CN106146663B - Novel antibody-drug conjugates labeled with unnatural amino acids and their preparation - Google Patents

Abstract

The present invention provides site-directed mutation-containing rituximab containing unnatural amino acid markers. Also provided are methods for site-directed mutagenesis and site-directed small molecule coupling of rituximab, the method comprising using gene codon extension technology to site-specifically introduce unnatural amino acids into rituximab genes, with the help of unnatural amino acids and modifiers, such as poly bifunctional linker DIBO ‑DOTA fixed-point connection, further realize the fixed-point coupling of rituximab and radioactive isotopes, such as Cu ⁶⁴ , through DOBO‑DOTA. The present invention further relates to the application of site-specific coupling small molecule rituximab, such as the application of imaging, tracing and therapeutic radioimmunoconjugates.

Description

Novel antibody-drug conjugates labeled with unnatural amino acids and their preparation

technical field

The invention belongs to the field of biopharmaceuticals, and relates to a method for point-fixed insertion of unnatural amino acids in proteins and a point-point coupling method for small molecule drugs. The method includes site-directed introduction of unnatural amino acids into proteins based on gene codon expansion technology, and site-specific linking of unnatural amino acids with effector small molecules.

Background technique

The following takes rituximab as an example to illustrate the background technology of the present invention, but the following content can not be understood as an acknowledgment of the prior art in any case, and it can not be considered that the present invention is only applicable to rituximab monoclonal antibody.

(1) Rituximab

The B lymphocyte antigen CD20 molecule is expressed on the surface of all B cells from late pre-B cells to mature B cells (Loken et al., 1987, Blood 70, 1316-1324). It is stably expressed in more than 90% of B-cell lymphomas , so it is very suitable as a target for targeted therapy (McLaughlin et al., 1998, Journal of clinical oncology: official journal of the American Society of Clinical Oncology 16, 2825-2833). CD20 monoclonal antibody has achieved satisfactory results in the treatment of B-cell lymphoma and chronic lymphocytic leukemia. The anti-CD20 monoclonal antibody Rituximab (C2B8)-rituximab, namely MabThera, prepared by IDEC in the United States for B-cell lymphoma, is the first antibody approved by the FDA for the treatment of B-cell lymphoma and the first to be marketed A human-mouse chimeric monoclonal antibody targeting CD20 on the surface of lymphoma and leukemia B cells. It is an IgG1κ immunoglobulin, the variable regions of the light and heavy chains are of murine origin, and the constant regions are of human origin (DrugBank DB0073). The heavy chain of rituximab is made up of 451 amino acids, and the light chain is made up of 213 amino acids, and its light chain sequence is as shown in SEQ ID NO:1, and its nucleotide sequence is as shown in SEQ ID NO:2; Heavy chain sequence As shown in SEQ ID NO:3, its nucleotide sequence is shown in SEQ ID NO:4. Its main mechanism of action includes antibody-mediated cell killing, complement-mediated cell killing and induction of tumor cell apoptosis.

Although rituximab has shown a good curative effect in clinical treatment, the total response rate is about 50%, the complete cure rate is no more than 20%, and recurrence and drug resistance often occur (McLaughlin et al., 1998, Journal of clinical oncology: official journal of the American Society of Clinical Oncology 16, 2825-2833; Coiffier et al., 2002, The New England journal of medicine 346, 235-242; Maloney et al., 1997, Blood 90, 2188-2195). Therefore, it is of great market value to develop more effective antibody drugs targeting CD20.

(2) Antibody-drug conjugates

Radiotherapy and chemotherapy are widely used clinically as the main means of tumor treatment. They mainly kill tumor cells by causing DNA double-strand breaks, inhibiting cell mitosis, and inhibiting the synthesis of DNA and related proteins. However, traditional radiotherapy and chemotherapy have a greater killing effect on normal cells, so research on improving drug efficacy and reducing side effects by coupling isotopes or chemotherapeutic drugs to targeting antibodies has been widely concerned.

Antibody drug conjugates are an emerging part of protein medicine, also known as immunoconjugates. Immunoconjugate drugs are composed of two parts: targeted monoclonal antibody components and cell killing components. According to different cell killing components, they are mainly divided into radioimmunotherapy drugs (conjugated isotopes), antibody drug conjugates ( Conjugated small molecule drugs), immunotoxins (conjugated bacterial toxins) and enzyme-linked antibody prodrug therapeutic drugs (conjugated enzymes) into four categories (Weiner et al., 2012, Cell 148, 1081-1084). Coupling the therapeutic drug with the monoclonal antibody, using the targeting of the antibody to deliver the therapeutic drug to the target cell accurately, can effectively increase the local drug concentration of the target cell and reduce the normal cell distribution of the drug in the body, thereby achieving high efficiency and low toxicity of the drug the therapeutic effect.

Take radioimmunoconjugate drugs as an example, which are currently widely used in clinical practice. Radioimmunoimaging drugs (radioimmunoimaging, RII) use monoclonal antibodies to transport diagnostic nuclides to the target site, and perform tomographic imaging and computer three-dimensional reconstruction through ECT to display the size and location of the tumor at the site; radioimmunoimaging Therapeutic drug (radiommunotherapy, RIT), that is, using antibodies as carriers to guide therapeutic nuclide to the tumor site for internal irradiation, has achieved outstanding clinical effects in lymphoma. Among them, two radioimmunotherapy drugs have passed the FDA's drug approval. Both of these drugs target CD20 for the treatment of relapsed or drug-resistant follicular lymphoma: one is 90Y-ibritumomab tiuxetan (Zevalin), and the other is 131I-tositumomab (Bexxar 2014 because it has the same effect as 90Y-ibritumomab tiuxetan). withdrawal from the market). Among them, the overall response rate of patients to Zevalin reached 80%, and the complete cure rate was about 30% (Goldsmith et al., 2010, Seminars in nuclear medicine 40, 122-135).

(3) Antibody drug conjugation technology

Traditional antibody conjugates usually use the amino group of the lysine side chain on the antibody or the sulfhydryl group after disulfide bond reduction as functional groups to couple small molecules. There are about 80 lysines and about 30 cysteines on the surface of the antibody, so the traditional modification method is non-specific and non-quantitative. However, the conjugates of antibody small molecules lack effective separation means, so the traditional coupling method is not suitable for quality control of large-scale production and preparation. There are many functional regions on the surface of the antibody, and even destroy the disulfide bond of the antibody. Random coupling may affect the binding of the antibody antigen, reduce the half-life of the antibody in the body, and affect antibody-mediated cell killing and complement-mediated cell killing. Killing effect, coupling too many small molecules will also increase the immunogenicity of antibody drugs.

(4) Gene codon expansion technology

In recent years, the genetic code expansion technology has developed rapidly. Using the amber stop codon as the sense codon, the designed unnatural amino acid can be introduced into the protein by introducing the corresponding orthogonal tRNA and aminoacyl tRNA synthetase. According to the properties of unnatural amino acids, proteins can be endowed with special functions. So far, this technology has successfully expressed dozens of unnatural amino acids on the surface of proteins. The unnatural amino acids involved have alkynyl groups and azide groups. Using these bioorthogonal groups, specific site-directed modification of proteins.

Facing the problem of indeterminate and indeterminate modification of existing antibody small molecule conjugated drugs, the application of gene codon extension technology to the preparation of existing antibody conjugates will effectively promote the development of antibody conjugates.

Invention content:

After pondering and researching the prior art, the inventor used the protein translation system of tRNA (tRNA ^Pyl ) and pyrrolysyl-tRNA synthetase (tRNA ^Pyl /PylRS) of Methanococcus ancient to insert unnatural amino acids into the surface of the protein , so as to obtain the target protein of site-directed mutation. Then, the site-directed mutagenesis protein is used as a raw material for further site-specific coupling, and is coupled with a tether or a small effector molecule, and the resulting conjugate is further coupled with a small effector molecule, such as a radionuclide, to obtain a single The product of site-specific coupling, in particular, the protein is rituximab.

Compared with other methods, the advantages of the present invention can be reflected in one or more of the following aspects:

1. Unnatural amino acids can be introduced at any position in the protein, thereby creating a raw material protein that can only be specifically modified at this position;

2. Using the unique active groups on unnatural amino acids can achieve efficient and specific modification purposes;

3. Site-specific conjugation at specific sites can bring better drug efficacy. Selecting the coupling in the non-functional area can effectively reduce the impact of drug coupling on protein activity, stability, and half-life; selecting coupling in the non-exposed area and fixing the number of modifications of small molecules can reduce potential immunogenicity , reduce the enzymatic hydrolysis in the body, thereby reducing the possibility of off-target conjugates;

4. Through the optimization of the modification conditions, the copper-free Click reaction mediated by cyclooctyne can be used to achieve high efficiency, no harm to the protein, and a simple and easy coupling reaction.

Specifically, in a specific embodiment of the present invention, there is provided a target protein that introduces an unnatural amino acid, such as rituximab, mainly through two steps: (1) constructing a protein containing Amber codon mutation encoding target protein, such as the vector of rituximab gene, (2) construct and obtain pXH-N ₃ plasmid, co-express steps (1) and (2) in a suitable host cell, and in Unnatural amino acids are added to the medium to obtain a mutated target protein, such as rituximab.

The principle of the mutation system is that the mutant tRNA ^Pyl /PylRS satisfies the following relationship: (1): the mutant tRNA ^Pyl cannot utilize the lysyl tRNA synthetase of the host cell, and can only be acylated by the mutant PylRS; ( 2): The mutant PylRS can only acylate tRNA ^Pyl and cannot acylate other tRNAs. Therefore, the relationship between the mutant tRNAPyl and PylRS is orthogonal, that is, the mutant PylRS can only acylate the mutant tRNA ^Pyl , and the mutant tRNA ^Pyl can only be acylated by the mutant PylRS, that is to say, the mutant tRNA ^Pyl and PylRS in the same plasmid are absolutely mutually specific. This orthogonal enzyme is the only enzyme that can acylate non-natural amino acids to this orthogonal tRNA, and can only acylate this tRNA, but not other tRNAs. The obtained orthogonal lysyl tRNA synthase/tRNA system makes Lys-azido of non-20 common amino acids, also known as: NAEK, corresponding to the amber codon, thereby introducing unnatural amino acids into the target protein at a fixed point, such as rituximab.

In a specific embodiment of the present invention, by inserting the rituximab gene with an amber codon into a vector such as pEF-1b, the vector obtained after the insertion and the pXH-N ₃ together are transiently Transfect the Freestyle293F cell line, then add unnatural amino acids such as NAEK to the medium, and finally obtain the target protein of site-directed mutation, such as mutant rituximab, from the medium supernatant.

In a specific embodiment of the present invention, the present invention provides a mutated recombinant protein or peptide that introduces unnatural amino acids site-specifically in mammalian cells, such as introducing unnatural amino acids NAEK at specific sites, and its characteristic azide The group can specifically react with the conjugate, such as a bifunctional linker, to undergo a Click reaction, thereby coupling the target small molecule to the target protein at a specific point.

In another embodiment of the present invention, a bifunctional tether DIBO-DOTA is provided. One end of the linker has cyclooctyne (DIBO), which can specifically recognize and couple to the azide group; the other end has a chelating group 1,4,7,10-tetraazacyclododecane-1,4, 7,10-tetraacetic acid (DOTA), this group can firmly chelate Y ⁹⁰ , Lu ¹⁷⁷ and other metal isotopes. For example, mutant rituximab and its site-specific conjugated small molecules,

In another embodiment of the present invention, the purified NAEK site-directed mutation target protein or peptide, such as rituximab, is subjected to a copper-free Click reaction with DIBO-DOTA, so that DIBO-DOTA passes through the rituximab Fixed-point and quantitative coupling of non-natural amino acids containing azide groups. Due to the high efficiency of the reaction, fixed-point and quantitative coupling of small molecules such as DIBO-DOTA target proteins or peptides can be obtained after simple desalting operations. . In vitro experiments preliminarily proved that rituximab still has biological activity after unnatural amino acid substitution and site-specific coupling of small molecules.

In one embodiment of the present invention, the target protein or peptide coupled with the linker DIBO-DOTA, such as rituximab, is reacted with a radioactive isotope, and then the radioactive isotope is quantitatively coupled to the target protein or peptide Among them, such as rituximab. The resulting antibody coupled with a radioactive isotope can be used for more effective radiographic imaging and radioactive targeted therapy.

In one embodiment of the present invention, the present invention selects a mammalian cell suspension expression system. The cells used in the system have been subjected to suspension acclimation, which can not only express the target protein in large quantities instantaneously, but also carry out large-scale fermentation and culture by constructing a stable line.

More specifically, the present invention provides:

1. A mutated polypeptide, the amino acid at at least one position of which is mutated into an unnatural amino acid, and the unnatural amino acid is:

Lys-azido (NAEK) as shown, or other unnatural amino acids containing azide structure.

2. The mutated polypeptide according to item 1, which is rituximab.

3. The mutated polypeptide according to item 2, wherein the mutation is located at any one or more positions in SEQ ID NO: 2 and/or SEQ ID NO: 4.

4. The mutated polypeptide as described in item 3, wherein the mutation site is selected from: K168 shown in SEQ ID NO: 2, A122 in SEQ ID NO: 4, or SEQ ID NO: 2 or SEQ ID NO : One or more of the sites on 4 that have less influence on the activity.

5. The mutated polypeptide as described in item 1, wherein the mutated amino acid is the Nth amino acid, and the connection mode of the mutated amino acid in the polypeptide is shown in the following formula:

Wherein, the direction from R1 to R2 is the N-terminal to C _- terminal direction of the amino acid sequence, R1 is the _1st to N- _1th amino acid residue in the polypeptide,

R ₂ is the amino acid residue from the N+1 position to the C-terminus in the polypeptide, and R ₄ is

6. The mutated polypeptide according to item 5, which is a mutated rituximab.

7. The mutated polypeptide according to item 6, wherein the N-th amino acid is the amino acid at any one or more positions in SEQ ID NO: 2 or SEQ ID NO: 4.

8. The mutated polypeptide according to item 7, wherein the mutation site is selected from: K168 shown in SEQ ID NO: 2, A122 in SEQ ID NO: 4, or SEQ ID NO: 2 or SEQ ID NO : One or more of other sites on 4 that have less effect on activity.

9. The mutant polypeptide as described in item 1, which comprises a bifunctional linking arm available for coupling azide and metal isotope, one end of said linking arm contains a cyclooctyne (DIBO) group, and the other end contains DOTA, so The structure of the dual-function connecting arm is shown in the following formula:

10. The mutated polypeptide according to item 9, wherein said mutated polypeptide is mutated rituximab.

11. The mutated polypeptide according to any one of the preceding items, whose heavy chain amino acid sequence is SEQ ID NO:10.

12. The mutated polypeptide according to any one of the preceding items, whose light chain amino acid sequence is SEQ ID NO:8.

13. The mutated polypeptide as described in item 1 after site-specific coupling, its structure is shown in the following formula:

Among them, where the Nth amino acid is mutated, R1 is the amino acid residue from the 1st to the N-1th position, R2 is the amino acid residue from the N+1st position to the C-terminal, and R3 is DOTA or other directly or indirectly providing functional Small molecule.

14. The mutated polypeptide after site-directed coupling according to item 13, wherein the polypeptide before mutation comprises the heavy chain with the amino acid sequence of SEQ ID NO: 2 and/or the light chain with the amino acid sequence of SEQ ID NO: 4.

15. The mutated polypeptide after site-directed coupling according to item 14, wherein R ₃ is DOTA, which chelates radioactive isotopes.

16. The mutated polypeptide after site-directed coupling according to item 15, the radioactive isotope is selected from Lu ¹⁷⁷ , Cu ⁶⁴ or Y ⁹⁰ .

17. The mutated polypeptide after site-directed coupling as described in any one of items 13-16, _R in the conjugate is selected from adriamycin (doxorubicin), monomethyl auristatin (Monomethyl auristatin E, MMAE).

18. A nucleic acid molecule encoding the mutated polypeptide of items 1-12.

19. A vector comprising the nucleic acid molecule of item 18.

20. A vector which is pXH-N3.

21. A pharmaceutical composition, which contains an effective amount of the mutated polypeptide described in any one of items 1-12, or the mutated polypeptide described in items 13-17 after site-directed conjugation.

22. The pharmaceutical composition as described in item 21, used in the preparation of medicines for radiographic imaging, treatment of intractable malignant tumors or tumor recurrence.

23. A microorganism comprising the carrier according to item 19 or 20.

24. The microorganism according to item 23, which is Escherichia coli.

25. An animal cell comprising the vector according to item 19 or 20.

26. The animal cell according to item 25, which is Freestyle293 cell line.

Description of drawings:

Figure 1: Expression and activity verification of MabThera in mammalian cells

A: Western Blot verification of antibody expression: the first lane is the empty vector transformation control, and the second lane is the expression of the vector inserted with the rituximab gene. The antibody shown in the figure was successfully expressed;

B: The effect of different expression vectors on the expression level: from left to right are pcDNA3.1, pEF-1b, pBudce4.1; pEF-1b vector is most conducive to the expression of antibodies;

C: Confocal fluorescent confocal verification of expressed antibody activity: from left to right, the antibodies acted on Jurket, Raji, and RamosRA1 cells respectively, in which Jurkat cells were CD20-negative cells, and as negative controls, Raji and Ramos RA1 cells were CD20-positive cells;

D: Flow cytometry analysis to verify the expression antibody activity: the antibody binding response value of CD20-negative cells Jurket in the M1 region, and the antibody binding corresponding value of CD20-positive cells Raji and Ramos RA cells in the M2 region;

Figure 2: Optimization and construction of pXH-N ₃ vector

A: Schematic diagram of pXH-N ₃ vector;

B: Western Blot to verify the effect of various types of promoters on the efficiency of unnatural amino acid insertion by promoting tRNA. The names of the promoters from left to right are: U1, mu6, H1, hu6, 7sk; The expression efficiency of natural amino-inserted protein is the highest;

C: At the cellular level, green fluorescent protein verifies the insertion efficiency of unnatural amino acids in each promoter; it is consistent with the results in Figure 2B;

D: The effect of different promoters on the read-through efficiency of the stop codon TAG, from left to right are U1, mu6, H1, hu6, 7sk promoters, the high read-through efficiency is equivalent to the high efficiency of unnatural amino acid insertion;

E: The influence of different numbers of tRNAs in tandem with 7sk promoter on the read-through efficiency of stop codon TAG, from left to right are 1, 2, 3, 4 and 5 7sk-tRNA tandem units respectively. It can be seen from the figure that under the 7sk promoter , the unnatural amino acid insertion efficiency of 4 tRNAs in series is the highest;

Figure 3: Expression of mutant rituximab

A. Schematic diagram of the crystal structure of the variable region of rituximab and CD20-binding short peptide (Ding Jianping et al. J. Biol. Chem. 2007, 282: 15073-15080); The three receptor binding sites LC-P15, LC-K168 and HC-A122 are potential unnatural amino acid insertion sites;

B. Western Blot verification of mutant protein expression, from left to right: WT Rituxan (without unnatural amino acid insertion type Rituxan); LC-P15 site mutation Rituxan without unnatural amino acid (UAA), plus UAA; HC-A122 Point mutation Rituxan does not add UAA, but adds UAA; LC-K168 site mutation Rituxan does not add UAA, but adds UAA; it can be seen from the figure that HC-A122 and LC-K168 sites have successfully inserted unnatural amino acids;

Figure 4: Condition optimization for the expression of mutant rituximab

A. Effects of different cell line pairs and transfection reagents on expression conditions: from left to right, the Freestyle293 cell line was transfected with 25kDa linear PEI, the Freestyle293 cell line was transfected with 40kDa branched PEI, and the Freestyle CHO cell line was transfected with 25kDa linear PEI transfection, Freestyle CHO cell line 40kDa branched chain PEI transfection; it can be seen from the figure that the expression of 293 cell line is significantly higher than that of CHO cell line under transient conditions, and the expression level of 40kDa branched chain PEI is better than that of 25kDa linear PEI the expression effect;

B. The effect of the ratio (mass ratio) of the total amount of transfected DNA to PEI on the transfection efficiency, as can be seen from the figure, when DNA:PEI=1:4, the expression efficiency is higher;

C. The total amount of fixed DNA, in the expression process of HC-A122 mutant protein, the influence of the ratio of transfection plasmid light chain, heavy chain and pXH-N ₃ on the expression efficiency;

D: The effect of adding additives (VPA and NaBut) and expression time on expression efficiency;

Figure 5: Mutant rituximab conjugated to DIBO-DOTA

A. The ESI-MS spectrum of two DIBO-DOTA conjugated at the fixed point and quantitatively of the intact antibody;

B. After reducing the antibody sample, the ESI-MS spectrum of the heavy chain coupled with 1 DIBO-DOTA was quantitatively determined;

C. The ESI-MS spectrum of the intact antibody non-specifically coupled DOTA-SCN;

D. After reducing the antibody sample, the ESI-MS spectrum of non-specific and non-quantitative coupling of one or several DOTA-SCNs on the light chain and/or heavy chain;

Figure 6: Site-specific coupling of DIBO-DOTA rituximab-labeled radioactive isotope

A: Paper thin-layer chromatography test of wild-type rituximab (RTX-WT) non-specific coupling Cu ⁶⁴ yield, left: before passing through PD-10 column; right: after passing through PD-10 column to remove remaining small molecules;

B: Paper thin-layer chromatography test of heavy chain A122 mutant rituximab (RTX-122) site-directed coupling Cu ⁶⁴ yield, left: before passing through PD-10 column; right: after passing through PD-10 column to remove remaining small molecules; In the illustration, a relatively pure (>98%) radioimmunoconjugate was obtained by treatment with a PD-10 column;

C: Autoradiography confirms the fixed-point coupling of radioisotopes. The left picture shows Coomassie brilliant blue staining, and the right picture shows radioactive gamma ray autoradiography. The corresponding positions of the two pictures are RTX-WT-DTT, RTX-WT from left to right +DTT, RTX-122-DTT, RTX-122+DTT, (DTT is dithiothreitol, which is a protein reducing agent); the diagram shows that both samples are successfully coupled with radioactive isotope Cu ⁶⁴ , and the RTX-WT sample is Non-site-specific conjugation, which has radioactive radiography in both the heavy chain and light chain, while RTX-122 is a site-specific conjugation, the mutation and coupling site is at the A122 position of the heavy chain, so only the heavy chain has radioactive radiography;

Figure 7: Micro-PET imaging of ⁶⁴ Cu antibody labeled with a specific point

Micro-PET imaging results of MatThera conjugated Cu ⁶⁴ tumor-bearing mice:

A: 18-hour Micro-PET imaging results, the left is the injection of 500uCi RTX-122-Cu ⁶⁴ tumor-bearing mice, the right is the injection of 500uCi RTX-122-Cu ⁶⁴ and 4 times the dose of cold antibody (unconjugated radioisotope) as Sealed tumor-bearing mice;

B: 60-hour Micro-PET imaging results;

The figure shows that with time, the radioimmunochemicals retain the tumor-targeting properties and are enriched to the tumor site at a higher level.

In order to better understand the present invention, the inventor uses examples to describe and illustrate specific experiments, wherein the examples are only for illustration and do not limit the protection scope of the present invention. Any variants or embodiments equivalent to the present invention are included in the present invention.

Embodiment 1: Construction of the gene vector comprising the rituximab of site-directed mutation

(1) Construction and acquisition of auxiliary plasmids

Through condition optimization and experimentation, it was determined that the tandem expression of the four tRNAs by the 7sk promoter (its sequence is shown in SEQ ID NO: 6) was optimal, and the pXH-N ₃ helper vector was constructed and obtained: the vector uses pUC19 as a template, By using the BamHI restriction site, introduce four tandem 7sk promoters and tRNA; then introduce the tRNA synthetase and its polyA termination signal under the control of the CMV strong promoter; use the EcorI restriction site to introduce the eukaryotic replication original f1 ori and SV40; the plasmid can express tRNA and tRNA synthetase that specifically recognize the unnatural amino acid NAEK.

(2) Obtaining the plasmid containing rituximab

The genes of rituximab (SEQ ID NO: 1 and 3) were obtained through whole gene synthesis. Then it was connected in pEF-1b (Life Technologies) eukaryotic expression vector to obtain the expression plasmids (pEF-RTX-HC-WT and pEF-RTX-LC-WT of wild-type rituximab, which express the heavy chain and light chain portion).

(3) Selection of site-directed mutation sites

According to the crystal structure of rituximab, the binding site of rituximab and its receptor, and taking into account information such as antigenic epitopes and enzymatic hydrolysis sites [Ding Jianping et al.J.Biol.Chem.2007,282:15073-15080], select Several suitable sites were selected for modification, which is mainly based on the following factors: 1. Amino acids are exposed on the surface of the protein to facilitate coupling; 2. It does not affect the normal binding of the antibody to the receptor; 3. It does not affect the properties of the antibody itself, such as stability sex, etc.;

Through more detailed literature review [Sondermann et al.Nature, 2000,406:267-273; Mulkerrin et al.J.Immunol.2000,164:4178-4184; Sun et al.J.Biol.Chem.2001, 276:16469-16477], the inventor selected the A122 position (HC-A122) of the heavy chain, the K168 (LC-K168) position of the light chain, and the P15 (LC-P15) as specific sites to carry out point mutations. MabThera is used as a raw material and it is coupled with a fixed-point small molecule.

(4) Primer design and mutation vector construction for site-directed mutagenesis

For position A122 of the heavy chain of MatThera, K168 and position P15 of the light chain, design primers that can mutate the codon encoding the amino acid into an amber codon, and the specific primers are shown in the table below.

Table 1: Mutation Primer List

Using a site-directed mutagenesis kit ( Lightning Site-Directed MutagenesisKits, Catalog #210518), according to the instructions, use the wild-type rituximab expression vectors pEF-RTX-HC-WT and pEF-RTX-LC-WT obtained in the above step (2) as templates to convert the rituximab heavy chain The amino acid codons at position A122, K168 and P15 of the light chain were mutated into amber stop codons, and expression plasmids (pEF-RTX-HC122, pEF-RTX-LC168, pEF-RTX -LC15), the mutation was verified by sequencing.

Example 2: Expression and purification of rituximab for site-directed mutation

The pXH-N ₃ plasmid constructed in the present invention contains tRNA (tRNA ^Pyl ) and pyrrolysyl-tRNA synthetase (pheRS) derived from ancient Methanococcus, and in expressing cells, amber stop codon (TAG) is used as an effective The sense codon can make the non-natural amino acid NAEK incorporated into the protein, thus causing the site-directed mutation of rituximab. Next, the inventors examined the incorporation possibility of NAEK and the production performance of the mutant protein.

1: Synthesis and identification of unnatural amino acid NAEK

The chemical synthesis reaction formula of unnatural amino acid Lys-azido is as follows

As described in the above formula, 2.3 mL of raw material 1 (2-bromoethanol) was dissolved in a mixed solution of 90 mL of acetone and 15 mL of water, 3.12 g of NaN was added, and the reaction was heated under reflux in an oil bath at 60° C. for 20 h. Cool to room temperature, remove acetone by rotary evaporation, extract with anhydrous ether (30 mL×8), dry over anhydrous Na2SO4, and remove solvent by rotary evaporation to obtain 2.62 g of product 2 as a colorless liquid.

Product 2 (500 mg, 5.74 mmol) was added to a solution of triphosgene (1.70 g, 5.74 mmol) in THF (10 ml). The reaction was stirred at 0°C for 8h, and the solvent was evaporated to dryness. The residue was dried under vacuum for 1 h to give product 3 as a colorless oil.

3 was dissolved in 1.5ml of THF and slowly added to a solution of Boc-Lys-OH (1.7g, 6.88mmol) in 1M NaOH (20ml)/THF (5ml). The reaction was stirred at 0°C for 12h and gradually warmed to room temperature. The reaction solution was cooled to 0°C again and the pH value of the reaction solution was adjusted to 2-3 with 1M hydrochloric acid solution at 0°C. The reaction solution was extracted with EtOAc (30 mL×5), and the organic layer was washed with 2×100 ml saturated brine. The organic layer was dried over anhydrous Na2SO4, filtered, and the solvent was removed by rotary evaporation to obtain 1.65 g of product 4 as a colorless viscous liquid without further purification.

Dissolve 4 in 15mL CH2Cl2, slowly add 15mL TFA dropwise under stirring, react at room temperature for 30min, distill off the solvent, dissolve the remaining liquid product in 5mL methanol, add 100mL ether, a large amount of white solid precipitates, filter and dry to obtain 1.38g white solid Final product 5.1H NMR(D2O):δ=1.22-1.45(m,4H),1.67-1.73(m,2H),2.99(m,2H),3.38(m,2H),3.70(m,1H) ,4.09(m,2H).13C NMR(D2O):δ＝21.4,28.4,29.6,39.5,53.4,56.2,57.8,116.0(TFA),153.1,162.3(TFA),172.9.HRMS:m/z calcd for C9H17N5O4[M]+:259.1281; found:259.1283, which proves that the obtained Lys-azido structure is correct. 2: Construction of pXH-N ₃

Through literature research, the high expression of tRNA ^Pyl is one of the key factors for the effective insertion of unnatural amino acids, and excessive expression will have obvious cytotoxic effects on host cells. Therefore, rational optimization of tRNA ^Pyl expression can be effective. Facilitate large-scale expression of mutant proteins.

(1) First fix the number of tRNAs (1), try different promoters by replacing them, including U1, mu6, H1, hu6 and 7sk promoters, green fluorescent protein (GFP39TAG) with the 39th amino acid as an unnatural amino acid ) as a reporter protein to verify the impact of different promoters on the insertion efficiency of unnatural amino acids, as shown in Figure 2-B, C; the results show that the 7sk (SEQ IDNO: 6) promoter can be the most effective among the selected promoters insertion of unnatural amino acids;

(2) The fixed promoter is 7sk promoter, by changing the number of tRNA series, including 1, 2, 3, 4 and 5, with firefly luciferase as the reporter gene and Renilla luciferase as the reference gene, the two The intergene contains a connecting arm containing a TAG stop codon, and the TAG read-through efficiency is used to detect the impact of different tRNA series numbers on the insertion efficiency of unnatural amino acids, as shown in Figure 2-E; the diagram shows that when four tRNAs are connected in series When , the insertion efficiency of unnatural amino acids is the highest;

(3) By cloning tRNA synthetase pheRS and 4 promoters 7sk-tRNA ^pyl into the same vector pUC19 by gene subcloning method (using BamHI restriction site) as shown in Figure 2-A, the auxiliary vector pXH- N ₃ , the sequence of which is shown in SEQ ID NO:5.

3: NAEK incorporation expression and purification of mutant rituximab

(1) Taking the mutant type (RTX-H122) expressing the A122 position of the heavy chain as an example: pEF-RTX-LC-WT (which expresses the light chain amino acid of the MabThera antibody) obtained in steps 2 and 4 of Example 1 and pEF -RTX-HC122 (which expresses the heavy chain amino acid of the MabThera antibody, and contains a point mutation at the No. 122 site), and the pXH-N ₃ of step 3 of Example 2 are mixed in a certain ratio, and then mixed with the transfection reagent PEI according to Mix in a certain proportion, add mammalian cells together, and add NAEK to a final concentration of 1mM at the same time, express at 100rpm, 37°C, 8% CO ₂ for several days and collect the culture supernatant;

(2) Subsequently, the expression cell line, transfection reagent, ratio of each carrier, ratio of carrier to transfection reagent, additive type, etc. were optimized for mutant expression conditions, as shown in Figure 4, and the expression condition was finally determined to be Freestyle293 cell line, 40kDa branched chain PEI transfection, the ratio of carrier to PEI is 1:4 (mass ratio), the ratio of three carriers pEF-RTX-LC-WT:pEF-RTX-HC122:pXH-N ₃ =1:1:3, add VPA, expressed for 7 days;

(3) The collected culture supernatant was centrifuged at 9000rpm at 4°C for 10min at high speed to remove cells and cell debris, then passed through a hollow fiber filtration system for microfiltration and ultrafiltration for further treatment and concentration (more than 40 times), and Ni-NTA-Bind Buffer exchange, centrifugation again to remove cell debris, Ni-NTA metal chelate affinity chromatography, fully washed with Ni-NTA-Wash buffer, and finally eluted with Ni-NTA-Elute buffer to obtain preliminary purified A sample of rituximab with a purity of about 95%. After the preliminary purified protein sample was changed by ultrafiltration for 4-5 times, it was changed to small molecule coupling buffer (20mM Tris-HCl pH=8.0, 200mM NaCl, treated three times by Chelex100 5×20cm, and the residual Metal ion).

4: Identification of mutant rituximab

(1) Design a control group, use the expression cells without NAEK as a control, and do the same transfection treatment;

(2) Collect the culture supernatant, directly add SDS-PAGE loading buffer to cook the sample, and perform SDS-PAGE electrophoresis and Western Blot analysis. Compared with the protein band at the target position, it can be clearly observed that the NAEK group expresses the full-length MabThera, as shown in Figure 3.

Example 3: Site-directed coupling of mutants to a bifunctional tether (DIBO-DOTA)

1: Synthesis and identification of bifunctional linker DIBO-DOTA:

Compound 1 (2.88g, 14.0mmol) was dissolved in 20mL of anhydrous CH2Cl2, under N2 protection, BF3 OEt2 (2.59mL, 21.0mmol) was slowly added, then the system was moved to a cold well at -10°C, and slowly added dropwise under stirring A CH2Cl2 solution of trimethylsilylated diazomethane (2.0 mol/Lin hexanes) (10.5 mL of trimethylsilylated diazomethane dissolved in 20 mL of anhydrous CH2Cl2) was added dropwise within 1 hour. After continuing to react at -10°C for 2-4 hours, the reaction solution was poured into 50 mL of ice water to quench the reaction, the organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (2×50 mL). The organic phases were combined and washed with saturated brine (2×40mL), dried over anhydrous sodium sulfate, concentrated by filtration, and separated on a silica gel column (petroleum ether: CH2Cl2, v/v, 2/1) to obtain a light yellow product 2 (2.22g, 72%).1H NMR (300MHz, CDCl3): δ8.26 (1H, q, J = 1.4, 6.6Hz), 7.13-7.43 (7H, m), 7.05 (2H, q, J = 3.8, 12.9Hz) ,4.06(2H,s).13C NMR(75MHz,CDCl3):δ196.6,136.9,136.3,135.4,133.8,133.1,132.4,131.4,130.6,129.3,128.8,128.0,127.3,126.9,48.4. MALDI HRMS: m /z 243.0767[M+Na+]. Calcd for C16H12NaO+: 243.0780.

Compound 2 (2.20g, 10mmol) was dissolved in a mixed solvent of EtOH and THF (1/1, v/v, 80mL), sodium borohydride (0.76g, 20mmol) was slowly added with stirring, and reacted overnight at room temperature. After the reaction was detected by TLC, 1 mL of acetic acid was slowly added dropwise to the system to quench the reaction. After removing the solvent by rotary evaporation, add 80mL CH2Cl2 to suspend, wash with saturated brine (3×80mL), dry over anhydrous sodium sulfate, filter and concentrate, and separate on a silica gel column (petroleum ether: ethyl acetate, v/v, 8/1; gradient The elution effect is not good) to obtain the white solid product 3 (2.00g, 91%). 1H NMR (300MHz, CDCl3): δ7.50 (1H, m), 7.14-7.30 (7H, m), 6.90 (2H, q ,J=2.7,12.0Hz),5.31(1H,q,J=6.3,10.0Hz),3.41(2H,m).13C NMR(75MHz,CDCl3):δ141.7,136.7,136.2,134.5,131.7,131.5, 130.1, 129.9, 129.3, 128.7, 127.4, 127.2, 126.9, 125.9, 74.4, 42.7. MALDI HRMS: m/z 245.0949[M+Na+]. Calcd for C16H14NaO+: 245.0937.

Compound 3 (1.11g, 5mmol) was dissolved in 25mL CHCl3, and bromine (0.26mL, 5mmol) was slowly added dropwise with stirring. Reaction at room temperature for 0.5h, TLC detection of raw materials after complete reaction, rotary evaporation to remove solvent, silica gel column separation (petroleum ether: CH2Cl2, v/v, 2/1, or gradient elution) to obtain yellow viscous liquid product 4 (1.11g , 58%). 1H NMR (300MHz, CDCl3): δ7.54-7.47 (2H, aromatics), 7.31-6.72 (6H, aromatics), 5.77 (1H, d, J = 5.4Hz, CHBr), 5.22 (1H, dd, J = 3.6, 15.9Hz, CHOH), 5.19 (1H, d, J = 5.4Hz, CHBr), 3.50 (1H, dd, J = 3.6, 15.9Hz, CH2), 2.75 (1H, dd, J = 3.6, 15.9Hz ,CH2).13C NMR(75MHz,CDCl3):δ141.3,140.0,137.2,134.0,133.4,131.5,131.3,130.9,127.8,126.2,123.7,121.3,76.5,70.0,62.3,32.2.MALDI HRMS:m/z40 .9313[M+Na+].Calcd for C16H14Br2NaO+: 402.9304.

Compound 4 (0.77g, 2mmol) was dissolved in 20mL of dry THF under N2 protection, and 2.0MLDA in THF (4mL, 8mmol) was slowly added dropwise under stirring. After reacting at room temperature for 0.5 h, slowly drop 0.5 mL of distilled water into the reaction system to quench the reaction. Remove the solvent by rotary evaporation, dissolve with 80ml DCM, wash twice with saturated brine, dry the oil phase, mix the sample, and separate on a silica gel column (petroleum ether: ethyl acetate, v/v, 8/1, or gradient elution) to obtain a white solid Product 5 (0.25 g, 57%). 1H NMR (300MHz, CDCl3): δ7.67 (1H, aromatics), 7.37-7.18 (7H, aromatics), 4.57 (1H, dd, J = 2.1, 14.7Hz, CHOH), 3.04 (1H, dd, J = 2.1, 14.7Hz, CH2), 2.86 (1H, dd, J=2.1, 14.7Hz, CH2).13C NMR (75MHz, CDCl3): δ154.5, 150.6, 128.6, 127.1, 1127.0, 126.0, 125.8, 125.1, 124.7, 123.0, 122.7, 121.7, 111.9, 109.6, 74.2, 47.7

Compound 5 (0.22g, 1mmol) was dissolved in 30mL CH2Cl2, and 0.4mL pyridine and phenyl p-nitrochloroformate (0.4g, 2mmol) were added. After reacting at room temperature overnight, TLC detected that raw material 5 was completely reacted, and the reaction solution was washed with saturated brine (2×40mL), dried over anhydrous sodium sulfate, concentrated by filtration, and separated on a silica gel column (petroleum ether: CH2Cl2, v/v, 2/ 1) The product 6 (0.34 g, 72%) was obtained as a white solid. 1H NMR (300MHz, CDCl3): δ8.23-8.18 (2H, aromatics), 7.56-7.54 (2H, aromatics), 7.46-7.18 (8H, aromatics), 5.52 (1H, dd, J＝3.9, 15.3Hz, CHOH), 3.26 (1H, dd, J=3.9, 15.3Hz, CH2), 2.97 (1H, dd, J=3.9, 15.3Hz, CH2); 13C NMR (75MHz, CDCl3): δ154.5, 150.7, 149.1, 148.7 ,129.0,127.4,127.3,126.7,126.5,125.5,125.2,124.3,124.0,122.6,122.4,120.8,120.6,120.2,112.2,108.5,80.6,44.8; MALDI HRMS:m/z408.Na][ .Calcd for C23H15NNaO5+: 408.0842.

Ethylenediamine (30 mg, 0.5 mmol) was dissolved in 10 mL (anhydrous) CH2Cl2, 300 μL triethylamine was added, under N2 protection, compound 6 (38 mg, 0.1 mmol) was added under stirring. Rinse the column with dichloromethane methanol system (30:1-5:1). Obtain compound 7.1HNMR (300MHz, CDCl3): δ7.51-7.50 (2H, aromatics), 7.35-7.28 (6H, aromatics), 5.50 (1H, s, CHOH), 3.26-3.15 (3H), 2.93-2.85 ( 3H); 13CNMR (75MHz, CDCl3): δ155.6, 152.0, 150.9, 129.8, 127.9, 127.8, 126.9, 126.1, 125.8, 123.7, 123.6, 121.2, 112.8, 109.9, 46.1, 43.6, 41.5.

2: NAEK site-directed mutein bifunctional tether quantitative coupling (see Figure 5)

The site-directed mutein containing NAEK, with the aid of the azide group on NAEK, realizes the copper-free catalyzed Click ligation reaction with DIBO-DOTA (a compound containing a cyclooctyne structure) through the ring strain of cyclooctyne. The copper-free catalytic connection reaction system is as follows:

RTX-122-NAEK (it is prepared by embodiment 2) 1ug/ul

DIBO-DOTA 1mM

Reaction conditions: 4°C, vertical suspension for 12 hours.

The results verified that the dual-functional linker DIBO-DOTA was coupled to MatThera in a fixed-point and quantitative manner (see Figure 5-A, B). After the above reaction conditions, more than 95% of the Rituxan could be coupled to the dual-functional linker within 12 hours. The reacted complex was desalted by ultrafiltration, and the solution was exchanged into an isotope coupling buffer (20 mM ammonium acetate-acetic acid pH=5.5, treated with Chelex100 3 times), and the obtained ligation product could be used as a site-specific labeling isotope.

Example 4: Site-directed coupling and identification of mutant radioisotopes

Positron emission tomography (PET) has increasingly become a key technology in tumor treatment, and Cu ⁶⁴ is less harmful to the human body due to its short half-life (~12 hours), and it is easier to form a stable combination with DOTA. Because of its moderate half-life (~6 days), Lu ¹⁷⁷ mainly releases beta rays and has a short radiation radius, so it is more suitable for the research and development of therapeutic radioimmunoconjugates.

1: Site-specific coupling of MabThera-Cu ⁶⁴ :

DIBO at one end of the bifunctional linker (DIBO-DOTA) undergoes a copper-free Click reaction with the azide group of the mutant antibody through an eight-membered ring octyne, and DOTA at the other end reacts with a radioactive metal isotope through chelation. The radioactive isotope Cu ⁶⁴ is produced by the Department of Nuclear Medicine, Peking University Cancer Hospital. The MatThera-Cu ⁶⁴ linkage reaction system is as follows:

RTX-122-DIBO-DOTA 100ug(1mg/ml)

Cu ⁶⁴ 2mCi

Reaction conditions: stand at 42°C for 1 hour.

The results were followed by Radio-TLC and SDS-PAGE analysis (as shown in Figure 6-A, B, and C), and it was confirmed that after isotope coupling, simple desalting by PD-10 column can obtain radioimmunoassay purity >98% radioimmunoconjugates;

2: Micro-PET imaging of MabThera-Cu ⁶⁴ in tumor-bearing mice

CD20 expression positive cells Ramos A1 cells were injected into severe immunodeficiency (Severe Combined Immune-deficiency, SCID) mice at 1×10 ⁷ /right underarm, and about two weeks later, 300-350uCi rituximab-Cu ⁶⁴ was injected into the tail vein Conjugates were also used as a blocking group, and 500ug of unconjugated radioactive substance rituximab was injected simultaneously as a blocking agent. At 18 hours and 60 hours after the injection, the mice were anesthetized with 1.5% isoflurane for Micro-PET imaging, as shown in FIG. 7 .

It can be seen from the imaging results that over time, radioactive substances are obviously enriched in the tumor site, and PET can show clear images of the tumor body.

Although the present invention has been described with the above embodiments, it should be understood that, without departing from the spirit of the present invention, the present invention can be further modified and changed, and these modifications and changes are within the protection scope of the present invention . For example, although the present application has described the coupling of rituximab to Cu ⁶⁴ as an example, it is obvious that the present invention should not be limited to the coupling of rituximab to Cu ⁶⁴ , and those skilled in the art can apply the present invention to any target protein coupling to any small molecular.

Claims (15)

1. the polypeptide of mutation, the amino acid at least one site is sported unnatural amino acid, the unnatural amino acid Are as follows:

Shown in Lys-azido (NAEK),

Wherein, the polypeptide is Rituximab,

Its light-chain amino acid sequence is as shown in SEQ ID NO:2, and heavy chain amino acid sequence is as shown in SEQ ID NO:4；

Wherein, the mutational site is selected from: A122 of SEQ ID NO:4.

2. a kind of conjugate, the polypeptide comprising mutation described in claim 1, and pair for coupling nitrine and metal isotope Cyclooctyne (DIBO) group is contained in function connects arm, described linking arm one end, and the other end contains DOTA, the difunctional linking arm Structure is shown below:

3. conjugate as claimed in claim 2, wherein the polypeptide of the mutation is the Rituximab of mutation.

4. conjugate as claimed in claim 2 or claim 3, wherein the polypeptide of the mutation and the following formula institute of difunctional connection arm configuration Show:

Wherein the position A122 of the heavy chain of the polypeptide of the mutation mutates, R₁For the 1st to the 121st amino acids residue, R₂It is 123 amino acid residues to C-terminal, R₃For DOTA or other directly or indirectly provide the small molecule of function.

5. conjugate as claimed in claim 4, R therein₃For DOTA, radioactive isotope is chelated.

6. conjugate as claimed in claim 5, the radioactive isotope is selected from Lu¹⁷⁷, Cu⁶⁴Or Y⁹⁰。

R 7. conjugate as claimed in claim 4, in the conjugate₃Selected from the auspicious statin of adriamycin or monomethyl Australia.

8. encoding the nucleic acid molecules of the polypeptide of mutation described in claim 1.

9. including the carrier of nucleic acid molecules according to any one of claims 8.

10. a kind of pharmaceutical composition, polypeptide or claim 2-7 containing a effective amount of mutation described in claim 1 Described in any item conjugates.

11. pharmaceutical composition as claimed in claim 10 preparation for Radionuclide imaging, treatment refractory malignancies or Purposes in the drug of tumor recurrence.

12. a kind of microorganism contains carrier as claimed in claim 9.

13. microorganism as claimed in claim 12 is Escherichia coli.

14. a kind of zooblast contains carrier as claimed in claim 9.

15. zooblast as claimed in claim 14 is Freestyle293 cell line.