KR101184869B1 - Novel formulations of tumour-associated peptides binding to human leukocyte antigen hla class i or ii molecules for vaccines - Google Patents

Abstract

The present invention relates to novel formulations of tumor-associated peptides that bind to human Leukocyte Antigen (HLA) class I or II molecules as vaccines for use in immunotherapy. In particular, the present invention relates to formulations for cancer immunity, inter alia renal cancer, brain cancer, glioma, in particular glioblastoma cancer. The present invention also relates to vaccine compositions that induce anti-tumor immune responses.

Description

NOVEL FORMULATIONS OF TUMOUR-ASSOCIATED PEPTIDES BINDING TO HUMAN LEUKOCYTE ANTIGEN (HLA) CLASS I OR II MOLECULES FOR VACCINES}

The present invention relates to novel formulations of tumor-associated peptides that bind to histology Leukocyte Antigen (HLA) class I or II molecules as vaccines for use in immunotherapy. In particular, the present invention relates to formulations for immunotherapy of cancer, in particular kidney cancer. The present invention also relates to vaccine compositions that induce anti-tumor immune responses.

For the purposes of the present invention, all documents cited herein are incorporated by reference in their entirety.

Injectable compositions comprising a diluent consisting of a peptide in powder form for vaccines of renal cell carcinoma and sodium hydrogen carbonate are disclosed in WO 2007/028573. However, this formulation has several disadvantages. The main disadvantage is the low solubility which makes it very difficult to apply in any application such as clinical use.

To dissolve the powder in the composition, the peptide and the diluent to be dissolved must be shaken vigorously for 3 minutes in a vial, put into an ultrasonic homogenizer for 1 minute and then shaken for another minute. The lack of the necessary equipment makes it impossible for all physicians to apply this treatment to their work, whereby the stored mixture may lack the homogeneity required for effective use.

Another problem with this composition is related to the fact that the resulting solution can only be used for about 30 minutes, given the rate of dissolution of the active ingredient using a given concentration of sodium hydrogen carbonate.

In addition, the properties of the formulations change over time, making safe use almost impossible. After storing the disclosed formulations for 12 months at different temperatures of −20 ° C. to + 25 ° C., using an ultrasonic homogenizer for several minutes did not produce a clear solution.

Thus, for safe and effective formulations, particularly for anticancer vaccines, there is a need for novel peptide formulations with improved solubility and improved moisturization of lyophilisate. The present invention satisfies this need.

In one preferred embodiment the invention provides 2 to 18, preferably less than 15, more preferably less than 13, even more preferably 2 to 12 peptides and even more preferably 3 to 12 tumor associated peptides ; Mannitol and poloxamer 188; Or mannitol and tween 80®, wherein each peptide is 8 to 22 amino acids, preferably 9 to 16 amino acids in length, at 90% acetic acid. The solubility of the peptide is at least 2.7 mg / mL, the weight ratio of peptide: mannitol: poloxamer 188 is from 1: 5: 1.5 (inclusive) to 1: 8: 2.2 (inclusive), and peptide: mannitol: twin 80 (registered trademark) ) Weight ratio is from 1: 2: 1.5 (inclusive) to 1: 8: 2.2 (inclusive).

In another preferred aspect of the invention, the invention provides a pharmaceutical composition according to claim 1 comprising two or more peptides, wherein the peptide is SEQ ID NO: 1 to SEQ ID NO: 10, or SEQ ID NO: 12 to At least one peptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 13, or an amino acid sequence selected from the group consisting of SEQ ID NO: 23, or Variants or salts, and further comprising mannitol and poloxamer 188, wherein the weight ratio of peptide: mannitol: poloxamer 188 is from 1: 5: 1.5 (inclusive) to 1: 8: 2.2 (inclusive).

In another preferred aspect of the invention, the invention provides a pharmaceutical composition according to claim 1 comprising two or more peptides, wherein the peptide is SEQ ID NO: 1 to SEQ ID NO: 10, or SEQ ID NO: 12 To an amino acid sequence selected from the group consisting of SEQ ID NO: 23, wherein the composition comprises one or more peptides comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 13, or Variants or salts thereof, and further comprising mannitol and tween 80®, wherein the weight ratio of peptide: mannitol: twin 80® is from 1: 2: 1.5 (inclusive) to 1: 8 : 2.2, inclusive.

An advantage of the formulations according to the invention is that they are suitable for stable formulations containing strong hydrophobic peptides (eg IMA-CHI-001; SEQ ID NO: 23). Such specific peptides are, for example, very insoluble in water and very weakly soluble in 90% acetic acid (solubility of about 2.9 mg / mL). Surprisingly, it is now possible to formulate a peptide according to SEQ ID NO: 23 with any other peptide of another formulation used for application to the organism.

The present invention thus contemplates 2-18 peptides, preferably less than 15, more preferably less than 13, even more preferably, having a peptide length of 8 to 22 amino acids, preferably 9 to 16 amino acids. Formulations for formulating 2-12, even more preferably 3-12 peptides are disclosed provided that the peptide exhibits a solubility in 90% acetic acid of at least 2.7 mg / mL, preferably 2.9 mg / mL And also mannitol and poloxamer 188, wherein the weight ratio of peptide: mannitol: poloxamer 188 is from 1: 5: 1.5 (inclusive) to 1: 8: 2.2 (inclusive).

In a preferred embodiment of the pharmaceutical composition according to the invention, the weight ratio of peptide: mannitol: poloxamer 188 is from 1: 0: 2 (inclusive) to 1: 0: 2.2 (inclusive).

Another preferred embodiment of the present invention provides the above pharmaceutical composition comprising two or more peptides, wherein the peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 12 to SEQ ID NO: 23, provided that the composition is And one or more peptides or variants thereof comprising SEQ ID NO: 12 or SEQ ID NO: 13, further comprising mannitol and Tween 80®, wherein the peptide: Mannitol: Twin 80® The preferred weight ratio of is 1: 5: 0.5 (inclusive) to 1: 5: 2 (inclusive).

Preferably, the peptides of the present invention have a total length of 9 to 16 amino acids. This peptide may comprise a non-peptide bond.

In one preferred embodiment, the pharmaceutical composition comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and also described in any one of SEQ ID NO: 3 to SEQ ID NO: 10 It further comprises one or more peptides consisting of amino acid sequences.

In certain preferred embodiments, the pharmaceutical composition may further comprise a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, provided that these peptides each satisfy the condition that they are not full length tumor associated polypeptides. In other embodiments, the pharmaceutical composition may further comprise a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 11.

In one preferred embodiment, the pharmaceutical composition comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12 and / or SEQ ID NO: 13, and further comprising any of SEQ ID NOs: 14 to SEQ ID NO: 23 It further comprises one or more peptides consisting of the amino acid sequences described. In certain preferred embodiments, such pharmaceutical compositions may further comprise a peptide comprising the amino acid sequence set forth in SEQ ID NO: 11, provided that these peptides each satisfy the condition that they are not full length tumor associated polypeptides. In other embodiments, the pharmaceutical composition may further comprise a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 11.

In certain embodiments, the peptide present in the composition is selected from tissue-, cancer-, and / or patient-specific peptides.

In a further preferred embodiment, the pharmaceutical composition comprises one or more suitable adjuvants. Adjuvants are substances that non-specifically enhance or enhance the immune response (e.g., immune responses mediated by helper-T cells (T _H ) against CTLs and antigens, and thus are agents of the invention). Is considered useful). Suitable adjuvants include 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligand, FLT3 ligand, GM-CSF , Interleukins, Interferon-alpha or Interferon Beta, including IC30, IC31, imiquimod (ALDARA), Recimiquimod, ImuFact IMP321, Interleukin-2, Interleukin-13, Interleukin-21, etc. -beta), or interferon such as PEGylated derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvimmune, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312 , Montanade ISA 206, Montanade ISA 50V, Montanade ISA-51, Water-in-oil and Oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® ) Vector systems, PLG and dextran microparticles, resiquimod, SRL172, virosomes and other viral agents Particles, YF-17D, VEGF traps, R848, beta-glucan, Pam3Cys, Aquila QS21 stills, including but not limited to saponins, mycobacterial extracts, synthetic bacterial cell wall analogs Other proprietary supplements include Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Some immune adjuvant specific to dendritic cells (eg MF59) and formulations thereof have been described previously (Dupuis M et al 1998; Allison 1998). Cytokines can also be used. Some cytokines are directly related to affecting the migration of dendritic cells into lymphoid tissues (eg, TNF-α) and increase the rate of growth of dendritic cells into effective antigen-providing cells for T-lymphocytes. (E.g., GM-CSF, interleukin-1 and interleukin-4) (US Pat. No. 5,849,589, incorporated herein by reference in its entirety), and also serve as an adjuvant (e.g., interleukin-12, interleukin- 15, interleukin-23, interleukin-7, IFN-alpha, IFN-beta) (Gabrilovich et al 1996).

CpG immunostimulatory oligonucleotides in vaccines are known to enhance the effects of adjuvants. Unless bound by any theory, CpG oligonucleotides work by activating the innate (non-obtaining) immune system primarily through toll-like receptors (TLR), such as TLR9. Activation of TLR9 initiated by CpG results in antigens for various antigens, including peptide or protein antigens, live or dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in prophylactic or therapeutic vaccines. Enhances specific humoral and cellular immune responses. More importantly, even without the help of CD4 T cells, it improves dendritic cell growth and differentiation, which increases the activation of T _H1 cells and increases the incidence of strong cytotoxic T-lymphocytes (CTLs). . Usually T _H1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvant such as alum or incomplete Freund's adjuvant (IFA) that promotes T _H2 bias. When CpG oligonucleotides are formulated or co-administered with other adjuvants or formulations, such as microparticles, nanoparticles, fat emulsions or similar formulations (which are necessary for causing a strong response when the antigen is relatively weak), Shows much stronger adjuvant activity. They can also accelerate the immune response and reduce antigen doses by approximately two orders of magnitude by similar antibody responses to the full dose vaccine without CpG in some experiments (Krieg et al 2006). US Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, nonnucleic acid adjuvants and antigens to induce antigen specific immune responses. The CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) manufactured by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules may be used, such as RNA binding TLR 7, TLR 8 and / or TLR 9.

Among other examples of useful adjuvants include dsRNA analogs such as chemically modified CpG (eg CpR, Idera), Poly (I: C), and AmpliGen, non-CpG bacterial DNA or RNA and immune active small molecules and cyclophosphamide, standing up Nitinib, Bezazumab, Celebrex, NCX-4016, Sildenafil, Tadalafil, Vardenafil, Sorafenib, Temozolomide, Temsirolimus, XL-999, CP-547632, Pazopanib, VEGF Trap, ZD2171 , AZD2171, anti-CTLA4 and SC58175, including but not limited to, these may act therapeutically and / or as adjuvant. The amounts and concentrations of auxiliaries and adducts useful in the present invention can be readily determined by one skilled in the art without more than necessary experimentation. Among the preferred adjuvants are dSLIM, interferon-alpha, -beta, CpG7909, IC31, imiquimod, resiquimod, PeviTer, RNA, tadalafil, temozolomide and juvium.

Among the preferred adjuvants are dSLIM, BCG, OK432, imiquimod, resiquimod, GMCSF, interferon-alpha, pebitter and jujuits, or combinations thereof.

In an embodiment of the preferred pharmaceutical composition according to the invention, the adjuvant is a colony such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), imiquimod, resquiquimod and interferon-alpha, and the like. It is selected from the group consisting of colony-stimulating factors.

In an embodiment of the preferred pharmaceutical composition according to the present invention, the adjuvant consists of colony growth factors such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), imiquimod and resiquimod, etc. Selected from the group.

In an embodiment of the preferred pharmaceutical composition according to the invention, the adjuvant is imiquimod or resiquimod.

Such compositions can be used for parenteral administration, such as subcutaneous, intradermal, intramuscular or oral administration. Peptides may also be administered in combination with substances that stimulate immunity, such as cytokines.

The pharmaceutical composition of the present invention can be used as an anticancer vaccine.

The present invention includes a kit comprising the pharmaceutical composition in a pre-prepared or separate container or glass bottle for at least one and / or immediate mixing of the peptides.

Preferred kits comprise (a) a container comprising a pharmaceutical composition in dissolved or lyophilized form according to the invention, (b) optionally a diluent or reconstitution solution for a lyophilized formulation and / or one or more adjuvants. Two containers, and (c) optionally, instructions for (i) use of solution, or (ii) reconstitution and / or use of the lyophilized formulation.

In addition, preferred kits in the present invention further comprise one or more of (i) buffer, (ii) diluent, (iii) filter, (iv) needle and (v) syringe.

The present invention provides pharmaceutical compositions useful as vaccines. The pharmaceutical composition includes various tumor related peptides (TUMAPs) in addition to a mixture of mannitol and Tween 80® or a mixture of mannitol and poloxamer 188 which provides the stability and solubility of the composition.

A "pharmaceutical composition" is a lyophilized formulation comprising peptide, mannitol and Tween 80® or peptide, mannitol and poloxamer 188, preferably a reconstituted liquid composition. As used herein, the term “pharmaceutical composition” may also refer to a lyophilized formulation indicating that the composition is in lyophilized form.

Preferably, the pharmaceutical composition comprises one or more of the peptides set forth in SEQ ID NOs: 1 to 10 found and located in primary renal cell cancer, or SEQ ID NOs: 11 to 22 found and located in primary glycoma cancer cells and At least one of the peptides described in 11-23.

In another preferred embodiment, the pharmaceutical composition comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 12 and / or SEQ ID NO: 13, and further comprising any of SEQ ID NOs: 14 to 23 Further comprises one or more peptides consisting of the amino acid sequences described.

Peptide sets include HLA class I and class II peptides. Preferably, the pharmaceutical composition of the invention comprises one or more other peptides or SEQ ID NOs: 12 comprising the peptides set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or SEQ ID NOs: 3-10; And / or the peptide set forth in SEQ ID NO: 13 and / or one or more other peptides comprising SEQ ID NOs: 11 and 14-23.

The pharmaceutical composition comprises a peptide in free form or in the form of a pharmaceutically acceptable salt.

As used herein, the term “pharmaceutically acceptable salts” refers to the disclosed peptide derivatives that are modified by making acid or base salts of the formulation. For example, acid salts are prepared from the free base (usually the neutral form of the drug has a neutral NH2 group) including the reaction with a suitable acid. Acids suitable for preparing acid salts are organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, Methanesulfonic acid, ethanesulfonic acid, P-toluenesulfonic acid, salicylic acid and the like, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. On the other hand, preparations of basic salts of acid residues which may be present in the peptide are prepared using pharmaceutically acceptable salts such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine and the like.

In a particularly preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate) or hydrochloric acid (chloride).

In an even more preferred embodiment according to the invention, the pharmaceutical composition comprises SEQ ID NO: 5 as a chloride and the other peptide as acetate.

The pharmaceutical compositions of the present invention may also contain one or more peptides that act as positive control peptides as immune markers for testing the effectiveness of intradermal dosing. Such exemplary peptides are derived from HBV core antigen (SEQ ID NO: 11).

In one particular embodiment, the pharmaceutical composition comprises eleven different peptides, each consisting of the amino acid sequences set forth in SEQ ID NOs: 1-11 (see Table 1). Preferably, each peptide in the pharmaceutical composition is present in an amount of about 1500 μg to about 75 μg, more preferred amount is about 1000 μg to about 750 μg, even more preferred amount is about 500 μg to about 600 μg, most The preferred amount is 578 μg. Preferably, each peptide is purified by HPLC and ion exchange chromatography and shown as white to off-white powder.

In another specific embodiment, the pharmaceutical composition comprises 12 different peptides, each consisting of the amino acid sequences set forth in SEQ ID NOs: 11-22 and SEQ ID NOs: 11-23 (see Table 1). Each peptide in the pharmaceutical composition is present in an amount of about 1500 μg to about 75 μg, more preferred amount is about 1000 μg to about 750 μg, even more preferred amount is about 500 μg to about 600 μg, most preferred amount is 578 μg to be. Preferably, each peptide is purified by HPLC and ion exchange chromatography and shown as white to off-white powder.

Preferred Peptides of the Pharmaceutical Compositions of the Invention Internal sequence ID antigen order SEQ ID NO: IMA-MMP-001 Substrate Metalloproteinase 7 SQDDIKGIQKLYGKRS One IMA-ADF-002 Adipophylline VMAGDIYSV 2 IMA-ADF-001 Adipophylline SVASTITGV 3 IMA-APO-001 Apolipoprotein L1 ALADGVQKV 4 IMA-CCN-001 CyClean D1 LLGATCMFV 5 IMA-GUC-001 GUCY1A3 SVFAGVVGV 6 IMA-K67-001 KIAA0367 ALFDGDPHL 7 IMA-MET-001 c-met Wonam gene YVDPVITSI 8 IMA-MUC-001 MUC1 STAPPVHNV 9 IMA-RGS-001 RGS-5 LAALPHSCL 10 IMA-HBV-001 HBV FLPSDFFPSV 11 IMA-BCA-002 Berebikan (NP_068767, 478-486) ALWAWPSEL 12

IMA-BIR-002 Baculovirus IAP Repeat-Containing 5 (NP_001159, 97-111) TLGEFLKLDRERAKN 13 IMA-CSP-001 Chondroitin Sulfate Proteoglycan 4 (NP_001888, 21-29) TMLARLASA 14 IMA-FABP7-001 Fatty acid binding protein 7, brain (NP_001437, 118-126) LTFGDVVAV 15 IMA-IGF2BP3-001 Insulin-like growth factor 2 mRNA binding protein 3 (NP_006538, 552-560) KIQEILTQV 16 IMA-MET-005 Met Wongam Gene (NP_000236, 651-667) TFSYVDPVITSISPKYG 17 IMA-NLGN4X-001 Neuroligin 4, X-linked (NP_065793, 131-139) NLDTLMTYV 18 IMA-NRCAM-001 Neuronal cell adhesion molecule (NP_001032209, 692-700) GLWHHQTEV 19 IMA-PTP-003 Protein Tyrosine Dephosphatase, Receptor Form, Z Polypeptide 1 (NP_002842, 195-203) AIIDGVESV 20 IMA-PTP-005 Protein Tyrosine Dephosphatase, Receptor Form, Z Polypeptide 1 (NP_002842, 1347-1355) KVFAGIPTV 21 IMA-TNC-001 Tenacin C (NP_002151, 3-11) AMTQLLAGV 22 IMA-CHI-001 SLWAGVVVL 23

The term "peptide" as used herein is generally used to designate a series of amino acid residues in which the alpha-amino and carbonyl groups of neighboring amino acids are linked by peptide bonds. Peptides are typically 9 amino acids long for MHC type I and longer (15 or 16 amino acids) for MHC type II but can have a minimum amino acid length of 8 and a maximum amino acid. The length of can be up to 16.

The term "oligopeptide" as used herein is generally used to designate a series of amino moieties in which the alpha-amino and carbonyl groups of neighboring amino acids are linked by peptide bonds. In the present invention, the length of the oligopeptide is not critical as long as the correct epitope is maintained. Oligopeptides are typically less than about 30 amino acids in length and more than about 14 amino acids in length.

The term "polypeptide" as used herein is generally used to designate a series of amino moieties in which the alpha-amino and carbonyl groups of neighboring amino acids are linked by peptide bonds. In the present invention, the length of the polypeptide is not critical as long as a suitable epitope is maintained. Unlike oligopeptides, the term polypeptide refers to protein molecules having residue lengths greater than about 30 residues.

If a peptide, oligopeptide, protein, or polynucleotide encoding such a molecule is capable of inducing an immune response, it is referred to as "immunogenic" (henceforth referred to as "immunogen"). In the present invention, immunogenicity is more specifically defined as the ability to induce a CTL-mediated response. Therefore, "immunogen" refers to a molecule capable of inducing an immune response, and in the present invention, refers to a molecule capable of inducing a CTL response.

T cell “epittop” refers to a short peptide molecule that binds to MHC molecule type I or type II and is subsequently recognized by T cells. T cell epitopes that bind class I MHC molecules are mostly 8 to 14 amino acids, in most cases 9 amino acids. T cell epitopes that bind class II MHC molecules are 12-30 amino acids in length. For epitopes that bind to MHC type II, the same T cell epitopes share the same common core segment, but the length of the carboxy- and amino-terminal contiguous sequences may be different, because the ends of these peptide molecules This is because the type I MHC molecule is in a peptide binding groove but is not buried in the structure of the peptide binding groove of the type II MHC molecule.

There are three different gene positions that encode class I MHC molecules: HLA-A, HLA-B, and HLA-C. HLA-Al, HLA-A2 and HLA-All are examples of other class I MHC molecules that can be expressed at these positions.

As used herein, the terms “part”, “segment” and “fragment” when used with a polypeptide refer to a contiguous sequence of residues, such as amino acid residues, which form a subset of the larger sequence. For example, when a polypeptide is treated by general endopeptidases such as trypsin or chymotrypsin, the oligopeptides resulting from the treatment represent the portion, segment or fragment of the polypeptide that starts. That is, any of the fragments may be naturally occurring as part of an amino acid sequence or may be segments, fragments or portions of SEQ ID NOs: 1 to 23 corresponding to the "parent" protein of SEQ ID NOs: 1 to 23, which are substantially identical. Or otherwise exactly the same). When used in connection with polynucleotides, these terms refer to products produced by treating polynucleotides with general endopeptidase.

According to the present invention, the terms "percent identity" or "percent identity" used when referring to a sequence are aligned by the sequence in which the sequence is to be compared ("comparative sequence") is described or claimed ("reference sequence"). Refers to a sequence which is then compared to the claimed or described sequence. Percent identity is determined according to the following formula:

Percent identity = 100 [I- (C / R)]

Wherein C refers to the number of differences between the reference sequence and the comparison sequence relative to the alignment length of the reference sequence and the comparison sequence, wherein

(i) each base or amino acid in the reference sequence that does not have a corresponding base or amino acid aligned in the comparative sequence,

(ii) each gap in the reference sequence, and

(iii) each aligned base or amino acid in the reference sequence different from the aligned base or amino acid in the comparative sequence constitutes a difference,

R is the number of bases or amino acids in the reference sequence relative to the length aligned by the comparative sequence, and any gaps created in the reference sequence are also counted as bases or amino acids.

If an alignment exists between the comparative sequence and the reference sequence, the alignment may occur even if the percent identity calculated as above is approximately above the stated minimum percent identity, even if the percent identity calculated above is less than the specified percent identity. The comparison sequence, if any, has the minimum percentage identity specified for the reference sequence.

Peptides in the pharmaceutical compositions of the invention may be altered by substituting one or more residues at different, possibly selective positions within the peptide chain. Such substitutions may have conservative properties, for example, when one amino acid is replaced by amino acids of similar structure and properties, such as when hydrophobic amino acids are replaced by other hydrophobic amino acids. More conservative is the replacement of amino acids having the same or similar size and chemical properties, eg leucine, with isoleucine. Studies of naturally occurring cognate protein sequence variations show that certain amino acid substitutions are more resistant than others, and show an interrelationship of similar size, charge, polarity and hydrophobicity between the original amino acid and its replacement, This is the basis for defining "conservative substitutions".

Conservative substitutions are defined herein as exchanges of one of the following five groups: group 1: small aliphatic, nonpolar or slightly polar residues (Ala, Asn, Thr, Pro, Gly); Group 2: polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3: polar, positively charged residues (His, Arg, Lys); Group 4: large aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); And group 5: large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions may include replacing amino acids with similar but somewhat different sizes, such as replacing alanine with an isoleucine residue. Highly non-conservative substitutions may include replacing acidic amino acids with polar, or even basic, ones. Such "radical" substitutions cannot be presumed to be potentially ineffective because chemical effects are not fully predictable and accidental effects may occur that are unpredictable from simple chemical principles.

Of course, such substitutions may include other than conventional L-amino acids. Thus, D-amino acids can substitute for L-amino acids found generally in the antigenic peptides of the invention, which are also included herein. In addition, amino acids having non-standard R-groups (ie, R groups other than those found in the usual twenty amino acids of natural proteins) may be used for alternative purposes to produce immunogens and immunogenic polypeptides according to the present invention. .

Where substitutions at one or more positions are found to produce peptides having substantially equivalent or greater antigenic activity as defined below, the combination of such substitutions may result in the combined substitution having an additive or synergistic effect on the antigenicity of the peptide. It will be tested to determine if it is caused. Up to four positions in a peptide are substituted at the same time.

Preferably, when the CTLs specific for the peptides of SEQ ID NOs: 1-23 are tested for the substituted peptide, the concentration of the peptide at which the substituted peptide achieves half the maximum rate of cytolysis against the background is about 1 mM. Or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, most preferably about 10 pM or less. It is also preferred that the substituted peptides are recognized by CTL from more than one individual, two or more, more preferably three individuals.

Mucin-1 (MUC1) is a high glycosylated type I transmembrane glycoprotein that is overexpressed on the cell surface of human adenocarcinoma such as breast and ovarian cancer. Aberrant deglycosylation usually occurs in cancer cells, which reveals an epitope in tumor cells that do not exist in normal cells. Moreover, MUC1 expression has been found in multiple myeloma and some B-cell non-Hodgkin's lymphomas. Some recent reports (Apostolopoulos V and McKenzie IF. Cellular mucins: targets for immunotherapy. Crit Rev. Immunol. 14: 293-309 (1994); Finn OJ, Jerome KR, Henderson RA, Pecher G, Domenech N, Magarian -Blander J, and Barratt-Boyes SM.MUC-1 epithelial tumor mucin-based immunity and cancer vaccines.Imunmun. Rev. 145: 61-89 (1995); Barnd 1989; Takahashi 1994; Noto 1997]) It has been demonstrated that pancreatic cells, cytotoxic MHC-unrestricted T cells in multiple myeloma tumors can recognize epitopes at the core of the MUC1 protein located in tandem repeats. Two HLA-A2-restricted T-cell epitopes derived from the MUC1 protein have been found (Brossart 1999, EP 1484397). One peptide is derived from the tandem repeat region of the MUC1 protein. The second peptide is located in the signal sequence of MUC1. In vivo induction of cytotoxic T-lymphocyte responses following vaccine with peptide-pulse dendritic cells has been successful in patients with advanced breast or ovarian cancer (Brossart 2000) (Wierecky 2005). For renal cell carcinoma, it has been reported that MUC1 expression is common in conventional tumors and is associated with tumor grade and stage. As for MUC1, protein overexpression is not correlated with mRAN overexpression.

Adipophylline is a marker for diseases associated with specifically differentiated cells and fat accumulating cells with fat stores (Heid 1998). Adipophylline occurs in a variety of cultured cell lines, including fibroblasts, endothelial and epithelial cells, and the like. However, the expression of adipophylline in tissues may be due to lactated epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of male reproductive organs, and fatty changes in hepatocytes resulting from fatty liver or alcoholic fatty cirrhosis. Confined to specific cell types (Heid 1998). Adipophylline is known to be overexpressed in colorectal cancer (Saha 2001), hepatocellular tumors (Kurokawa 2004), and renal cell carcinoma (Young 2001).

c-Met encodes a heterodimeric transmembranous receptor consisting of α-chains that have tyrosine kinase activity and are linked by disulfide bonds to β-subunits (Bottaro 1991; Rubin 1993) . Both subunits are expressed on the cell surface, heavy β-subunits are involved in ligand, hepatocyte growth factor (HGF) binding, and α-subunits have intracellular domains that mediate the activation of other signal transduction pathways. . c-Met signaling is involved in the regulation of organ regeneration, embryonic development, hematopoiesis, muscle development, and the migration and adsorption of normally activated B-cells and monocytes as demonstrated in liver and kidney (Zarnegar 1995; Naldini 1991; Montesano 1998; Schmidt 1995; Uehara 1995; Bladt 1995; Takayama 1996; Mizuno 1993; van, V 1997; Beilmann 2000). Numerous studies have also suggested that c-Met overexpression is involved in malignant transformation and invasiveness of malignant cells.

c-Met mediates the multifunctional and potential carcinogenic activities of HGF / scattering factors, including cell growth, motility, survival, promotion of extracellular matrix lysis and angiogenesis (Bottaro 1991; Rubin 1993; Zarnegar 1995). The binding of HGF to receptors induces autophosphorylation of c-Met, which triggers downstream signal transduction events such as ras, phosphatidylinositol 3′-kinase, phospholipase C _γ , and cleavage-activated protein kinase related pathways. Activate it. The c-Met gene is predominantly expressed in epithelial cells and overexpressed in several malignant tissues and cell lines. More reports have shown that non-epithelial cells such as hematopoietic cells, neurons and skeletal muscle cells respond to HGF and that hematopoietic malignancies such as multiple myeloma, Hodgkin's lymphoma, leukemia and lymphoma express c-Met protein. . Abnormal control of the invasive growth phenotype by oncogenic active c-Met provoked by C-Met-activating mutations, c-Met amplification / overexpression, and HGF / c-Met self-secreting rings is invasive to malignant tumors To be given transitive properties. In particular, constitutive activation of c-Met in HGF overexpressed transgenic mice promotes wide tumorigenesis.

The regulator of G-protein signal pathway 5 (RGS5) is a negative regulator of the heterotrimeric G-protein signal pathway, but its in vivo function is still difficult to define. RGS proteins consist of a family of molecules that have a variety of homogeneous catalytic functions but vary in tissue distribution. They stimulate the intrinsic guanosine triphosphatase (GTPase) activity of activated Gα subunits and thus accelerate the inactivation of G-proteins. Thus, RGS molecules inhibit signaling pathways downstream of G protein bound receptors (De 2000). Recently, the induction of regulators of G-protein signaling pathway 5 in pericytes has been shown to be consistent with active vascular remodeling in tumor angiogenesis. In mouse models of pancreatic islet cell carcinoma and high angiogenic astrocytomas, overexpression of RGS5 has been reported in perivascular cells during angiogenic switch periods with active vascular remodeling. Overexpression is limited to cancer vessels as compared to normal islet of Langerhans. However, RGS5 is also upregulated during ovulation or wound healing (Berger 2005).

Expression of RGS5 is increased in RCC (Rae 2000). In another study, strong expression of RGS5 was seen by RT-PCR in all tested RCCs, and expression in normal kidneys was very weak or undetectable (6.6 vs 1 when tested by real time PCR). Cancer endothelial cells were the major site of RGS5 in RCC (Furuya 2004). RGS5 is also known as an isovascular endothelial cell marker in hepatocellular tumors (Chen 2004).

Apolipoprotein L1 (APOL1) is a secretory high density lipoprotein that binds to apolipoprotein A-I. Apolipoprotein A-I is a relatively abundant plasma protein and the major apoprotein of HDL. It is involved in the formation of most cholesteryl esters in plasma and also promotes the outflow of cholesterol from cells. Apolipoprotein L1 plays a role not only in the reverse transport of cholesterol from the surrounding cells to the liver but also in the exchange of fat and in the body. Plasma proteins are single chain polypeptides with an approximately 40 kDa apparent molecular mass (Duchateau 1997; Duchateau 2001). APOL1 cDNA was isolated from an activated endothelial cDNA library and was found to be upregulated by TNF-α, a potent pro-inflammatory cytokine (Monajemi 2002).

KIAA0367 was discovered in the Kazusa cDNA project, which aims to uncover unknown long human transcripts encoding putative proteins (Ohara 1997). Although the function of a protein with an estimated 820 amino acids of KIAA0367 has not been determined, it binds to small hydrophobic molecules and binds to several nucleotide exchange factors and several BCL2 / adenovirus E1B 19-kDa protein-interacting protein 2 (BNIP-2 Has a CRAL-TRIO fat binding domain profile at the C-terminus. BNIP-2 is involved in the regulation of various cell functions such as cell morphology, migration, endocytosis and cell cycle progression (Zhou 2005). KIAA0367 is located on chromosome 9q21. This position is described as a common target of homozygous deletion (Gursky 2001; Weber 2001) or loss of heterozygocity (Louhelainen 2000; Tripathi 2003) in several tumors.

Soluble guanylate cyclise (sGC) is a heterodimeric protein consisting of alpha and beta subunits (one heme-group) that promotes the conversion of GTP to secondary messenger cGMP and nitric oxide And nitro vasodilator drugs (Zabel 1998). GUCY1A3 and GUCY1B3 are overexpressed in human glycoma. Cellular nucleic acid transfer infection of antisense GUCY1A3 or GUCY1B3 in nude mouse experiments reduced vascularization and cancer growth. This may be due to the fact that VEGF is induced by cGMP (Saino 2004). GUCY1A3 promotes the migration of tumor cells in mouse mammary tumor cell lines (Jadeski 2003).

Cyclin D1 belongs to the highly conserved cyclin family and, more particularly, to the cyclin D subfamily (Xiong1991; Lew 1991). Cyclin acts as a regulator of cyclin-dependent kinases (CDKs). Different cyclins exhibit distinct expression and degradation patterns that contribute to the transient regulation of each mitotic event (Deshpande 2005). Cyclin D1 acts as a regulator subunit of CDK4 or CDK6 whose activity is required for cell cycle G1 / S metastasis and binds together to form a complex. CCND1, together with CDK4 and CDK6, form a serine / threonine kinase completease complex and impart substrate specificity to the complex (Bates 1994). The protein was found to interact with the tumor suppressor protein Rb (Loden 2002) and the expression of this gene is positively regulated by Rb (Halaban 1999). Mutations, amplification and overexpression of this gene, which can alter cell cycle progression, are frequently observed in various tumors and may contribute to tumorigenesis (Hedberg 1999; Vasef1999; Troussard 2000).

CSPG4 (chondroitin sulfate proteoglycan) refers to the integral membrane chondroitin sulfate proteoglycan. It is known as an early cell surface melanoma progression marker and is known to be involved in stimulation of cell proliferation, migration and invasion. CSPG4 is strongly expressed in at least 90% of human melanoma. Although CSPG4 is not strictly tumor specific, tumor-reactive CD4 + T-cell responses in melanoma patients and healthy individuals in the absence of _autoimmunity recognize CSPG4 _693-709 on melanoma cells expressing HLA-DR11 (Erfurt et al., 2007).

In addition, the expression of CSPG4 in some normal tissues, such as endothelial cells, chondrocytes, striated muscle cells, specific basal keratinocytes in the envelope and hair follicles, in addition to activated perivascular cells, is described (Campoli et al. , 2004).

During response to angiogenesis and CNS pathology, CSPG4 cells with high motility are recruited to sites that undergo rapid morphological changes and where blood vessel growth and repair occurs. CSPG4 is overexpressed by peripheral cells on tumor cells and malignant brain tumor vessels (Chekenya and Pilkington, 2002). Cell transplantation of CSPG4-positive human glycoma cell lines into immunodeficient nude mouse brains showed that these tumors had a higher microvascular density when compared to the control group, indicating that CSPG4 expression is a function and structure of host-derived tumor vessels. Have been shown to regulate all of them (Brekke et al., 2006). Xerograft experiments of GBM biopsy data into nude rats revealed that CSPG4 is primarily associated with blood vessels on the bottom membrane components of peripheral cells and tumor vessels, and expression is associated with high cell proliferation regions (Chekenya et al. ., 2002a). In addition, CSPG4 expression was consistent with tumor progression in the glycoma transplant model (Wiranowska et al., 2006).

CSPG4 is expressed differently, such as higher expression in higher grade glyomas than in low grade glycomas (Chekenya et al. 1999). High expression of GSP4 is associated with increased α3β1 integrin / PI3K signal activation and multidrug resistance mediated by downstream targets and promotes cell survival (Chekenya et al., 2008).

FABP7: Fatty acid binding proteins (FABPs) are 14-15 kDa cytoplasmic proteins that have been shown to be involved in fatty acid (FA) uptake, transport and targeting. They are known to increase the solubility of fatty acids in the cytoplasm when transporting fatty acids between membrane compartments and when transporting fatty acids to nuclear targets (Glatz et al., 2002). Fatty acid binding proteins can regulate the concentration of fatty acids, thereby affecting various cellular functions such as enzyme activity, gene expression, cell growth and differentiation (Glatz and Storch 2001).

FABP7 mRNA is expressed in tissues of neuroepithelial origin and in malignant glycoma tumors (WHO grades III and IV). This gene is in the range of chromosome band 6q22-23, which also includes the prototypical gene c-myc, which often suffers from dysplastic loss in malignant glyomas. Analysis of malignant glycoma cell lines shows that FABP7 is often expressed with neuroglial fibrin protein (GFAP), which suggests the potential for cells of origin of malignant glycoma to express these two proteins normally or as a result of tumor formation. It may also be a stellate glial progenitor (Godbout et al., 1998). FABP7 protein shows moderate to strong nuclear and cytoplasmic expression in GBM. Gnuoma cells transfected with FABP7 show five times greater migration than cells in the control group. Thus, the shorter overall survival associated with FABP7 overexpression, which is particularly pronounced in GBM, may be due to increased migration and invasion of tumor cells into the surrounding brain parenchyma (Liang et al., 2005). More analysis of the distribution of FABP7 in astrocytic tumors shows increased levels of FABP7 at the site of tumor infiltration, suggesting an important role that FABP7 leads to infiltration of tumor cells into surrounding brain tissue (Mita et al. al., 2007). FABP7 shows various expression levels and intracellular locations in glial tissues and all grades of astrocytoma tumors. Nevertheless, in particular, the nuclear position of FABP7 appears to be related to the invasive phenotype of glycoma cells, and after EGFR activation, the intranuclear migration pathway of FABP7 is detected, which is associated with an inferior prognosis in EGFR positive GBM. It appears to be associated with the EGFR pathway. In addition, no immune response of nuclear FABP7 was detected in Grade I astrocytoma (Liang et al., 2006; Kaloshi et al., 2007).

X-linked neurorigin 4 is a member of the cell adhesion protein family and appears to play a role in the maturation and function of neuronal synapses. Members of the neurorigin family are N-terminal signal peptides, esterase-like domains with two alternating splicing sites, small sequence regions of low sequence identity in front of the transmembrane domain, and short cytoplasm with highly conserved C-terminus Have a structural organization associated with the part. Relatively high levels of neuroligin 4 mRNA are found in the heart. Lower expression is found in liver, skeletal muscle and pancreas, and little neuroligin 4 mRNA is detected in brain, placenta, lung and kidney (Bolliger et al., 2001).

Mutations in the X-linked NLGN4 gene are potential causes of autism spectrum disorders, and mutations have been reported in several patients with autism, Asperger's syndrome and mental retardation (Jamain et al., 2003; Laumonnier etal., 2004; Lawson- Yuen et al., 2008).

Several associations between NLGN4X and cancer have been described: In gastrointestinal stromal tumors, overexpression of NLGN4X was found in children and young adults than in older adults (Prakash et al., 2005).

Tenacin C: The extracellular matrix surrounding the tumor cells is different from the extracellular matrix of normal tissue. Tenasin C (TNC) has high migration activity such as embryo growth (Bartsch et al., 1992), wound healing (Mackie et al., 1998) and tumor processes (Chiquet-Ehrismann 1993; Chiquet-Ehrismann and Chiquet, 2003). It is an extracellular matrix protein that is highly upregulated in a process that is closely related to. In addition, TNC is overexpressed in tumor vessels with high proliferative capacity, indicating that TNC is involved in neoplastic angiogenesis (Kim et al., 2000). In the normal human brain, expression of TNC is rarely detected, whereas expression in malignant glioma is high (Bourdon et al., 1983). TNC-expression may be associated with hypoxia (lal et al., 2001), TGF-beta 1 (Hau et al., 2006), when high-grade glycoma is provided with a mechanism to invade healthy parenchyma, or human GMB cells. It can be induced by gastrin (Kucharczak et al., 2001), which significantly regulates migration. TNC downregulates tropomyosin-1, thus destabilizing actin stress fibers. It also downregulates the Wnt inhibitor Dickkopf1. Decreased tropomyosin-1 expression and increased Wnt signal are closely related to transformation and tumorigenesis, so TNC specifically modulates these signaling pathways to enhance the proliferation of glycoma cells (Ruiz et al., 2004). .

In perivascular cell staining around tumor supply vessels, TNC is found in GBM tissue, but less frequently in WHO grades II and III gliomas, where the intensity of TNC staining is associated with tumor grade and the strongest staining Show the poorest prognosis (Herold-Mende et al., 2002). TNC also aids in the generation of stem cell parasites in the subventricular zone (SVZ), while regulating growth factors that signal neural stem cell development. The main effect of TNC on cells in SVZ is the regulation of developmental processes (Garcion et al., 2004). TNC is the strongest direct inducer of human neural stem cell (NSC) migration. Thus, ECM made from tumors provides an environment in which NSC fragrances can spread to tumor cells (Ziu et al., 2006).

Neuronal cell adhesion molecules (NRCAMs) are neural transmembrane cell adhesion molecules with multiple immunoglobulin-like C2-type and fibronectin type-III domains. This is achieved by forming mutual or heterophilic interactions with other IgCAMs (Volkmer et al., 1996; Sakurai et al., 1997; Zacharias et al., 1999), inducing, generating and spasming neurons (Grumet et al., 1991; Morales et al., 1993; Stoeckli and Landmesser, 1995; Perrin et al., 2001; Sakurai et al., 2001). Ankyrin binding NRCAM (Davis and Bennett, 1994) is upregulated in vascular producing endothelial cells, suggesting a possible role in vascular production and angiogenesis (Aitkenhead et al., 2002).

NRCAM is a target gene of β-catenin and flacoglobin-LEF / TCF complexes that contribute to tumorigenesis (Conacci-Sorrell et al., 2002). The extracellular domain of NRCAM can be removed from the cell surface by metalloproteinase-like activity. This removed domain can activate several signaling pathways, enhance cell motility and cause tumorigenesis in mice (Conacci-Sorrell et al., 2005).

NRCAM is upregulated compared to normal brain in anaplastic astrocytoma and GBM tumor tissue, and increased levels are associated with invasive behavior (Sehgal et al., 1998). Antisense RNA against NRCAMs reduces the tumorigenic capacity of human GBM cells (Sehgal et al., 1999).

IGF2BP3 is a member of the insulin-like growth factor-II mRAN binding protein family, which participates in mRNA localization, turnover and translational regulation. Proteins have several KH (K- homologue) domains, which are important for RNA binding and are known to be involved in RNA synthesis and metabolism. Expression has been described to occur mostly in embryonic development and in some tumors. Thus, IGF2BP3 is considered to be a stromal protein (Liao et al., 2005). Transcripts of IGF2BP3 are present at high levels in many cancer tissues compared to control tissues, suggesting that IGF2BP3 protein plays a functional role in the proliferation of transformed cells. This hypothesis is supported by the finding that only one non-tumoral human tissue expressing a transcript of IGF2BP3 is the human placenta, a tissue characterized by cell growth and proliferation (Mueller-Pillasch et al., 1997).

For example, IGF2BP3 is expressed in clear cell RCC samples and this expression is associated with the progressive stage and grade of the primary tumor. In addition, positive IGF2BP3 expression is associated with a 5-10 fold increase in the risk of distant metastasis and an increased risk of death from RCC of 42% to 50% (Hoffmann et al., 2008; Jiang et al., 2006; Jiang et al., 2008).

IGF2BP3 is also highly expressed in pancreatic cancer. Two studies show that after immunostaining, IGF2BP3 expression was not detected in non-tumor pancreatic tissues, whereas in pancreatic cancer tissues, IGF2BP3 was expressed in 90% or more of the samples. It has also been shown that the higher the stage of cancer, the higher its expression level (Yantiss et al., 2005; Yantiss et al., 2008).

IGF2BP3 expression has also been shown to be significantly increased in high grade urinary epithelial tumors, whereas it is mostly absent in benign tumors or low grade urinary epithelial tumors. In addition, compared to patients with IGF2BP3 negative tumors, patients with IGF2BP3-positive tumors showed lower progression-free survival and disease-free survival (Li et al., 2008; Sitnikova et al., 2008; Zheng et al., 2008).

Brevican (BCAN) is a member of the brain-specific lectican family of chondroitin sulfate proteoglycans. Two BCAN homologs have been reported: full-length homologous secreted into the extracellular matrix and shorter homologous with sequences expected to be glycophosphatidylinositol (GPI) anchors. The secreted allogene is expressed high from birth to 8 years of age and downregulated to age 20 with low levels maintained in the normal adult cortex. GPI isoforms are expressed at uniformly low levels throughout development (Gary et al., 2000). BCAN belongs to the family of proteoglycans and has been described as a barrier molecule that prevents the movement of cells and nerves in the adult nervous system (Viapiano and Matthews, 2006). In vivo, BCAN is expressed around the border of the occipital flow (Jaworski and Fager, 2000), which is a major upregulatory component of nerve scars that develop after nerve injury (Jaworski et al., 1999).

BCAN shows dramatic upregulation that can detect a sevenfold increase in normal levels in glioma. BCAN mRNA was not detected in adult human cortex samples that died without neurological complications. In contrast, BCAN mRNA was detected in one of every 27 human glycoma surgical samples, suggesting that BCAN may be a specific selective marker in glycoma (Jaworski et al., 1996).

Protein Tyrosine Dephosphatase, Receptor Type, Zetal (PTPRZ1, PTP-ξ) -PTPRZ1 is a member of the family of receptor type protein tyrosine dephosphorase enzymes, which are two cytosolic tyrosine-protein dephosphate domains, and alpha-carbonate dehydration domains and five It is a type I membrane protein that crosses a membrane once with a Ronectin type-III domain. Expression of this gene was found in gastric cancer cells (Wu et al., 2006), breast cancer (Perez-Pinera et al., 2007), remyelination oligodendrocytes in multiple sclerosis (Harroch et al., 2002) and hypoxic environments. It is induced by human embryonic kidney cells (Wang et al., 2005).

Both protein and transcription are overexpressed in glioblastoma cells to promote genomic DNA cell amplification in haptotactic migration (Lu et al., 2005) and glioblastoma (Mulholland et al., 2006).

Chitinase 3-like 2 (CHI3L2)-CHI3L2 was originally found in chondrocytes and is upregulated, for example in arthritis (Steck et al., 2002). Although the protein is not well described yet, it is likely to be secreted into the extracellular space. It is often described as the target antigen of rheumatoid arthritis. Experimentally, antiangiogenesis by siRNA nucleic acid transfer infection (VEGF-A) in human glycoma cell lines leads to upregulation of CHI3L2.

Survivin (BIRC5)-Expression of BIC5 (survivin), a member of the family of cell death proteins (IAPs), is increased in fetal tissue and in various human cancers. Frost beans appear to be able to regulate cell proliferation and cell death regulation. In particular, very high levels of frostbin expression are detected in glioblastoma (Angileri et al., 2008). Frost bean overexpression of brain glycoma has been shown to play an important role in malignant proliferation, anti-cell death and angiogenesis (Zhen et al., 2005; Liu et al., 2006). Especially in glioblastoma and other tumors, frost bean expression appears to be significantly correlated with malignant grade (highest frost bean expression in glioblastoma) and short survival (Kajiwara et al. , 2003; Saito et al., 2007; Uematsu et al., 2005; Mellai et al., 2008; Grunda et al., 2006; Xie et al., 2006; Sasaki et al., 2002; Chakravarti et al. , 2002).

MMP family proteins are not only involved in extracellular matrix destruction in normal physiological processes such as embryonic development, reproduction, and tissue remodeling, but also in disease progression such as arthritis and metastasis (Mott 2004). . Matrix metalloproteinase protein 7 (MMP7) is secreted as an inactive protein with a molecular weight of 29.6 kDa and is activated upon degradation by extracellular protease. The molecular weight of the activating enzyme is 19.1 kDa and binds two zinc ions and two calcium ions per subunit (Miyazaki 1990; Browner 1995). MMP7 degrades gelatin, fibronectin and casein, which differ from most members of the MMP7 family in that there is no conserved C-terminal protein domain (Gaire 1994). MMP7 is known to be frequently overexpressed in malignant tissues and is known to help tumor cell invasion in vivo (Wang 2005).

In many types of cancer, these proteins can be targets of tumor specific immune responses.

Hepatitis B virus nuclear antigen peptide HBV-001 is not derived from antigens associated with endogenous human tumors, but from nonhepatitis virus nuclear antigens. First, it is possible to quantitatively compare the progress of T-cell responses induced by tumor-associated peptides (TUMAPs) used in the pharmaceutical compositions of the present invention, thereby enabling to show anticancer effects. Second, it provides the function of an important positive control in the absence of a T-cell response in the patient. Third, it allows monitoring of the immunocompatibility of the patient.

Hepatitis B virus (HBV) infection is one of the leading causes of liver disease and affects about 350 million people worldwide (Rehermann 2005). Because of the ease of horizontal and vertical delivery and the potential for chronic disease that can lead to cirrhosis and hepatocellular carcinoma, HBV has a great impact on public health systems in many countries. The HBV genome (Orevisani 2002) consists of partial double stranded circular DNA. HBV virions combine core protein HBc with other proteins to produce nucleocapsids, which are surrounded by an envelope containing lipids and surface protein HBs (also called envelope proteins). Antigen antigenic determinants associated with HBc and HBs are represented by HBcAg and HBsAg, respectively. These antigens are associated with serologic reactions, which are antibody responses in the blood of patients, which is the most useful antigen-antibody system for the diagnosis of HBV infection. HBc can be a new foreign antigen to all individuals without a history of previous HBV infection. As an immunological peptide well known as this antigen (Bertoletti 1993; Livingston 1997), one 10-amino acid length peptide from HBcAg was selected as a positive control antigen in IMA. Induction of HBc peptide specific CTLs is used as a marker for patient immunocompatibility and successful vaccines.

The pharmaceutical compositions of the present invention can be used for parenteral administration, such as subcutaneous, intradermal, intraperitoneal, intravenous, intramuscular or oral administration. To this end, the peptide is dissolved or suspended in a pharmaceutically available, preferably aqueous carrier. In addition, the composition may have additives such as antioxidant components, preservatives, buffers, binders, blasting agents, diluents and flavorings and the like. The peptide can also be administered with an immune stimulant such as cytokine. An extensive list of additives that can be used in such compositions is described, for example, in A. kibbe, Handbook of Pharmaceutical Excipients, 2. Ed 2000, American Pharmaceutical Association and pharmaceutical press. The compositions of the present invention can be used for the inhibition, prevention and / or treatment of adenomas or cancer diseases.

The pharmaceutical composition of the present invention is a patient having an adenomatous or cancerous disease associated with peptides or antigens of SEQ ID NOs: 1 to 10, or brain cancer associated with peptides or antigens of SEQ ID NOs: 11 to 22 or 11 to 23, In particular, it may be administered to a patient suffering from glyoma, in particular glyblastoma cancer disease. Peptides in the pharmaceutical composition cause a T-cell mediated immune response in the patient. As mentioned above, in the case of renal cell carcinoma, the pharmaceutical composition according to the invention comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, and also SEQ ID NOs: 3 to At least one additional tumor associated peptide comprising the sequence of amino acids of SEQ ID NO: 10. In the case of brain cancer, in particular in the case of glycoma, in particular in the case of glioblastoma cancer, the pharmaceutical composition comprises the sequence of SEQ ID NO: 12 and / or SEQ ID NO: 13, and also SEQ ID NOs: 11 and 14 At least one additional tumor associated peptide comprising a sequence of amino acids from 22 to 23 and / or 23.

Preferably, the peptides present in the pharmaceutical composition of the present invention have a total length of 9 to 100, preferably 9 to 30, most preferably 9 to 16 amino acids. In addition, at least one peptide according to any of SEQ ID NO: 1 to SEQ ID NO: 23 may comprise a non-peptide bond.

Preferred pharmaceutical compositions of the invention (preferably in renal cell cancer vaccines) comprise a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2, other embodiments include SEQ ID NOs: 3 to At least one peptide consisting of the amino acid sequence of SEQ ID NO: 11.

More preferred pharmaceutical compositions of the invention (in the case of brain cancer, in particular for gliomas, especially for glioblastoma cancer vaccines) comprise a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and / or SEQ ID NO: 13 , Another embodiment comprises at least one peptide consisting of the amino acid sequences of SEQ ID NO: 11 to SEQ ID NO: 22 and / or 23.

Peptides may be tagged and may be fusion proteins or hybrid molecules. Peptides having the sequences set forth herein are expected to stimulate CD8 ⁺ CTL. However, stimulation is more efficient with the help of CD4 ⁺ T-cells. Thus, compartments of fusion partners or hybrid molecules provide adequate epitopes that stimulate CD4 ⁺ T-cells. The action of epitopes to stimulate CD4 ⁺ is well known and includes the same ones found in tetanus toxin. In a more preferred embodiment, the peptide has an N-terminal amino acid, which is in a fusion protein, in particular the HNL-DR antigen related constant chain (Ii). In one embodiment, a peptide of the invention is a fused protein of a truncated human protein or protein fragment and other polypeptide moiety if the human moiety has one or more amino acid sequences of the invention.

Peptides useful in pharmaceutical compositions may be very pure, combined with immuno-stimulatory adjuvants, used with immuno-stimulatory cytokines, or as liposomes, micro-, nanoparticles, micelles, emulsions, gels can be processed with systems such as gels. Peptide vaccines should generally be supplemented and GM-CSF is preferred (human GM-CSF is currently a Sargramostim®, available as Berlex from Bayer HealthCare Pharmaceuticals). Other suitable adjuvants are Aquila QS21 stymulon (Aquila Biotech, Worcester, MA, USA) derived from saponins, mycobacterial extracts and synthetic bacterial cell wall analogs, and libido detox. The same proprietary adjuvant is available: Quil A, another saponin derived adjuvant may also be used (Superfos, Denmark), and other adjuvant such as Freund's is also useful. Alternatively, it is also useful to provide a peptide that is bound with the met (see WO 95/18145 and Longenecker et al (1993) Ann. NY Acad. Sci. 690,276-291). Substances including toll-like receptor agonists (TLR agonists) having their function, preferably TLRs, because they are defined as substances that increase the immune response to the antigen (MedlinePlus® Medical Dictionary, NIH) Substances that act in conjunction with 3, 7, 8, and 9, including but not limited to protamine-stable RNA, CpG-oligonucleotides, CpR-oligonucleotides, bacterial DNA, imidazoquinolines, and the like. Other materials may also be used.

Nitric oxide synthase (iNOS), arginase (ARG1), indoleami-2,3-dioxygenase (IDO), vascular endothelial growth factor Vascular endothelial growth factor receptor 1 (VEGFR-1), vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), TGF-beta receptor I (TGF- Substances known to enhance immune responses, including but not limited to beta-RI), and the like. Such inhibitors may be, for example, monoclonal antibodies that bind to molecules or small molecules. Examples of small molecule and monoclonal antibodies that have inhibitory functions against the factors mentioned above, and thus have an effect of increasing immune responses, are 1-MT, NCX-4016, rofecoxib, cerebrex, BEC, ABH, nor-NOHA , SB-505124, SD-208, LY580276, AMD3100, acitinib, bevacizumab, JSI-124, CPA-7, XL-999, ZD2171, panofanib, CP-547632 and VEGF traps.

In addition, agents that reduce the number of regulatory T-cells (CD 4+, CD25 +, FoxP3 +) are suitable as adjuvants. This includes, for example, cyclophosphamide (cytosan), ONTAK (denyleukin diphytox), sunnytinib, anti-CTLA-4 (MDX-010, CP-675206), anti-CD25, anti-CCL22 , And anti-GITR.

The amount of peptide of the preferred pharmaceutical composition of the present invention is 0.1 to 100 mg, more preferably on the order of 0.1 to 1 mg, most preferably from 300 to 800 μg per 500 μL of solution. The term "about" refers to +/- 10 percent of a given number, unless otherwise specified. Those skilled in the art will be able to determine the amount of peptide depending on several factors such as, for example, the immune status of each patient and or the amount of TUMAP according to each particular type of cancer.

The pharmaceutical compositions of the present invention provide formulations with much improved solubility and moisturizing properties of lyophilisates compared to previously known compositions. This was accomplished with the use of special excipients. In this way, the pharmaceutical compositions of the invention and variants thereof, which consist of peptides of SEQ ID NOs: 1 to 10 or SEQ ID NOs: 1 to 11, have been developed, which have excellent storage at -20 ° C, + 5 ° C, + 25 ° C. It shows stability and is easily redissolved.

The term "storage stability" refers to the percentage of by-products not to exceed 5 percent in two years. In addition, the term "stability" is one in which certain properties such as solubility, clarity of the optical phase of dissolution, and the number of particles in dissolution do not change to a perceptible degree over time.

The term “easy to re-dissolve” refers to the freeze-dried being completely dissolved without the use of an ultrasonic homogenizer for a few seconds to two minutes through the use of a buffer or other binder. In addition, the composition can be readily provided to a patient in need of treatment via i.d., less preferably s.c. The resulting solution should have a pH between 2.7 and 9.

In another embodiment, the pharmaceutical compositions of the present invention may include sugars, sugar alcohols, amino acids such as glycine, azinin, glutamic acid, and the like as structure formers. The sugar can be monosaccharide, disaccharide, trisaccharide. The sugar may be used independently or may be used in combination with a sugar alcohol. Examples of sugars include glucose, mannose, galactose, fructose or sorbose or disaccharides such as saccharose, sucrose and lactose. lactose, maltose or trehalose, and trisaccharide is an example of raffinose. Sugar alcohols are, for example, mannitose. Preferred ingredients are saccharose, sucrose, lactose, maltose, trehalose, mannit and / or sorbit, and mannitol mannitol) is more preferred.

In addition, the pharmaceutical composition of the present invention is a synthetic preservative such as an antioxidant such as ascorbic acid or glutathione, phenol, m-cresol, methyl- or propylparaben, clobutanol, thiomersal or benzalkonium chloride, stabilizer , Saccharose, sucrose, lactose, lactose, maltose, trehalose, mannitose, mannit and / or sorbit, Previously mentioned substances such as mannit and / or lactose and polyethylene glycol (PEG), ie PEG 3000, 3350, 4000 or 6000, or cyclodextrins, ie hydroxypropyl-β-cyclodextrins , Sulfobutylethyl-β-cyclodextrin or γ cyclodextrin, or dextran or poloxamer, ie poloxaomer 407®, poloxamer 188, or Tween 20®, Tween 80® Physiological As it may comprise agents that are resistant (see literature [Handbook of Pharmaceutical Excipients, 5thed., Edited by Raymond Rowe, Paul Sheskey and Sian Owen, Pharmaceutical Press (2006)]). In a preferred embodiment, the pharmaceutical composition of the invention comprises one or more resistant excipients selected from the group consisting of antioxidants, structure formers and stabilizers.

The present invention provides enhanced stability of formulations comprising at least two peptide sets, mannitol and poloxamer 188, according to a certain proportion of SEQ ID NOs: 1-10 or SEQ ID NOs: 1-11, The present invention relates to the discovery that it leads to a composition that can be well dissolved even if there is no. Moreover, this solvation takes only a few seconds. According to the specifications of the freeze-dried product, when combined with 4.2% sodium bicarbonate solution, a clear or milky solution should be observed. The redissolution properties of the pharmaceutical compositions of the present invention can be reproduced after two years of storage of the drug at -20 ° C, + 5 ° C and + 25 ° C.

Moreover, the present invention is directed to SEQ ID NO: 11 to SEQ ID NO: 22, SEQ ID NO: 11 to SEQ ID NO: 23, SEQ ID NO: 12 to SEQ ID NO: 22 or SEQ ID NO: 12 to SEQ ID NO: A formulation comprising at least four peptide sets according to: 23, wherein a composition is easily dissolved due to a certain proportion of mannitol and poloxamer 188. According to the specification of the freeze-dried product, when combined with 4.2% sodium bicarbonate solution, a clear or milky solution should be observed. Each peptide in the formulations described is stable for at least 3 months after storage at -20 ° C, + 5 ° C and + 25 ° C.

In a preferred embodiment, the pharmaceutical compositions of the present invention should have a ratio of peptide: mannitol: twin 80® (by weight) from 1: 2: 1.5 (inclusive) to 1: 8: 2.2 (inclusive). For other preferred proportions included in the present invention, see the table below. A particularly preferred ratio is 1: 5: 2. Another preferred ratio is 1: 8: 2.

In another preferred embodiment, the pharmaceutical compositions of the present invention should have a ratio of peptide: mannitol: poloxamer 188 (by weight) from 1: 5: 1.5 (inclusive) to 1: 8: 2.2 (inclusive). For the preferred ratios included in the present invention, see the table below. Preferred ratio is 1: 5: 1.

Another particularly preferred peptide: mannitol: poloxamer 188 (by weight) ratio is 1: 8: 2. More preferred compositions comprise a mixture of peptide: mannitol: poloxamer 188 in a weight ratio of 1: 5: 2 (see Example 2). Another ratio includes peptide: mannitol: poloxamer 188 in a weight ratio of 1: 0: 2 to 1: 2: 2.2.

Other embodiments of the present invention include the following weight ratios:

Peptide Mannitol Poloxamer 188 Peptide Mannitol Poloxamer 188 One 5 1.5 One 8 1.5 One 5 1.6 One 8 1.6 One 5 1.7 One 8 1.7 One 5 1.8 One 8 1.8 One 5 1.9 One 8 1.9 One 5 2 One 8 2 One 5 2.1 One 8 2.1 One 5 2.2 One 8 2.2 One 6 1.5 One 5.5 1.5 One 6 1.6 One 5.5 1.6 One 6 1.7 One 5.5 1.7 One 6 1.8 One 5.5 1.8 One 6 1.9 One 5.5 1.9 One 6 2 One 5.5 2 One 6 2.1 One 5.5 2.1 One 6 2.2 One 5.5 2.2 One 7 1.5 One 6.5 1.5 One 7 1.6 One 6.5 1.6 One 7 1.7 One 6.5 1.7 One 7 1.8 One 6.5 1.8 One 7 1.9 One 6.5 1.9 One 7 2 One 6.5 2 One 7 2.1 One 6.5 2.1 One 7 2.2 One 6.5 2.2 One 7.5 1.5 One 0 2 One 7.5 1.6 One 0 2.1 One 7.5 1.7 One 0 2.2 One 7.5 1.8 Preferred for making IMA950 One 7.5 1.9 One 5 0.5 One 7.5 2 One 5 0.6 One 7.5 2.1 One 5 0.7 One 7.5 2.2 One 5 0.8 One 5 0.9 One 5 One One 5 1.1 One 5 1.2 One 5 1.3 One 5 1.4

Peptide Mannitol Tween 80 (registered trademark) Peptide Mannitol Tween 80 (registered trademark) Peptide Mannitol Tween 80 (registered trademark) One 2 1.5 One 5 1.5 One 8 1.5 One 2 1.6 One 5 1.6 One 8 1.6 One 2 1.7 One 5 1.7 One 8 1.7 One 2 1.8 One 5 1.8 One 8 1.8 One 2 1.9 One 5 1.9 One 8 1.9 One 2 2 One 5 2 One 8 2 One 2 2.1 One 5 2.1 One 8 2.1 One 2 2.2 One 5 2.2 One 8 2.2 One 3 1.5 One 6 1.5 One 2,1 1.5 One 3 1.6 One 6 1.6 One 2,1 1.6 One 3 1.7 One 6 1.7 One 2,1 1.7 One 3 1.8 One 6 1.8 One 2.1 1.8 One 3 1.9 One 6 1.9 One 2.1 1.9 One 3 2 One 6 2 One 2.1 2 One 3 2.1 One 6 2.1 One 2.1 2.1 One 3 2.2 One 6 2.2 One 2.1 2.2 One 4 1.5 One 7 1.5 One 4 1.6 One 7 1.6 One 4 1.7 One 7 1.7 One 4 1.8 One 7 1.8 One 4 1.9 One 7 1.9 One 4 2 One 7 2 One 4 2.1 One 7 2.1 One 4 2.2 One 7 2.2

The pharmaceutical composition of the present invention may be freeze dried. The freeze-dried can be reconstituted into a hydration composition by addition of a hydration solvent. Preferably, the hydration composition may be administered directly to the patient. Accordingly, another embodiment of the present invention is a hydrated pharmaceutical composition of the disclosed formulations made by reconstitution of the lyophilisate with a hydration solvent.

The permissible pH range for intravenous and intramuscular administration is from pH 2 to 12, but subcutaneous administration reduces the rate of dissolution in vivo, thus increasing the potential for radiation at the injection site, thus increasing the range of pH from 2.7 to 9.0. (Strickley Robert G., Pharm. Res., 21, NO: 2, 201-230 (2004)).

Preferred pharmaceutical compositions of the present invention have a pH value of 7 to 9, more preferred values are 8 to 9, even more preferred values are 8.3 to 8.7.

The pharmaceutical compositions of the present invention are physiologically resistant, can be easily produced, can be easily metered, and have a good storage stability with regard to concentrations, degradation products and aggregates during storage. The formulations are stably stored for 2 years in a freezer at -20 ° C, in a refrigerator at + 2 ° C to 8 ° C, and even at room temperature (+ 25 ° C) and at 60% relative humidity.

The pharmaceutical composition of the present invention should preferably be sterile and formulated for in vivo administration.

The present invention provides a method of eliciting an immune response in a subject comprising administering a pharmaceutical composition of the present invention to a subject in need thereof, wherein the peptides, variants and salts are effective in causing the immune response. It is administered in an effective amount. The present invention further provides the use of the pharmaceutical composition of the invention in a medicament for administration to a subject for eliciting an immune response against cancer. Also included is the use of the pharmaceutical composition of the invention for the treatment of cancer, preferably renal cancer, and the use in the manufacture of a medicament for treating cancer by administering to a subject for eliciting an immune response against the cancer.

Peptides used in pharmaceutical compositions are preferably pure or essentially pure and preferably essentially homogeneous (ie no contaminants such as peptides or proteins, etc. are included). By “essentially pure” is meant at least 90% HPLC purity test results, more preferably at least 95% purity results, in the preparation of the peptide. By “essentially uniform” is meant a peptide solution comprising at least 99% of the peptide by weight of the peptide solution, at least by weight in the preparation of the peptide.

The present invention includes a kit comprising:

(a) a container comprising the pharmaceutical composition as described above in solution or lyophilized form,

(b) optionally, a second container comprising a diluent or reconstitution solution for lyophilisate, and

(c) Optionally, instructions for use of (i) solution or (ii) reconstitution and / or use of freeze-dried formulations.

The kit may further comprise one or more of the following: (i) buffer, (ii) diluent, (iii) filter, (iv) needle, or (v) syringe. The container is preferably a bottle, vial, syringe or test tube, which may be a multipurpose container. The pharmaceutical composition is preferably freeze dried.

Preferably, the kit of the present invention is contained in a suitable container with instructions for reconstitution and / or use of the freeze-dried formulations of the present invention. Suitable containers include the following. Examples include bottles, vials (eg, double chamber vials), syringes (such as double chamber syringes), test tubes, and the like. The container may be made of various materials such as glass or plastic. Preferably, the kit and / or container includes instructions for use for the container or for matters relating to the container for reconstitution and / or use. For example, the label may say that the lyophilized formulation should be reconstituted to the appropriate peptide concentration as described above. The label may further indicate that the formulation is useful or intended for subcutaneous administration.

The container containing this formulation may be a multipurpose vial that allows for repeated administration of the reconstituted formulation (eg, two to six administrations). The kit may further have a second container containing a suitable diluent (eg, sodium bicarbonate solution).

When mixing the diluent and the lyophilized formulation, the final peptide concentration of the reconstituted formulation is preferably at least 0.15 mg / mL / peptide (= 75 μg), preferably 3 mg / mL / peptide (= 1500 μg) Should not exceed The kit may further comprise other materials suitable from a commercial and user standpoint, such as buffers, diluents, filters, needles, syringes, and packages containing instructions for use.

Kits of the invention may comprise one container containing a formulation of a pharmaceutical composition according to the invention with or without other ingredients (e.g., other compounds or pharmaceutical compositions of other compounds), or It may also include a separate container.

Preferably, the kit of the present invention comprises a formulation of the invention with a second compound (e.g., an adjuvant (e.g., GM-CSF, chemotherapy drug, natural product, hormone or antagonist, antiangiogenic agent or inhibitor, cell death). Packaged for simultaneous administration use with an inducer or chelating agent) or their pharmaceutical compositions The components of the kit may be pre-combined or contained in different containers prior to administration to the patient. The kit is provided containing one or more liquid solutions, preferably aqueous liquid solutions, more preferably sterile aqueous solutions The components of the kits are preferably provided in a separate container. It may be provided as a solid which can be converted into a liquid state by adding a suitable solvent.

The container of the treatment kit may be a vial, test tube, flask, bottle, syringe, or any type of container that can hold a solid or liquid. Usually, when there is more than one component, the kit includes a second vial or other container that allows for separate metering. The kit may also include another container containing a pharmaceutically acceptable liquid. Preferably, the treatment kit comprises an instrument (e.g., one or more needles, syringes, eye vials, pipettes, etc.) that allows for the input of the agents of the invention that are components of the kit.

The formulations are intended to allow for the introduction of peptides into acceptable routes such as oral (digestive tract), nasal, eye drop administration, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Subcutaneous administration is preferred and most preferred is intradermal administration using an infusion pump.

Assay test procedure:

Homogeneity, purity, peptide content is determined by analytical RP-HPLC chromatography. The detection wavelength was 220 nm.

Stability Test (Test Formulation Stability):

a) renal cell carcinoma vaccine:

Various freeze-drieds were tested for stability of each peptide through analytical HPLC tests at + 25 ° C and + 40 ° C. Stress tests were conducted at + 25 ° C and + 40 ° C to identify trends in which formulations were more stable at ambient or elevated temperatures.

Peptides according to SEQ ID NOs: 1 to 10 were lyophilized without the excipients, adding mannitol and poloxamer 188 (Lutrol F68®) or Tween 80®.

The following test formulations were made and stability evaluated at + 25 ° C and + 40 ° C through analytical HPLC tests:

Formulation 1: Peptides without any excipients,

Formulation 2: Peptide: Mannitol: Lutrol F68® (Poloxamer 188) (weight ratio 1: 5: 2),

Formulation 3: Peptide: Mannitol: Tween 80® (weight ratio 1: 5: 2) and

Formulation 4: Peptide: Mannitol: Tween 80® (weight ratio 1: 5: 1).

Each formulation was subjected to initial HPLC measurements prior to storage in the room thermostat. It was completely dissolved in 30% acetic acid and measured by RP-HPLC by repeated crystals. In order to be able to compare the results of each measurement with each other, lyophilized β-naphthyl alanine as a peptide was used as an internal standard.

After 7, 14, 21 and 28 days, the other two vials were removed from the room thermostat and evaluated for stability.

Data evaluation was performed with repeated measurements of one vial. Four different data points were obtained using two different vials at one specific time point and at a specific temperature. These values were used to calculate the standard deviation shown by the error bars in the relevant plot.

As a result of the experiment, the stability of the formulation of each peptide containing mannitol and Lutrol F68® can be compared with the experimental results of the formulation without any excipients. Formulations containing Tween 80® showed a higher degree of degradation of IMA-RGS-001, which was particularly pronounced at + 40 ° C., in particular an increase in signal for the IMA-ADF-001 peptide. This increase may also be due to the dissolution of the impurity resulting from degradation of the peptide.

b) glioblastoma cell cancer vaccine

Various freeze-drieds were tested to see the solubility and stability of the peptides formed in the mixture.

The peptides according to SEQ ID NOs: 11-23 were lyophilized with the addition of mannitol / poloxamer 188 (Rutrol F68®) and mannitol / Twin 80® without other excipients.

The following experimental formulations were made and tested for solubility in different solutions and stability at + 25 ° C and + 40 ° C.

Formulation 1: Peptides without any excipients,

Formulation 2: Peptide: Mannitol: Lutrol F68® (Poloxamer 188) (weight ratio 1: 5: 2),

Formulation 3: Peptide: Mannitol: Tween 80® (weight ratio 1: 5: 2).

The stability of both formulations was equally good. Solubility with Formulation 2 resulted in a clear, colorless mixture, and often at high concentrations Formulation 2 produced some deposits. When using Formulation 1, dissolution was not possible.

As a result of the experiment, the stability of the combination formulation of peptide with mannitol / rutrol F68® and mannitol / twin 80® can be compared with that of the formulation without any excipients. Formulations without any excipients did not dissolve in a suitable solution such as sodium hydrogen carbonate (4.2%).

Calculation of efficacy for manufacturing IMA 901:

The efficiency of T-cell activation for poloxamer 188 and mannitol excipients was tested. The inert excipients included in the IMA 901 formulation are Poloxamer 188 (Rutrol F68®) and mannitol. These two excipients were used in Phase 1 clinical trials to enhance the stability and solubility of IMA 901. It was not included in the formulation of but was included in phase 2 clinical trials In this study, the efficiency of T-cell activation by these substances was tested in mouse models subjected to peptide immunization with murine model peptides. No toxic effects of Samer 188 (Rutrol F68®) and mannitol were observed. T-cell activation was analyzed 9 days after in vitro immunization by tetramer staining and phagocyte analyzer. Trademark) (Rutrol F68®) / mannitol to the immune complex does not alter the activation efficiency of CD8 + T cells.

Principle of examination

Immunization: For activation of native CD8 + T cells, Ova, an immunogen H2-Kb binding peptide with a supplement (CpG deoxyoligonucleotide), commonly used in mouse immunization, contained in 4.2% bicarbonate buffer _257-264 (SIINFEKL) was injected into the dermal route in a female mouse model of 8-12 weeks of age (strain C57BL / 6, H2b, 3 mice per group). Injection solutions with and without poloxamer 188 (Rutrol F68®) (16.5 mg / ml) and mannitol (41.3 mg / ml) were compared. The concentration of excipients was as planned for the IMA 901 injection. Positive controls were immunized via the subcutaneous route with CpG dissolved in Titermax® Classic (Sigma-Aldrich).

Tetramer Staining: After 9 days mice were euthanized and Ova _257-264 -specific T cells in spleen were analyzed in vitro by tetramer staining and phagocyte analyzer. Tetramer technology enables specific and sensitive detection of T-cells with compatible T-cell receptors.

Results: No toxic effects of Poloxamer 188 (Rutrol F68® BASF, Rubischchafen, Germany) / mannitol were observed, either bent or systemically at the injection site. The positive control group, CpG group, and CpG / poloxamer 188 / mannitol group showed significantly higher frequency of peptide-specific T-cells when compared to the negative control group (p <0.05). There was no significant correlation between the CpG-only group and the CpG / poloxamer 188 / mannitol group.

The possibility of an artificially observed positive population was ruled out by tetramer staining of propagated peptide-specific cells after 5 days in vitro stimulation with peptide (data not shown).

In conclusion, the presence of poloxamer 188 (Rutrol F68®) / mannitol in the immune mixture does not change the immune response caused by peptides in mice, and therefore poloxamer 188 (Rutrol F68®) No adverse or adverse effects on the effectiveness and stability of immunotherapy based on peptides of mannitol are expected.

Materials and methods

Peptides used in other formulations were synthesized and provided by Bachem AG, Switzerland.

Example 1:

Depending on the nature of the solubility, a stock solution containing each peptide was prepared by dissolving the peptides using a suitable solvent (see Table 2). After addition of 10% of 1.47 mL of acetic acid, peptides with higher concentrations of 1.47 mL (peptides 5 to 10) and 7.34 mL of peptide solutions 1 to 4 were combined. This mixture was vortexed for 2 minutes and treated for 1 minute by an ultrasonic bath. A clear result of about 1 mL was transferred to a glass bottle, followed by the addition of a mixture of 19.2 mg mannitol and 50 μL of Tween 80 stock solution (77 mg Tween 80® dissolved in 30% acetic acid). In about a few minutes the solution was frozen at minus 40 degrees Celsius and lyophilized for 14 hours at 0.06 mbar, which was then dried for 6 hours at 0.003 mbar (see Table 3).

Peptides Used in Example 1 Peptide Sequence Identification Number: Peptide Peptide Content (%) Amount [mg] Solvent [ml] 4 IMA-CCN-001 95.5 16.65 7.57 (50% HAc) 6 IMA-GUC-001 88.8 17.72 7.50 (90% HAc) 3 IMA-ADF-001 91.7 17.35 7.58 (10% HAc) 2 IMA-ADF-002 94.0 16.95 7.58 (10% HAc) 10 IMA-RGS-001 89.4 17.66 1.50 (10% HAc) 8 IMA-MET-001 91.8 17.68 1.55 (50% HAc) 9 IMA-MUC-001 90.6 17.84 1.54 (10% HAc) One IMA-MMP-001 89.6 17.92 1.53 (WFI) 4 IMA-APO-001 92.1 17.76 1.56 (WFI) 7 IMA-K67-001 92.4 17.22 1.52 (WFI)

Freeze Drying Parameters for Example 1 process time pressure Temperature freezing 3:00 Atmospheric pressure -40 ℃ First drying 0:10 0.06 mbar -39 ℃ First drying 3:00 0.06 mbar -20 ℃ First drying 10:00 0.06 mbar + 0 ℃ First drying 6:00 0.06 mbar + 20 ℃ 2nd drying 3:00 0.003 mbar + 20 ℃ 2nd drying 6:00 0.003 mbar +25 ℃

Example 2:

A GMP lot was produced with mannitol and poloxamer 188 and 2.000 vials containing 578 μg of each peptide per bottle. This formulation comprises peptide, mannitol and poloxamer 188 in a ratio of 1: 5: 2 [w: w: w].

Each lyophilized peptide was weighed into different vials according to the amounts shown in Table 4. After weighing, all peptides were dissolved in specific solvents.

Peptides Used in Example 2 Peptide Sequence Identification Number Peptide Total amount [mg] Peptide Content [%] Brutto amount [mg] Solvent [ml] 4 IMA-CCN-001 1156.00 91.6% 1262.01 550 (50% HAc) 6 IMA-RGS-001 1156.00 83.2% 1389.42 110 (10% HAc) 3 IMA-GUC-001 1156.00 92.9% 1244.35 550 (90% HAc) 2 IMA-MET-001 1156.00 92.6% 1248.38 110 (50% HAc) 10 IMA-ADF-001 1156.00 93.3% 1239.01 540 (10% HAc) 8 IMA-ADF-002 1156.00 95.5% 1210.47 540 (10% HAc) 9 IMA-MUC-001 1156.00 87.2% 1325.69 110 (10% HAc) One IMA-MMP-001 1156.00 88.6% 1304.74 110 (WFI) 4 IMA-APO-001 1156.00 95.0% 1216.84 110 (WFI) 7 IMA-K67-001 1156.00 88.3% 1309.17 110 (WFI)

Because of the different solubility of different peptides, they must be dissolved starting with peptide 1 in the order shown in Table 4. Because of the difference in solubility of each peptide relative to the final volume of the bulk solution, different amounts and concentrations of acetic acid solution and water used for injection (WFI) were used to dissolve the peptide. In order to increase solubility, each bottle was subjected to strong shaking for up to 5 minutes and ultrasound for up to 5 minutes if necessary. See Table 4 for the amounts and concentrations used.

After the peptide has dissolved, the solution should be mixed starting from No. 1 in the order given in Table 4. The solution is collected in sterile glass containers. Each peptide bottle is washed with 105 ml of acetic acid (30%), this solution is added to the mixture of peptide solutions and stirred for 5 minutes.

23.1 g of poloxamer 188 (Rutrol F68®) and 57.8 g of mannitol are added to the peptide mixture and the entire solution is stirred for another 5 minutes.

The bulk solution containing 10 peptides and excipients was sterile filtered through a pore 0.22 μm filter. 1.485 ml of the solution was placed in sterile 2R glass tubes under sterile conditions, under an inert nitrogen environment, sealed and transferred to a freeze dryer for freeze drying. Freeze drying (see Table 5) consists of freezing bottles at −45 ° C., raising the temperature stepwise to + 20 ° C. (major drying step) and passing the final drying step at + 25 ° C. When all of this was complete, the freeze drier was returned to atmospheric pressure using sterile filtered dry nitrogen.

Lyophilization Parameters Used in Example 2 number step Hours [minutes] Temperature [Celsius] Pressure [mbar] One loading 3 -45 2 frozen 180 -45 3 Dry state 15 -45 1,50E-01 4 Dry state 300 -20 1,50E-01 5 Dry state 480 0 1,50E-01 6 Dry state 360 20 1,50E-01 7 Dry state 60 20 1,50E-01 8 after drying 15 2 5,00E-03 9 after drying 180 20 5,00E-03 10 after drying 120 25 5,00E-03 11 after drying 240 25 5,00E-03 12 Before air inlet One 25 8,00E + 02 13 seal 5 25 8,00E + 02 14 Nitrogen inflow One 25 1,00E + 03 15 Save 3 5 1,00E + 03

Redissolution Procedure: For clinical trials, the formulation described above was dissolved in 700 l of sodium bicarbonate (4.2%). Remove the plastic cap from the vial. At least 30 minutes before re-dissolution, remove the sodium bicarbonate from the refrigerator and open the pack to ensure that the temperature of the contents is room temperature at the time of injection. Prepare syringes and needles for reconstitution. Aseptic technique is used to collect 700 L of diluent for reconstitution of the freeze-dried product. To dissolve the lyophilisate, the bottle and the diluent should be shaken vigorously for 1 minute, to see if the solution is clear, otherwise shake for 1 more minute to confirm that the solution is clear. Ten to thirty minutes after GM-CSF injection, a new injection is used to inject 500 L of the reconstituted formulation into the dermis using the same injection site. Injection should be made within 1 hour after reconstitution. The dissolved lyophilisate can be stored for 1 hour after reconstitution at aseptic room temperature.

Stability testing after reconstitution showed that almost all peptides remained stable enough after reconstitution. However, two specific peptides, IMA-CCN-001 and IMA-RGS-001, were found to have reduced stability (see Table 6a). Since this peptide contains cystine, time dependent dimerization occurs.

The influence of different pH values (pH 8.5) and the success of peptide immunization with high dose carbonate buffers are demonstrated in mouse models. Activity efficiency was tested using a standard ⁵¹ Cr release assay by using standard immunological model peptides in other injection solutions. The results were confirmed by flow cytometry after tetramer staining. Overall, when comparing the activation efficiency of the dermal immunity at pH 7.5, 8.5 (the pH of the IMA901 diluent) and pH9.5, it is confirmed that there is no significant difference.

In addition, high ionic strength did not reduce the observed immune response when compared to isotonic injection buffer. No toxic effect was observed in the immunization of the injection solution with pH 7.5, 8.5 and IMA 901 diluent, but this toxic effect was evident when the pH 9.5 injection solution was used (lesion of necrotic skin at the topical injection site). This result secures that the selected buffer is suitable as a diluent of IMA 901.

Example 3:

Stock solutions of each peptide were prepared by dissolving the peptides in the appropriate solvent according to their dissolution properties (see Table 6b).

Peptides Used in Example 3 Peptide
number: Peptide Peptide Purity (%) Amount [mg] Solvent [ml] 23 IMA-CHI-001 99,7 23.19 11,560 (90% HAc) 15 IMA-FABP7-001 92,0 25.13 4,624 (90% HAc) 17 IMA-MET-005 93,1 24.83 3,853 (50% HAc) 18 IMA-NLGN4X-001 90,3 25.60 5,780 (50% HAc) 22 IMA-TNC-001 94,0 24.60 5,780 (30% HAc) 20 IMA-PTP-003 96,3 24.01 2,890 (20% HAc) 12 IMA-BCA-002 97,7 23.66 3,303 (10% HAc) 13 IMA-BIR-002 96,0 24.08 2,312 (10% HAc) 14 IMA-CSP-001 98,1 23.57 5,780 (10% HAc) 16 IMA-IGF2BP3-001 94,6 24.44 2,890 (10% HAc) 19 IMA-NRCAM-001 96,0 24.08 2,312 (10% HAc) 21 IMA-PTP-005 97,0 23.84 2,312 (10% HAc) 11 IMA-HBV-001 99,0 23.35 4,624 (WFI) -/- Cleaning volume -/- -/- 1,800 (30% HAc) -/- Filling volume -/- -/- 0,180 (WFI)

Because of the different solubility of different peptides, they must be dissolved starting with peptide 1 in the order shown in Table 4. Because of the difference in solubility of each peptide relative to the final volume of the bulk solution, different amounts and concentrations of acetic acid solution and water used for injection (WFI) were used to dissolve the peptide. To increase solubility, each bottle was subjected to strong shaking for up to 5 minutes, and up to 5 minutes of ultrasound when needed. See Table 6 for the amounts and concentrations used.

After the peptide has dissolved, the solution should be mixed starting from No. 1 in the order given in Table 6. The solution is collected in a glass container containing a stir bar. Finally, this solution is added to the peptide solution mixture and stirred for at least 5 minutes.

601.1 mg of poloxamer 188 (Rutrol F68®) and 1502.8 mg of mannitol are added to the peptide mixture and the entire solution is stirred for another 5 minutes.

The bulk solution containing 13 peptides and excipients was sterile filtered through a pore 0.22 μm filter. 1.500 ml of this solution was placed in a 2R glass tube, sealed and transferred to a freeze dryer for freeze drying. Freeze drying (see Table 7) consists of freezing the bottles at −45 ° C., raising the temperature stepwise to + 20 ° C. (main drying step) and passing the final drying step at + 25 ° C. When all of this was complete, the freeze drier was returned to atmospheric pressure using sterile filtered dry nitrogen.

Lyophilization Parameters Used in Example 3 number step Hours [minutes] Temperature [Celsius] Pressure [mbar] One loading 5 -45 2 Primary drying 10 -45 0,50E-01 3 Primary drying 360 -20 0,50E-01 4 Primary drying 480 0 0,50E-01 5 Primary drying 360 20 0,50E-01 6 Primary drying 60 20 0,50E-01 7 Secondary drying 15 20 5,00E-03 8 Secondary drying 180 20 5,00E-03 9 Secondary drying 120 25 5,00E-03 10 Secondary drying 240 25 5,00E-03 11 Nitrogen inflow One 25 1,00E + 03 12 seal 5 25 1,00E + 03

Remelting procedure:

For clinical trials, the formulation described above was dissolved in 700 l of sodium bicarbonate (4.2%). To dissolve the lyophilisate, the bottle and the diluent should be shaken vigorously for 1 minute, to see if the solution is clear, otherwise shake for 1 more minute to confirm that the solution is clear. Injection of 500 μL of solution should be done within 1 hour after reconstitution. The dissolved lyophilisate can be stored for 1 hour after reconstitution at aseptic room temperature.

Stability tests after reconstitution showed that almost all peptides remained stable enough after reconstitution (see Table 8).

HPLC results on stability of use after reconstitution with 4.2% sodium bicarbonate. Comparison between formulations with and without excipients Peptide first 2 hours 4 hours 6 hours Peptide [%] Peptide [%] Peptide [%] Peptide [%] NRCAM-001 + Mannitol / rutrol 100.0 99.9 100.0 99.5 No additives 100.0 99.8 100.2 95.6 BIR-002 + Mannitol / rutrol 100.0 100.8 101.2 100.9 No additives 100.0 100.6 101.4 96.2 CSP-001 + Mannitol / rutrol 100.0 100.0 100.1 99.6 No additives 100.0 99.2 99.7 95.0 PTP-003 + Mannitol / rutrol 100.0 100.3 100.5 100.1 No additives 100.0 100.4 100.9 96.1 IGF2BP3-001 + Mannitol / rutrol 100.0 100.4 100.6 100.7 No additives 100.0 100.0 101.2 96.0 PTP-005 + Mannitol / rutrol 100.0 100.1 100.3 99.8 No additives 100.0 99.9 100.4 95.8 TNC-001 + Mannitol / rutrol 100.0 100.5 100.6 100.2 No additives 100.0 100.0 100.4 95.7 MET-005 + Mannitol / rutrol 100.0 100.5 100.7 100.4 No additives 100.0 100.3 100.7 96.0 FABP7-001 + Mannitol / rutrol 100.0 100.9 100.7 100.6 No additives 100.0 100.5 100.9 96.3 NLGN4X-001 + Mannitol / rutrol 100.0 100.8 100.9 100.5 No additives 100.0 99.9 100.5 95.7 HBV-001 + Mannitol / rutrol 100.0 100.3 100.6 100.3 No additives 100.0 100.2 100.6 95.8 CHI-001 + Mannitol / rutrol 100.0 100.0 100.7 100.2 No additives 100.0 102.2 101.3 90.6 BCA-002 + Mannitol / rutrol 100.0 99.8 99.6 98.9 No additives 100.0 99.9 100.1 95.4

Example 4:

Stock solutions of each peptide were prepared by dissolving the peptides in their appropriate solvents according to their dissolution properties (see Table 9).

Peptides Used in Example 4 Peptide Sequence Identification Number: Peptide Peptide Purity (%) Amount [mg] Solvent [ml] 23 IMA-FABP7-001 92,0 28,27 4,335 (90% HAc) 15 IMA-MET-005 94,0 27,67 4,335 (50% HAc) 17 IMA-NLGN4X-001 91,0 28,58 6,503 (50% HAc) 18 IMA-TNC-001 94,0 27,67 3,251 (50% HAc) 22 IMA-PTP-003 97,0 26,81 3,251 (20% HAc) 20 IMA-BCA-002 98,0 26,54 3,251 (10% HAc) 12 IMA-BIR-002 96,0 27,09 3,251 (10% HAc) 13 IMA-CSP-001 91,0 28,58 5,202 (10% HAc) 14 IMA-IGF2BP3-001 95,0 27,38 5,202 (10% HAc) 16 IMA-NRCAM-001 96,0 26,54 3,251 (10% HAc) 19 IMA-PTP-005 97,0 26,81 3,251 (10% HAc) 21 IMA-HBV-001 99,0 26,27 5,202 (WFI) -/- Cleaning volume -/- -/- 3,375 (30% HAc) -/- Filling volume -/- -/- 13,839 (WFI)

Because of the different solubility of different peptides, they must be dissolved starting with peptide 1 in the order shown in Table 4. Because of the difference in solubility of each peptide relative to the final volume of the bulk solution, different amounts and concentrations of acetic acid solution and water used for injection (WFI) were used to dissolve the peptide. To increase solubility, each bottle was subjected to strong shaking for up to 5 minutes, and up to 5 minutes of ultrasound when needed. See Table 9 for the amounts and concentrations used.

After the peptide has dissolved, the solution should be mixed starting with the peptide of SEQ ID NO: 23 in the order given in Table 9. The solution is collected in a glass container containing a stir bar. Finally, this solution is added to the peptide solution mixture and stirred for at least 5 minutes.

624.2 mg of poloxamer 188 (Rutrol F68®) and 1560.6 mg of mannitol are added to the peptide mixture and the entire solution is stirred for another 5 minutes.

The bulk solution containing 12 peptides and excipients was sterile filtered through a pore 0.22 μm filter. 1.500 ml of this solution was placed in a 2R glass tube, sealed and transferred to a freeze dryer for freeze drying. Freeze drying (see Table 10) consists of freezing bottles at −45 ° C., raising the temperature stepwise to + 20 ° C. (main drying step) and passing the final drying step at + 25 ° C. When all of this was complete, the freeze drier was returned to atmospheric pressure using sterile filtered dry nitrogen.

Freeze Drying Parameters for Example 4 number step Hours [minutes] Temperature [Celsius] Pressure [mbar] One loading 5 -45 2 Primary drying 10 -45 0,50E-01 3 Primary drying 360 -20 0,50E-01 4 Primary drying 480 0 0,50E-01 5 Primary drying 360 20 0,50E-01 6 Primary drying 60 20 0,50E-01 7 Secondary drying 15 20 5,00E-03 8 Secondary drying 180 20 5,00E-03 9 Secondary drying 120 25 5,00E-03 10 Secondary drying 240 25 5,00E-03 11 Nitrogen inflow One 25 1,00E + 03 12 seal 5 25 1,00E + 03

Stability tests conducted at different temperatures showed that all peptides remained sufficiently stable in adverse environments (+ 25 ° C.) (see Table 11).

HPLC results for stability of peptide in formulation for 3 months at 25 ° C +/- 2 ° C Peptide first[%] 1M [first%] 1M [first%] 3M [first%] NRCAM-001 100.0 97.90 96.73 97.59 BIR-002 100.0 97.49 96.24 98.93 CSP-001 100.0 98.04 96.99 98.71 PTP-003 100.0 98.29 97.11 98.29 IGF2BP3-001 100.0 98.01 96.77 97.34 PTP-005 100.0 98.16 97.10 98.30 TNC-001 100.0 97.86 96.80 97.21 MET-005 100.0 97.49 96.13 97.21 FABP7-001 100.0 98.43 97.71 98.67 NLGN4X-001 100.0 97.90 96.30 96.41 HBV-001 100.0 97.83 96.47 97.50 BCA-002 100.0 98.20 97.05 97.88

HPLC results for stability of peptide in formulation for 3 months at 5 ° C +/- 3 ° C Peptide first[%] 1M [first%] 1M [first%] 3M [first%] NRCAM-001 100.0 99.13 98.17 98.79 BIR-002 100.0 98.53 97.28 100.01 CSP-001 100.0 98.99 98.05 99.81 PTP-003 100.0 99.07 97.95 98.68 IGF2BP3-001 100.0 99.00 97.92 98.57 PTP-005 100.0 98.93 97.79 98.67 TNC-001 100.0 99.02 98.10 98.72 MET-005 100.0 99.06 97.92 98.59 FABP7-001 100.0 99.08 98.15 98.66 NLGN4X-001 100.0 99.12 98.24 98.84 HBV-001 100.0 98.87 97.76 98.47 BCA-002 100.0 99.13 98.04 98.73

HPLC results for stability of peptide in formulation for 3 months at -20 ° C +/- 5 ° C Peptide first[%] 1M [first%] 1M [first%] 3M [first%] NRCAM-001 100.0 99.23 97.86 98.91 BIR-002 100.0 98.43 96.98 100.14 CSP-001 100.0 98.99 97.61 99.61 PTP-003 100.0 99.11 97.48 98.57 IGF2BP3-001 100.0 99.10 97.54 98.42 PTP-005 100.0 98.99 97.41 98.34 TNC-001 100.0 99.01 97.61 98.57 MET-005 100.0 99.14 97.54 99.00 FABP7-001 100.0 99.09 97.60 98.39 NLGN4X-001 100.0 99.05 97.60 98.62 HBV-001 100.0 98.93 97.39 98.34 BCA-002 100.0 99.17 97.61 98.61

1: Analytical HPLC chromatogram of peptides (SEQ ID NOs: 1-11) containing beta-naphthylalanine (NAL) as internal standard.

2: Stability of Formulation 1 (control) without any excipients at + 25 ° C. FIG.

3: Stability of Formulation 2 according to the invention with mannitol and Lutrol F68® poloxamer 188) at + 25 ° C. FIG.

4: Stability of Formulation 3 according to the invention with mannitol and Tween 80® at + 25 ° C. FIG.

5: Stability of Formulation 4 according to the invention with mannitol and Tween 80® at + 25 ° C. FIG.

6: Stability of Formulation 1 (control) without any excipients at + 40 ° C. FIG.

7: Stability of Formulation 2 according to the invention with mannitol and Lutrol F68® (poloxamer 188) at + 40 ° C. FIG.

8: Stability of Formulation 3 according to the invention with mannitol and Tween 80® at + 40 ° C. FIG.

9: Stability of Formulation 4 according to the invention with mannitol and Tween 80® at + 40 ° C. FIG.

10: Effect of poloxamer 188 (Luprol F68® from BASF, Rubischshafen, Germany) and mannitol on in vivo CD8 + T cell initiation. Mice were sacrificed 9 days after immunization, splenocytes were collected, stained with tetramer and analyzed by flow cytometry. The proportion of tetramer positive cells among CD8 + T cells was indicated by an error bar showing the standard deviation of the mean. All three groups of peptide immunized significantly differed from the negative control according to analysis by the T-test of both unpaired students (p <0.05). There was no significant difference in the groups with and without poloxamer 188 (Rutrol F68®) / mannitol (p = 0.49).

11 is the manufacturing process described in Example 2. FIG.

Figure 12: Analytical HPLC chromatogram of peptides (SEQ ID NOs: 11-23) containing beta-naphthylalanine (NAL) as internal standard.

13: "In-use" stability of peptides of SEQ ID NOs: 11-23 with mannitol / rutrol F68®.

Figure 14: "in use" stability of the peptides of SEQ ID NOs: 11-23 without any excipients.

15: Stability of peptides of SEQ ID NOs 11-22 with mannitol / rutrol F68® at + 25 ° C. FIG.

16: Stability of peptides of SEQ ID NOs: 11-22 with mannitol / rutrol F68® at + 5 ° C. FIG.

17: Stability of peptides of SEQ ID NOs 11-22 with mannitol / rutrol F68® at −20 ° C. FIG.

18: 24 months stability data at −20 ° C. for the formulation shown in Example 2. FIG.

19: 24 months stability data at + 5 ° C. for the formulation shown in Example 2. FIG.

20: 24 months stability data at + 25 ° C. for the formulations shown in Example 2. FIG.

The sequence listing shows the peptides (SEQ ID NOs 1 to 23) used in the formulations according to the invention.

                     SEQUENCE LISTING <110> immatics biotechnologies GmbH <120> Novel formulations of tumour-associated peptides binding to        human leukocyte antigen (HLA) class I or II molecules for        vaccines <130> I31370 <150> US 61 / 047,669 <151> 2008-04-24 <150> EP 08008292.8 <151> 2008-04-30 <160> 23 <170> PatentIn version 3.4 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Ser Gln Asp Asp Ile Lys Gly Ile Gln Lys Leu Tyr Gly Lys Arg Ser 1 5 10 15 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Val Met Ala Gly Asp Ile Tyr Ser Val 1 5 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Ser Val Ala Ser Thr Ile Thr Gly Val 1 5 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Ala Leu Ala Asp Gly Val Gln Lys Val 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Leu Leu Gly Ala Thr Cys Met Phe Val 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Ser Val Phe Ala Gly Val Val Gly Val 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Ala Leu Phe Asp Gly Asp Pro His Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Tyr Val Asp Pro Val Ile Thr Ser Ile 1 5 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Ser Thr Ala Pro Pro Val His Asn Val 1 5 <210> 10 <211> 9 <212> PRT <213> Homo sapiens <400> 10 Leu Ala Ala Leu Pro His Ser Cys Leu 1 5 <210> 11 <211> 10 <212> PRT <213> Homo sapiens <400> 11 Phe Leu Pro Ser Asp Phe Phe Pro Ser Val 1 5 10 <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400> 12 Ala Leu Trp Ala Trp Pro Ser Glu Leu 1 5 <210> 13 <211> 15 <212> PRT <213> Homo sapiens <400> 13 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn 1 5 10 15 <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Thr Met Leu Ala Arg Leu Ala Ser Ala 1 5 <210> 15 <211> 9 <212> PRT <213> Homo sapiens <400> 15 Leu Thr Phe Gly Asp Val Val Ala Val 1 5 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Lys Ile Gln Glu Ile Leu Thr Gln Val 1 5 <210> 17 <211> 17 <212> PRT <213> Homo sapiens <400> 17 Thr Phe Ser Tyr Val Asp Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr 1 5 10 15 Gly      <210> 18 <211> 9 <212> PRT <213> Homo sapiens <400> 18 Asn Leu Asp Thr Leu Met Thr Tyr Val 1 5 <210> 19 <211> 9 <212> PRT <213> Homo sapiens <400> 19 Gly Leu Trp His His Gln Thr Glu Val 1 5 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <400> 20 Ala Ile Ile Asp Gly Val Glu Ser Val 1 5 <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Lys Val Phe Ala Gly Ile Pro Thr Val 1 5 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Ala Met Thr Gln Leu Leu Ala Gly Val 1 5 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 Ser Leu Trp Ala Gly Val Val Val Leu 1 5  

Claims (15)

2 to 18 tumor associated peptides, and Mannitol and poloxamer 188 or mannitol and polyoxyethylene (20) sorbitan monooleate (Twin 80 (registered trademark)) As comprising a pharmaceutical composition for inducing an anti-tumor immune response, Each peptide has a length of from 8 to 22 amino acids, The peptide exhibits a solubility of at least 2.7 mg / ml in 90% acetic acid, The weight ratio of peptide to mannitol to poloxamer 188 is 1: 5: 1.5 or 1: 8: 2.2 or 1: 5: 1.5 to 1: 8: 2.2, The weight ratio of the peptide to mannitol to polyoxyethylene (20) sorbitan monooleate (Tween 80®) is 1: 2: 1.5 or 1: 8: 2.2 or 1: 2: 1.5 to 1: 8: 2.2, the pharmaceutical composition. 2 to 18 tumor associated peptides, and Poloxamer 188 or mannitol and polyoxyethylene (20) sorbitan monooleate (Twin 80 (registered trademark)) As comprising a pharmaceutical composition for inducing an anti-tumor immune response, Each peptide has a length of from 8 to 22 amino acids, The peptide exhibits a solubility of at least 2.7 mg / ml in 90% acetic acid, The weight ratio of peptide to poloxamer 188 is 1: 2 or 1: 2.2 or 1: 2 to 1: 2.2, The weight ratio of the peptide to mannitol to polyoxyethylene (20) sorbitan monooleate (Tween 80®) is 1: 2: 1.5 or 1: 8: 2.2 or 1: 2: 1.5 to 1: 8: 2.2, the pharmaceutical composition. The method of claim 1, SEQ ID NO: 1 to SEQ ID NO: 10, or SEQ ID NO: 12 to SEQ ID NO: 23, two or more peptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, Sequence At least one peptide, or variant or salt thereof, comprising SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 13, and additionally mannitol and poloxamer 188, wherein peptide to mannitol versus poloxamer 188 Pharmaceutical composition having a weight ratio of 1: 5: 1.5 or 1: 8: 2.2 or 1: 5: 1.5 to 1: 8: 2.2. The method of claim 2, SEQ ID NO: 1 to SEQ ID NO: 10, or SEQ ID NO: 12 to SEQ ID NO: 23, two or more peptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, Sequence At least one peptide, or variant or salt thereof, comprising SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 13, and additionally poloxamer 188, wherein the weight ratio of peptide to poloxamer 188 Pharmaceutical compositions wherein 1: 2 or 1: 2.2 or 1: 2 to 1: 2.2. The method of claim 1, SEQ ID NO: 1 to SEQ ID NO: 10, or SEQ ID NO: 12 to SEQ ID NO: 23, two or more peptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, Sequence At least one peptide comprising SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 13, or a variant or salt thereof, and further mannitol and polyoxyethylene (20) sorbitan monooleate (Twin 80 ( Trademark), wherein the weight ratio of peptide to mannitol to polyoxyethylene (20) sorbitan monooleate (Tween 80®) is 1: 2: 1.5 or 1: 8: 2.2 or 1: Pharmaceutical composition wherein 2: 1.5 to 1: 8: 2.2. 6. The method according to any one of claims 1 to 5, Pharmaceutical composition wherein the peptide has a total length of 9 to 16 amino acids. 3. The method according to claim 1 or 2, A pharmaceutical composition wherein at least one peptide comprises a non-peptide bond. 3. The method according to claim 1 or 2, A peptide comprising an amino acid sequence having SEQ ID NO: 1, SEQ ID NO: 2 or both, and further comprising one or more peptides consisting of the amino acid sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 10 Pharmaceutical composition. 3. The method according to claim 1 or 2, A peptide consisting of an amino acid sequence having SEQ ID NO: 12, SEQ ID NO: 13 or both, and further comprising one or more peptides consisting of the amino acid sequence of any one of SEQ ID NO: 14 to SEQ ID NO: 23 Pharmaceutical composition. 3. The method according to claim 1 or 2, A pharmaceutical composition further comprising a peptide comprising the amino acid sequence of SEQ ID NO: 11, wherein the peptide is not each full length tumor associated polypeptide. 3. The method according to claim 1 or 2, A pharmaceutical composition wherein the amount of peptide present in the pharmaceutical composition is tissue, cancer or patient-specific. 3. The method according to claim 1 or 2, A pharmaceutical composition further comprising one or more adjuvants. An anticancer vaccine comprising the pharmaceutical composition according to claim 1. (a) a container containing the pharmaceutical composition according to claim 1 in the form of a solution or lyophilized; (b) optionally, a second container containing a diluent, or reconstitution solution, one or more adjuvants or combinations thereof for a lyophilized formulation; And (c) optionally, instructions for (i) use of a solution, or (ii) reconstitution of the freeze-dried formulation, use of the freeze-dried formulation, or both. Including, kit. 15. The method of claim 14, and a kit comprising (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, and (v) a syringe.

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