/mouse IGF-1 standards (Diagnosic Systems Laboratories). Significant differences were determined by t-test using a two-tailed distribution (unpaired, equal variance). Figure 18, panel A shows the evaluation of compounds in hypophysectomized rats. Single or daily doses were given subcutaneously to rats (n=5 to 7 per group). Animals were weighed, anesthetized, bled and dosed (when appropriate) sequentially daily. Shows the effect on placebo treatment, wild-type hGH (hGH), His-tagged hGH ((his)hGH) and hGH polypeptide treatment comprising p-acetylphenylalanine covalently linked to 30kDa PEG at positions 35 and 92 weight results. Figure 18, Panel B - graph showing the effect on circulating plasma IGF-1 levels following single dose administration of a PEGylated hGH polypeptide comprising a non-naturally encoded amino acid. Lines represent standard deviation. In Figure 18, panel A, the body weight gain at day 9 of the 30KPEG-pAF35(his)hGH compound was statistically different (p<0.0005) from the 30KPEG-pAF92(his)hGH compound (where greater weight gain was observed ).

Figure 18, Panel C shows the evaluation of compounds in hypophysectomized rats. Rats (n=11 per group) were given either a bolus dose or a daily dose subcutaneously. Animals were weighed, anesthetized, bled and dosed (when appropriate) sequentially daily. Body weight results are shown for placebo treatment, wild-type hGH (hGH), and hGH polypeptides comprising the unnatural amino acid p-acetylphenylalanine covalently linked to a 30 kDa PEG at positions 92, 134, 145, 131 and 143. Figure 18, Panel D - Graph showing single dose administration of PEGylated hGH polypeptide comprising non-naturally encoded amino acids (positions 92, 134, 145, 131, 143) compared to placebo treatment and wild-type hGH effect on circulating plasma IGF-1 levels. Figure 18, panel E shows mean (+/- S.D.) plasma concentrations corresponding to PEGylated hGH polypeptides comprising non-naturally encoded amino acids (positions 92, 134, 145, 131, 143). Plasma samples were collected at the indicated time intervals and assayed for the injected compound. Lines represent standard deviation.

Example 31

Human clinical trial of safety and/or efficacy of PEGylated hGH comprising non-naturally encoded amino acids.

Objective To compare subcutaneously administered PEGylated recombinant human hGH comprising non-naturally encoded amino acids with one or more commercially available hGH products (including, but not limited to, Humatrope ^™ (Eli Lilly & Co.), Nutropin ^™ (Genentech ), Norditropin ^TM (Novo-Nordisk), Genotropin ^TM (Pfizer) and Saizen/Serostim ^TM (Serono)) safety and pharmacokinetics were compared.

Patients Eighteen healthy volunteers aged between 20 and 40 years and weighing between 60 and 90 kg were enrolled in the study. Subjects will not have any clinically significant abnormal laboratory values for blood or plasma chemistry and urology screening, HIV screening, and hepatitis B surface antigen. They should not have any of the following signs: hypertension; history of any primary hematologic disease; history of significant hepatic, renal, cardiovascular, gastrointestinal, genitourinary, metabolic, neurological disease; anemia or epilepsy (seizure known sensitivity to bacterial or mammalian-derived products, PEG, or human serum albumin; habitual heavy drinking of caffeinated beverages; participation in any other clinical trial or previous blood transfusion or donation within 30 days of starting the study have been exposed to hGH within 3 months of study initiation; have been ill within 7 days of study initiation; and had significant abnormalities on pre-study physical examination or clinical laboratory assessments within 14 days of study initiation. All subjects were evaluable for safety and all blood samples for pharmacokinetic analysis were collected as scheduled. All studies were conducted according to institutional ethics committee approval and patient consent.

Study Design This will be a Phase I, single center, open-label, randomized, two-stage crossover study in healthy male volunteers. Eighteen subjects were randomly assigned to two treatment sequence groups (9 subjects/group). Using equal doses of PEGylated hGH containing non-naturally encoded amino acids and selected commercial products, GH was injected subcutaneously in the upper thigh as a bolus over two separate dosing sessions. The dosage and frequency of administration of commercially available products should be as indicated on the package label. Additional dosing, frequency of dosing, or other desired parameters using commercially available products can be added to the study by including additional groups of subjects. Each dosing period was separated by a 14-day washout period. Subjects were confined to the study center for 12 hours before and 72 hours after each two dosing periods, but not between dosing periods. If there are additional dosing, frequency, or other parameters to be tested for PEGylated hGH, additional groups of subjects can be added. Various GH formulations approved for human use were available for this study. Humatrope ^™ (Eli Lilly & Co.), Nutropin ^™ (Genentech), Norditropin ^™ (Novo-Nordisk), Genotropin ^™ (Pfizer) and Saizen/Serostim ^™ (Serono) are commercially available GH products approved for human use. The experimental formulation of hGH was PEGylated hGH containing non-naturally encoded amino acids.

Blood sampling Serial blood was drawn by direct venipuncture before and after hGH administration. Venous blood samples (5 mL) for determination of serum GH concentrations were obtained approximately 30, 20, and 10 minutes before dosing (3 baseline samples) and at the following approximate times after dosing: 30 minutes and 1, 2, 5, 8, 12 , 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample was divided into two aliquots. All serum samples were stored at -20°C. Load serum samples on dry ice. Fasting clinical laboratory tests (hematology, serum chemistry and urinalysis) were performed prior to the initial dosing on Day 1 (immediately), on the morning of Day 4, prior to dosing on Day 16 (immediately) and on the morning of Day 19.

Bioanalytical Methods ELISA panel procedures (Diagnostic Svstems Laboratory [DSL], Webster TX) were used to determine serum GH concentrations.

Safety Assays Vital signs were recorded prior to each dose (Day 1 and Day 16) and 6, 24, 48, and 72 hours after each dose. Safety measures were based on the incidence and type of adverse events and changes from baseline in clinical laboratory tests. In addition, changes in vital sign measurements (including blood pressure) and physical examination results from before the study were assessed.

Data Analysis Post-dose serum concentrations were determined by subtracting the mean baseline GH concentration (determined by averaging GH levels from triplicate samples taken 30, 20, and 10 minutes prior to dosing) from each post-dose value. Concentration values were corrected for baseline GH concentrations prior to dosing. If the pre-dose serum GH concentration was below the quantified level of the assay, it was not included in the calculation of the mean. Pharmacokinetic parameters were determined from serum concentration data corrected for baseline GH concentrations before dosing. Pharmacokinetic parameters were calculated by model-independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of BIOAVL's software. The following pharmacokinetic parameters were determined: peak serum concentration (C _max ); time to peak serum concentration (t _max ); concentration-time from 0 to time of last blood sampling (AUC _0-72 ) calculated using trapezoidal integration rule Area under the curve; and Terminal elimination half-life (t _1/2 ) calculated via elimination rate constant. Elimination rate constants were estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time curve. The mean, standard deviation (SD) and coefficient of variation (CV) of the pharmacokinetic parameters were calculated for each treatment. Calculate the ratio of the mean value of the parameter (preserved formula/non-preserved formula).

Safety Results The incidence of adverse events was evenly distributed among the treatment groups. There were no clinically significant changes from baseline or pre-study clinical laboratory tests or blood pressure, and no significant changes from pre-study on physical examination results and vital sign measurements. The safety profiles of the two treatment groups should be expressed as similar.

Pharmacokinetic Results All 18 subjects were administered a single dose of one or more commercially available hGH products including, but not limited to, Humatrope ^™ (Eli Lilly & Co. ), Nutropin ^TM (Genentech), Norditropin ^TM (Novo-Nordisk), Genotropin ^TM (Pfizer), and Saizen/Serostim ^TM (Serono)) mean serum GH concentration-time profiles (uncorrected baseline GH content) compared with those containing unnatural The encoded amino acids were compared with PEGylated hGH. All subjects should have pre-dose baseline GH concentrations within the normal physiological range. Pharmacokinetic parameters were determined from serum data corrected for mean baseline predose GH concentrations, and _Cmax and _tmax were determined. The mean t _max of selected clinical comparators (Humatrope ^TM (Eli Lilly & Co.), Nutropin ^TM (Genentech), Norditropin ^TM (Novo-Nordisk), Genotropin ^TM (Pfizer) and Saizen/Serostim ^TM (Serono)) was significantly shorter Mean t _max for PEGylated hGH containing non-naturally encoded amino acids. The terminal half-life values of commercially available hGH products were significantly shorter compared to the terminal half-life of PEGylated hGH comprising non-naturally encoded amino acids.

Although this study was conducted in healthy male subjects, similar absorption profiles and safety profiles can be expected in other patient populations; such as male or female patients with cancer or chronic renal failure, pediatric renal failure patients, autologous Patients in stored procedures or scheduled for elective surgery. In conclusion, a single subcutaneously administered dose of PEGylated hGH comprising a non-naturally encoded amino acid would be safe and well tolerated by healthy male subjects. Based on the incidence of comparative adverse events, clinical laboratory values, vital signs and physical examination results, the safety profiles of commercially available forms of hGH and PEGylated hGH comprising non-naturally encoded amino acids will be equivalent. PEGylated hGH comprising non-naturally encoded amino acids potentially offers greater clinical utility to patients and healthcare providers.

Example 32

In the following examples, ^a hGH and PEG- ^a hGH are hGH and PEGylated hGH of SEQ ID NO: 2 with p-acetylphenylalanine at position 35, respectively, and in which PEG is passed through to Oxime linkage formed by acetylphenylalanine, where PEG is a linear 30kDa PEG.

STAT5 phosphorylation assay

Interaction of hGH with its receptor leads to tyrosine phosphorylation of Signal Transducer and Activator of Transcription Family Member (STAT5) in the human IM-9 lymphoid cell line. Concentrations of ^ahGH and PEG- ^αhGH were determined by activating IM-9 cells with increasing concentrations of ahGH and PEG-αhGH followed by intracellular immunofluorescence staining for ^phospho - ^STAT5 (requires 50% maximal STAT5 Phosphorylation (EC ₅₀ )). According to the World Health Organization (WHO) EC ₅₀ of hGH as 1.0, the relative EC ₅₀ of ^a hGH compared with WHO hGH was 1.1, and the relative EC ₅₀ of PEG- ^α hGH was 10.9.

Example 33 :

Proliferation test

PEG- ^α hGH activity was measured in vitro by means of cell lines that proliferated in response to growth hormone. An example of such a cell line is the rat GHR[L43R]/BAF3 cell clone called 2E2-2B12-F4, which was obtained by stably transfecting a small Cell line derived from murine BAF3 cells. The conversion of Leu-43 to Arg-43 in the rat receptor makes the rat GHR more "human-like" and thus enables efficient hGH binding. Bromodeoxyuridine (BrdU) labeling was employed to activate isolated rat GHR[L43R]/BAF3 cells to provide a high-resolution, radiation-free method for the quantification of growth hormone-induced proliferation. Setting the EC ₅₀ of WHO hGH as consistent, the EC ₅₀ of ^a hGH and PEG- ^α hGH compared with WHO hGH were 1.06±0.29 (n=11) and 5.37±1.23 (n=9), respectively.

Example 34.

Efficacy study of PEGylated hGH

Preclinical efficacy studies were conducted in a growth hormone deficient rat model. In this model, the pituitary gland is surgically removed at approximately 4 weeks of age. The body weight of these hypophysectomized rats was monitored postoperatively and animals showing a body weight gain of less than 7.5 g over the 7 day period prior to the start of the study were considered successfully hypophysectomized and randomized into treatment groups. Animals showing greater than 2.5 g body weight loss were considered unhealthy and excluded from the study. The study began approximately 3 weeks post-surgery.

Animals were dosed weekly (ie, on days 0 and 7) with placebo or increasing doses of PEG- ^α hGH. An additional treatment group took daily genotropin or a placebo. Body weight and blood samples were taken daily prior to dosing. All animals died on day 14. Figure 28 shows plasma IGF-1 levels throughout the study period. A robust, dose-dependent increase in IGF-1 was observed.

A dose-dependent increase in IGF-1 Cmax was observed after each dosing interval. Curve fitting of the Cmax values from the first dose estimated the minimum (Eo) and maximum (Emax) IGF-1 Cmax to be 136.7 and 1,136.2 ng/mL, respectively, and determined an _ED50 of 0.136 mg/kg (Table 8).

Plasma IGF-1 levels also showed a clear dose-dependent increase when expressed as AUC. For this parameter, the AUC was expressed as the AUC above the effective IGF-1 plasma level. Previous work showed that sustained IGF-1 concentrations of 34 ng/mL above baseline resulted in increased body weight in hypophysectomized rats. In the current study, the baseline IGF-1 level determined from the placebo group throughout the study was 128 ng/mL. An increase of 34 ng/mL above 128 ng/mL was estimated to be an effective IGF-1 level of 162 ng/mL. Therefore an AUC above 162 ng/mL is intact. By this analysis, a dose-dependent increase in AUC above the effective level was observed after each dosing interval. Curve fitting of the AUC values from the first administration estimated Eo and Emax IGF-1 AUC to be 0 and 5,029.8 ngXhr/mL, respectively, and the _ED50 of said parameters was determined to be 0.909 mg/kg (Table 8) .

The duration of induction of IGF-1 levels above the estimated effective level after the first dose is determined. At the higher dose, IGF-1 levels did not return to baseline by the second dose. In these cases, the calculated terminal slope was used to extrapolate IGF-1 concentrations to effective levels. From this estimate, a dose-dependent increase in the duration of IGF-1 concentrations above effective levels is evident. Curve fitting estimated the minimum and maximum durations as 0 and 8.84 days, respectively, and the _ED50 for these parameters was determined to be 0.173 mg/kg (Table 8).

Bone growth was used as an additional potency measure to calculate PEG- ^a hGH _ED50 . Tibias were harvested from each animal at the end of the study and the immersion fixed in 10% neutral buffered formalin prior to radiographing. The results are shown in Figure 29. There was a significant dose-dependent increase in tibia length in PEG- ^α hGH treated animals. In addition, there was a dose-saving effect compared to animals treated daily with Gincoin. The amount of genpronine administered over a 7-day interval was equivalent to 2.1 mg/kg of PEG- ^α hGH provided weekly (ie, Day 0 and Day 7). PEG- ^α hGH at 0.42 to 1.0 mg/kg showed induction of increased tibial length similar to that of genpronin. This represents a dose saving effect of greater than or equal to 50% for PEG- ^α hGH compared to genpronil.

PEG- ^α hGH also induced a dose-dependent increase in body weight (Fig. 30). Furthermore, a dose-sparing effect of PEG- ^α hGH over genpronil was evident, as was the induction of increased tibial length. Daily treatment with 0.3 mg/kg/day of Gintropine induced a 27.74 (+/- 7.92)% change in body weight starting at Day 0 and at Day 14. PEG- ^α hGH administered in equivalent amounts to GH (ie 2.1 mg/kg on day 0 and day 7) induced greater body weight gain compared to genknil. This dose of PEG- ^α hGH resulted in a 33.66 (+/-7.55)% change in body weight starting at day 0 and at day 14. The percent change in body weight induced by PEG- ^α hGH administered at the lower dose of 1.0 mg/kg on Days 0 and 7 induced a body weight change of 27.02 (+/- 3.97) % on Day 14. This resulted in a dose saving effect of approximately 50% for PEG- ^α hGH. Weekly administration of non-PEGylated GH at a dose of 2.1 mg/kg failed to induce body weight gain.

Curve fitting of percent body weight change on day 14 estimated the minimum and maximum percent body weight change (induced by PEG- ^α hGH) to be 7.17 and 50.65, respectively. From this curve, the _ED50 of said parameter was determined to be 1.097 mg/kg (Table 8).

Table 8. Results of efficacy studies using PEG- ^a hGH

Pharmacodynamic parameters (effects) Treatment Range of effect (Eo to Emax) _ED50 (mg/kg)

IGF-1 induction: IGF-1 Cmax After a single subcutaneous dose 136.7 to 1,136.2 mg/mL 0.136 IGF-1 AUG above effective level After a single subcutaneous dose 0 to 5,029.8ngXhr/mL 0.909 IGF-1 duration above effective levels After a single subcutaneous dose 0 to 8.84 days 0.173 tibial length Day 14, after 2 weekly subcutaneous doses 30.52 to 33.67mm 0.424 percent change in body weight Day 14, after 2 weekly subcutaneous doses 7.17 to 50.65% 1.097

The same plasma samples collected for IGF-1 concentration determinations were also used to assay PEG- ^α hGH concentrations. The ELISA assay used was specific for hGH, which showed a lower but still robust response to PEG- ^α hGH, and was not used to detect rat GH. In Figure 31 a PEG- ^α hGH plasma concentration-time profile is shown. A dose-dependent increase in PEG- ^a hGHCmax occurred. The duration of PEG- ^α hGH in circulation and total PEG- ^α hGH AUC increased in a dose-dependent manner. Daily administered genpronine was undetectable in plasma, probably because of rapid clearance. The PEG- ^α hGH exposure results support the dose-dependent efficacy shown above.

The above study in hypophysectomized rats has shown the following:

1. PEG- ^a hGH increases IGF-1 plasma concentration in a dose-dependent manner. IGF-1 Cmax above effective levels, as well as IGF-1 AUC and duration, increased in a dose-dependent manner.

2. The length and weight of the tibia increase depending on the dose.

3. Demonstrate a dose savings of greater than or equal to 50% compared to daily doses of Gincoin.

4. PEG- ^α hGH showed dose-dependent increases in Cmax, AUC, and persistence in circulation, which correlated with the above pharmacodynamic effects.

Example 35

Pharmacokinetic Studies

Rat Pharmacokinetics

The plasma concentration versus time profile of PEG- ^α hGH administered at various doses in a rat disease model is shown in Example 34 above.

Primate Pharmacokinetics

The pharmacokinetic properties of the PEG- ^α hGH type (PEG- ^α (met)hGH) differing only in the molecule containing methionine in the N-terminal position were evaluated in macaques ( FIG. 32 ).

Single injections of PEG- ^α (met)hGH were administered at subcutaneous or intravenous doses of 0.75 mg/kg or 0.15 mg/kg, respectively. The apparent terminal half-life was 12.23 (+/- 1.72) hours compared to 7.05 (+/- 0.47) hours in rats with PEG- ^α hGH. The half-life for intravenous administration was 6.42 (+/- 0.51) hours. The dose-corrected AUC values were 362,443 and 312,440 ngXhrXkg/mL/mg for subcutaneous and intravenous administration, respectively. The biological activity by subcutaneous administration was calculated to be 116%.

Example 36

Rationale for proposed doses and regimens in clinical protocols

The optimal administration of PEGylated GH in humans was determined by the following studies in animals.

Rat Efficacy Studies—The dose savings observed in the preclinical efficacy studies (ie, comparable potency to Genadrol, but with a lower dose of PEG ^-α hGH) was used to estimate the dose required for biological activity in humans.

Pharmacokinetic assessment in rats and macaques - Estimation of pharmacokinetic parameters in humans using the allometric scale.

Single-dose acute safety studies in rats and macaques were used to assess exposure and determine the NOAEL (No Observed Adverse Effect Level).

One-Month Repeated Dose GLP Safety Study in Rats - Safety was assessed after one month of exposure to doses 2, 6 and 20 times higher than the maximum recommended human dose (MRHD). For pediatric GHD, MRHD = 0.36 mg/kg/week.

One-Month Repeated Dose GLP Safety Study in Rhesus Monkeys - Safety was assessed after one month of exposure to doses 2, 10 and 30 times higher than the maximum recommended human dose (MRHD). For pediatric GHD, MRHD = 0.36 mg/kg/week.

A six-month repeated-dose GLP safety study in rats. Safety was assessed after dosing exposures for six months such that exposures in rats were between 1 and 2 times and 10 times those observed at the MRHD in humans.

A six-month repeated-dose GLP safety study in rhesus monkeys. Safety was assessed after dosing exposures for six months such that exposures in rhesus monkeys were between 1 and 2 times and 10 times those observed at the MRHD in humans.

It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes made therefrom will readily occur to those skilled in the art, and will be included in the spirit and scope of the application and within the scope of the appended claims. All publications, patents, patent applications and/or other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent The application and/or other documents are hereby incorporated by reference for all purposes.

Table 9: Sequences cited.

SEQID# sequence name 1 The full-length amino acid sequence of hGH

2 The mature amino acid sequence of hGH (isoform 1) 3 20-kDa hGH variant in which hGH residues 32 to 46 are deleted twenty one Nucleotide sequence of full-length hGH twenty two Nucleotide sequence of mature hGH

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Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn

275 280 285

Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg

290 295 300

Leu

305

<210>12

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>12

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Ash Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>13

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>13

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>14

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>14

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Val His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>15

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>15

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Ser His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>16

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>16

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Cys His

145 150 155 160

Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>17

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>17

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Thr His

145 150 155 160

Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>18

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>18

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Gly His

145 150 155 160

Tyr Leu Gly Val Asp Val Ile Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>19

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>19

Met Asp Glu Phe Glu MetIle Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Arg Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Val Ile His

145 150 155 160

Tyr Asp Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>20

<211>306

<212>PRT

<213> Methanococcus jannazii

<400>20

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr

145 150 155 160

Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210>21

<211>654

<212>DNA

<213> Homo sapiens

<400>21

atggctacag gctcccggac gtccctgctc ctggcttttg gcctgctctg cctgccctgg 60

cttcaagagg gcagtgcctt cccaaccatt cccttatcca ggctttttga caacgctatg 120

ctccgcgccc atcgtctgca ccagctggcc tttgacacct accagagtt tgaagaagcc 180

tatatcccaa aggaacagaa gtattcattc ctgcagaacc cccagacctc cctctgtttc 240

tcagagtcta ttccgacacc ctccaacagg gaggaaacac aacagaaatc caacctagag 300

ctgctccgca tctccctgct gctcatccag tcgtggctgg agcccgtgca gttcctcagg 360

agtgtcttcg ccaacagcct ggtgtacggc gcctctgaca gcaacgtcta tgacctccta 420

aaggacctag aggaaggcat ccaaacgctg atggggaggc tggaagatgg cagcccccgg 480

actgggcaga tcttcaagca gacctacagc aagttcgaca caaactcaca caacgatgac 540

gcactactca agaactacgg gctgctctac tgcttcagga aggacatgga caaggtcgag 600

acattcctgc gcatcgtgca gtgccgctct gtggagggca gctgtggctt ctag 654

<210>22

<211>576

<212>DNA

<213> Homo sapiens

<400>22

ttcccaacca ttcccttatc caggcttttt gacaacgcta tgctccgcgc ccatcgtctg 60

caccagctgg cctttgacac ctaccaggag tttgaagaag cctatatccc aaaggaacag 120

aagtattcat tcctgcagaa cccccagacc tccctctgtt tctcagagtc tattccgaca 180

ccctccaaca gggaggaaac acaacagaaa tccaacctag agctgctccg catctccctg 240

ctgctcatcc agtcgtggct ggagcccgtg cagttcctca gaggtgtctt cgccaacagc 300

ctggtgtacg gcgcctctga cagcaacgtc tatgacctcc taaaggacct agaggaaggc 360

atccaaacgc tgatggggag gctggaagat ggcagccccc ggactgggca gatcttcaag 420

cagacctaca gcaagttcga cacaaactca cacaacgatg acgcactact caagaactac 480

gggctgctct actgcttcag gaaggacatg gacaaggtcg agacattcct gcgcatcgtg 540

cagtgccgct ctgtggaggg cagctgtggc ttctag 576

Claims (79)

1. hormonal composition, it comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.

2. hormonal composition according to claim 1, wherein said GH are human growth hormone (hGH).

3. hormonal composition according to claim 2, wherein said hGH comprise at least about 80% with the identical sequence of SEQ ID NO:2.

4. hormonal composition according to claim 2, wherein said hGH comprises the sequence of SEQ ID NO:2.

5. hormonal composition according to claim 1 and 2, wherein said GH comprise non-naturally encoded amino acid (NEAA).

6. hormonal composition according to claim 5, it is included in the oxime key between described NEAA and the described water-soluble polymers.

7. hormonal composition according to claim 6, wherein said NEAA comprises carbonyl.

8. hormonal composition according to claim 7, wherein said NEAA comprises ketone group.

9. hormonal composition according to claim 8, wherein said NEAA are to acetylphenylalanine.

10. hormonal composition according to claim 9 wherein replaces acetylphenylalanine with described in the position corresponding with the position 35 of SEQ ID NO:2.

11. hormonal composition according to claim 1 and 2, wherein said water-soluble polymers comprise polyoxyethylene glycol (PEG).

12. hormonal composition according to claim 11, wherein said PEG are linear PEG.

13. hormonal composition according to claim 12, wherein said PEG have the molecular weight between about 0.1kDa and the about 100kDa.

14. hormonal composition according to claim 12, wherein said PEG have the molecular weight between about 1kDa and the about 60kDa.

15. hormonal composition according to claim 12, wherein said PEG have the molecular weight between about 20kDa and the about 40kDa.

16. hormonal composition according to claim 12, wherein said PEG has the molecular weight of about 30kDa.

17. hormonal composition according to claim 11, wherein said PEG are branch PEG.

18. hormonal composition according to claim 17, wherein said PEG have the molecular weight between about 1kDa and the about 100kDa.

19. hormonal composition according to claim 17, wherein said PEG have the molecular weight between about 30kDa and the about 50kDa.

20. hormonal composition according to claim 17, wherein said PEG has the molecular weight of about 40kDa.

21. hormonal composition according to claim 7, wherein said water-soluble polymers comprises PEG.

22. hormonal composition according to claim 21, wherein said PEG are linear PEG.

23. hormonal composition according to claim 21, wherein said PEG have the molecular weight between about 0.1kDa and the about 100kDa.

24. hormonal composition according to claim 21, wherein said PEG have the molecular weight between about 1kDa and the about 60kDa.

25. hormonal composition according to claim 21, wherein said PEG have the molecular weight between about 20kDa and the about 40kDa.

26. hormonal composition according to claim 21, wherein said PEG has the molecular weight of about 30kDa.

27. hormonal composition according to claim 7, wherein said PEG are branch PEG.

28. hormonal composition according to claim 28, wherein PEG has the molecular weight between about 1kDa and the about 100kDa.

29. hormonal composition according to claim 28, wherein said PEG have the molecular weight between about 30kDa and the about 50kDa.

30. hormonal composition according to claim 28, wherein said PEG has the molecular weight of about 40kDa.

31. hormonal composition according to claim 10, wherein said water-soluble polymers are PEG.

32. hormonal composition according to claim 31, wherein said PEG are linear PEG.

33. hormonal composition according to claim 32, wherein said PEG have the molecular weight between about 0.1kDa and the about 100kDa.

34. hormonal composition according to claim 32, wherein said PEG have the molecular weight between about 1kDa and the about 60kDa.

35. hormonal composition according to claim 32, wherein said PEG have the molecular weight between about 20kDa and the about 40kDa.

36. hormonal composition according to claim 32, wherein said PEG has the molecular weight of about 30kDa.

37. hormonal composition according to claim 32, wherein said GH are hGH.

38. according to the described hormonal composition of claim 37, wherein said hGH comprises SEQ ID NO:2.

39. hormonal composition according to claim 1, it comprises the GH that is connected with a plurality of water-soluble polymerss by a plurality of covalent linkage, and at least one in the wherein said covalent linkage is the oxime key.

40. according to the described hormonal composition of claim 39, wherein said GH is human growth hormone (hGH).

41. according to the described hormonal composition of claim 40, wherein said hGH comprises at least about 80% sequence identical with SEQ IDNO:2.

42. hormonal composition according to claim 2, wherein said hGH comprises the sequence of SEQ ID NO:2.

43. according to claim 39 or 41 described hormonal compositions, wherein said GH comprises a plurality of NEAA.

44. hormonal composition, it comprises the hGH that is connected by the linear PEG of oxime key and at least one, and wherein said hGH comprises the aminoacid sequence of SEQ ID NO:2 and comprises the NEAA that at least one replaces in one or more positions that are selected from the group that is made up of following each residue position: residue 1-5,6-33,34-74,75-96,97-105,106-129,130-153,154-183 and 184-191.

45. according to the described hormonal composition of claim 44, wherein replace with described NEAA in one or more positions that are selected from the group that forms by following each residue position: before the position 1 (promptly, at the N end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191 and 192 (that is, at proteinic carboxyl terminales).

46., wherein replace with described NEAA: position 35,92,131,134,143 and 145 in one or more positions that are selected from the group that forms by following each residue position according to the described hormonal composition of claim 44.

47., wherein replace with described NEAA: position 30,35,74,92,103,143 and 145 in one or more positions that are selected from the group that forms by following each residue position according to the described hormonal composition of claim 44.

48., wherein replace with described NEAA: position 35,92,143 and 145 in one or more positions that are selected from the group that forms by following each residue position according to the described hormonal composition of claim 44.

49. according to the described hormonal composition of claim 44, it is included in the NEAA that replaces on the position 35.

50. according to claim 44,45,46,47,48 or 49 described hormonal compositions, it is included as the NEAA to acetylphenylalanine.

51. according to the described hormonal composition of claim 50, wherein said PEG has the molecular weight between about 0.1kDa and the about 100kDa.

52. according to the described hormonal composition of claim 50, wherein said PEG has the molecular weight between about 1kDa and the about 60kDa.

53. according to the described hormonal composition of claim 50, wherein said PEG has the molecular weight between about 20kDa and the about 40kDa.

54. according to the described hormonal composition of claim 50, wherein said linear PEG has the molecular weight of about 30kDa.

55. the method for the GH that a manufacturing is connected with water-soluble polymers by the oxime key, it comprises makes the GH that comprises the NEAA that contains carbonyl contact with the PEG oxyamine being suitable for forming under the condition of oxime key.

56. according to the described method of claim 55, wherein said NEAA contains ketone group.

57. according to the described method of claim 56, wherein said NEAA is to acetylphenylalanine.

58., wherein (for example, acetylphenylalanine is replaced with described corresponding to the position of the amino acid among the SEQ IDNO:2 35 in hGH) at described GH according to the described method of claim 57.

59. according to the described method of claim 55, wherein said PEG oxyamine is mono methoxy PEG (MPEG) oxyamine.

60. according to the described method of claim 59, wherein said MPEG oxyamine is linear.

61. according to the described method of claim 60, the molecular weight of wherein said PEG is about 20 to 40kDa.

62. according to the described method of claim 61, the molecular weight of wherein said PEG is about 30kDa.

63. according to the described method of claim 62, wherein said MPEG oxyamine is mono methoxy-PEG-2-amino oxygen base ethamine carbamate hydrochloride of linear 30kDa.

64. according to the described method of claim 55, it further comprises makes the GH that comprises NEAA by the following method, described method comprises: will

(i) coding GH nucleic acid (wherein said nucleic acid has been modified so that for incorporating into of described NEAA providing signal) and

(ii) described NEAA introduces in the organism, and wherein said organism can be incorporated described NEAA in the protein in response to the described signal of described (i) nucleic acid.

65. according to the described method of claim 55, wherein said condition comprises:

(i) described GH is mixed with MPEG to produce the MPEG-GH mixture, wherein the ratio of MPEG: GH is about 5 to 10,

(ii) the pH value is about 4 to 6; With

(iii) at room temperature with the soft stir about of described MPEG-GH mixture 10 to 50 hours.

66. according to the described method of claim 55, it further comprises the described GH of purifying.

67. according to the described method of claim 56, wherein the GH that obtains by described method is at least about 99% pure.

68. a medical composition, it comprises:

(i) hormonal composition, it comprises the tethelin that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key; With

(ii) pharmaceutically acceptable vehicle.

69. according to the described medical composition of claim 68, wherein said GH is hGH.

70. according to claim 68 or 69 described medical compositions, wherein said composition comprises pharmaceutically acceptable injection prescription liquid.

71. according to the described medical composition of claim 69, wherein said GH comprises NEAA.

72. according to the described medical composition of claim 71, wherein said water-soluble polymers comprises PEG.

73. according to the described medical composition of claim 71, wherein said PEG is linear PEG.

74. according to the described medical composition of claim 73, wherein said PEG is the linear PEG of about 30kDa, and described GH is corresponding to the position of the amino acid 35 among the SEQ ID NO:2 hGH through acetylphenylalanine is replaced, and wherein described to acetylphenylalanine and described PEG between the described oxime key of formation.

75. a methods of treatment, it comprises throws the individuality that gives the needs treatment with the hormonal composition of significant quantity, and described hormonal composition comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, and wherein said covalent linkage is the oxime key.

76. according to the described method of claim 75, wherein said individuality suffers from the illness that is selected from the group that is made up of following illness: the paediatrics growth hormone deficiency, special send out property of short and small stature, the Childhood outbreak grownup's growth hormone deficiency, the grownup's growth hormone deficiency or the Secondary cases growth hormone deficiency of Adulthood outbreak.

77. methods of treatment, it comprises throws the individuality that gives the needs treatment with the hormonal composition of significant quantity, described hormonal composition comprises the tethelin (GH) that is connected with at least one water-soluble polymers by covalent linkage, wherein said water-soluble polymers is a linear polymer, and wherein with weekly only once, whenever biweekly or mensal frequency throw and to give described hormonal composition.

78. a hormonal composition, it comprises GH, wherein has average serum transformation period at least about 12 hours as described GH when Mammals is given in subcutaneous throwing.

79. a hormonal composition, it comprises the GH that is connected with PEG by the oxime key, wherein when the average serum transformation period of described GH when Mammals is given in subcutaneous throwing be comprise no described PEG GH composition serum half-life at least about 7 times.

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