Method for specific covalent coupling of monoclonal antibody drug by using Z structural domain

Abstract

The invention discloses a method for specific covalent coupling of a monoclonal antibody drug by using a Z structural domain, which comprises the following steps: introducing non-natural amino acid fluorosulfonic acid-L-tyrosine (FSY) capable of being covalently cross-linked at a specific site of the Z structural domain by a genetic code expansion technology, and converting non-covalent interaction between the Z structural domain and an IgG monoclonal antibody drug into covalent interaction; the binding force of the two molecules is enhanced, the stability is good under the physiological condition, stable and irreversible binding is realized, and the stability and uniformity of the coupled molecules can be remarkably improved. The method can be used as an XDC (antibody coupling medicine, antibody coupling probe, antibody coupling diagnostic reagent and the like) platform method, and is compatible with various loads such as medicine molecules, imaging molecules, nucleic acid modification and the like.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to a method for covalently coupling a monoclonal antibody drug with a Z domain. (II) BACKGROUND

[0002] Monoclonal antibody drugs have significantly improved the treatment of diseases and reduced the risk of adverse reactions due to their precise targeting and long-acting characteristics, and have become the core treatment for cancer, immune diseases and other fields. In order to further improve the targeting delivery efficiency of tumor monoclonal antibody drugs and reduce the side effects of small molecule drugs, current research is focusing on the site-directed modification of antibodies and the development of new antibody drug conjugates.

[0003] Traditional non-specific chemical conjugation methods (such as lysine or cysteine modification) are simple to operate and do not require additional modification of antibodies, so they are widely used in the early development of antibody conjugate drugs (ADCs). However, the conjugation sites are random, leading to uneven distribution of drug-antibody ratio (DAR), which can cause differences in pharmacokinetics and immunogenicity risks. In contrast, genetically engineered cysteines (such as THIOMAB) can achieve site-specific conjugation with uniform DAR, but require structural modification of the antibody, which is more complex in terms of process complexity. The introduction of non-natural amino acids can provide highly specific reaction sites for precise conjugation, but it relies on a special expression system and orthogonal tRNA / synthetase system, which has a higher production cost. Enzyme-catalyzed site-directed modification methods (such as transglutaminase, Sortase A, glycosyltransferase, etc.) have high selectivity and mild reaction conditions, which can achieve efficient modification on the natural antibody skeleton and reduce the damage to the activity of the antibody, but are often limited by enzyme recognition sequences, and the process stability for large-scale production still needs to be optimized. Overall, non-specific conjugation methods are mature and easy to implement, while site-specific conjugation methods are more conducive to obtaining ADCs with uniform structure, safety and more controllable efficacy, representing the future direction of development.

[0004] Protein A, derived from the cell wall of Staphylococcus aureus, has the ability to bind to immunoglobulins (IgG) (Materials, 1996, 9(12): 994). This binding property makes protein A important in biotechnological applications such as affinity chromatography, and optimized binding domains are expected to be applied to a wider range of biotechnologies (Analytica Chimica Acta, 2022, 1189: 338907). The Z domain, derived from Staphylococcus aureus protein A, has the ability to bind to the constant region of IgG Fc (Kd = 10 nM), but this non-covalent binding is a reversible process, affecting its application effectiveness (Proceedings of the National Academy of Sciences, 2015, 112: 4552-4557; Journal of Controlled Release, 2021, 330: 161-172). Compared to the entire antibody conjugation modification, the Z domain contains only 59 amino acids, has a small molecular weight and simple structure, and can serve as an "antibody binding adapter," making the modification and conjugation of the payload molecule more efficient and convenient, and simplifying the modification of related payload molecules. Traditional binding of Protein A or Z domain to IgG antibody Fc is non-covalent and reversible, and may dissociate during elution, in vivo environment, or pharmacokinetic processes, affecting stability. Therefore, it is necessary to find the optimal covalent cross-linking site to provide a rapid, efficient, and uniform conjugation method, offering new insights for the development of XDC.

[0005] Genetic Code Expansion (GCE) is an advanced genetic engineering technology that aims to increase the diversity of an organism's genetic code through specific means (Journal of the American Chemical Society, 2019, 141:7698-7703; Cell, 2020, 182:85-97; Journal of the American Chemical Society, 2021, 143:16377-16382; Chem, 2022, 8:2766-2783). The basic principle of genetic code expansion technology is to utilize the help of biological orthogonal pairs—aminoacyl-tRNA synthetase (aaRS) / transfer RNA (tRNA) (i.e., the aaRS / tRNA pair)—to enable nonsense codons (usually amber codons UAG) to perform meaningful coding functions and introduce unnatural amino acids (UAA) at the corresponding sites (Chemical Reviews, 2024, 124:11544-11584). In this way, the diversity of the genetic code can be increased, thereby endowing proteins with new functions or properties. Genetic code extension technology can be used to study the function and mechanism of action of proteins. By introducing UAA, precise labeling and regulation of protein molecules can be achieved (Chem, 2019, 5: 2955-2968; Nature Communications, 2021, 12: 7039; Chemical Communications, 2022, 58: 6861-6864; ACS Central Science, 2023, 9: 1241-1251; Chem, 2024, 5: 2955-2968; Chimia, 2024, 78: 22-31; Chem, 2024, 10: 1868-1884).

[0006] However, genetic code expansion technology faces challenges in selecting suitable sites for introducing non-natural amino acids. First, sites must be chosen that do not affect the protein's natural function, avoiding changes in conformation or activity due to the introduction of non-natural amino acids. Second, the selected sites must be located at protein-protein interaction interfaces, facilitating covalent cross-linking. Therefore, both introduction and cross-linking sites exhibit specificity. Finally, the selected codons (such as stop codons TAG and TAA) should be easily inserted into the target gene without affecting normal gene expression and translation. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a method for covalently conjugating monoclonal antibody drugs using the Z domain. This invention utilizes genetic code expansion technology to develop covalently conjugable non-natural amino acids, replacing the amino acid at a suitable site in the Z domain. Then, based on a novel click chemistry reaction—the sulfofluoride exchange reaction (SuFEx reaction)—a nucleophile (such as an amine, alcohol, or water) attacks the sulfur atom in the sulfofluoride of the amino acid and replaces the fluorine atom, forming a stable SN bond. The non-natural amino acid fluorosulfonic acid-L-tyrosine (FSY) is introduced, thereby obtaining a Z domain (hereinafter referred to as Z) capable of covalently binding IgG. This method transforms the non-covalent interaction between the Z domain and IgG-type monoclonal antibody drugs into a covalent interaction, significantly enhancing the binding affinity and maintaining good stability under physiological conditions.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a method for specific covalent coupling of a Z-domain to a monoclonal antibody drug. The method includes the following steps: 1) introducing a stop codon TAG at a specific site of the amino acid sequence of the Z-domain protein using site-directed mutagenesis; 2) using genetic code expansion technology, introducing a non-natural amino acid at the TAG site during protein induction expression in step 1) to obtain a mutant Z-domain protein containing the non-natural amino acid; 3) co-incubating the mutant Z-domain protein with an IgG monoclonal antibody drug at 37°C and pH 9 for 2-24 hours (preferably 12 hours) to achieve specific covalent coupling between the Z-domain and the IgG monoclonal antibody drug.

[0010] Furthermore, the Z-domain protein is derived from protein A of Staphylococcus aureus, and its amino acid sequence is shown in SEQ ID NO. 1.

[0011] Furthermore, the Z-domain protein-specific sites for introducing non-natural amino acids include sites 11, 14, and 24 of the amino acid sequence shown in SEQ ID NO.1.

[0012] Furthermore, the mutant Z-domain protein is a pure protein solution extracted from recombinant E. coli DH10B after the amino acid at the specific site of the Z-domain protein is mutated to the stop codon TAG and a plasmid carrying an aminoacylated tRNA synthetase / tRNA system capable of recognizing non-natural amino acids (preferably FSY) is co-transformed with the plasmid. The recombinant E. coli is then induced with arabinose and expressed with the addition of non-natural amino acids.

[0013] Furthermore, the non-natural amino acid is fluorosulfonic acid-L-tyrosine (FSY), with the structure shown below:

[0014]

[0015] Furthermore, the fluorosulfonic acid-L-tyrosine is prepared according to the following steps:

[0016] (1) N-(tert-butyloxycarbonyl)-L-tyrosine tert-butyl ester was dissolved in acetonitrile, and triethylamine was added to activate it at room temperature for 10 min. Then 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-trifluoromethanesulfonate was added. The mixture was stirred at room temperature for 6 h. During this period, the reaction progress was monitored by TLC (thin-layer chromatography). The developing solvent was ethyl acetate and petroleum ether in a volume ratio of 1:5. After the reaction was completed, the system was rotary evaporated to dryness. After redissolving in ethyl acetate, it was extracted with 10% citric acid aqueous solution, ddH2O and saturated NaCl aqueous solution in sequence. Each solution was extracted twice. The organic phase was dried with anhydrous Na2SO4, filtered and rotary evaporated to remove ethyl acetate. Silica gel column chromatography (silica gel 200-300 mesh, column height 15 cm, diameter 305 mm) was performed. The eluent was ethyl acetate and petroleum ether in a volume ratio of 1:15. Gravity elution was used. All eluent was collected and rotary evaporated until no liquid flowed out, resulting in a yellow oily substance.

[0017] (2) Dissolve the yellow oily substance in dichloromethane, add trifluoroacetic acid, stir overnight at room temperature, monitor the reaction progress by TLC, and use ethyl acetate and petroleum ether in a volume ratio of 1:8 as the developing solvent; after the reaction is completed, remove the dichloromethane by rotary evaporation, add methanol to dissolve the product, and then use a dropper to add it dropwise to cold diethyl ether (0℃). Centrifuge at 3000 rpm for 10 min, discard the supernatant, and dry the precipitate under vacuum to obtain the white solid product fluorosulfonic acid-L-tyrosine (FSY).

[0018] Further, in step (1), the molar ratio of N-(tert-butoxycarbonyl)-L-tyrosine tert-butyl ester to triethylamine is 1:1-3 (preferably 1:2); the molar ratio of N-(tert-butoxycarbonyl)-L-tyrosine tert-butyl ester to 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-trifluoromethanesulfonate is 1:1-3 (preferably 1:1.5); and the volume of acetonitrile used is 1-5 mL / mmol (preferably 1.7 mL / mmol) based on the amount of N-(tert-butoxycarbonyl)-L-tyrosine tert-butyl ester.

[0019] Further, in step (2), the volume of dichloromethane used is 1-5 mL / mmol (preferably 3.5 mL / mmol) based on the amount of the yellow oily substance; the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, the volume ratio of dichloromethane to methanol is 1:0.05, and the volume ratio of dichloromethane to cold diethyl ether is 1:3.

[0020] Furthermore, the IgG class of monoclonal antibody drugs includes cetuximab and pembrolizumab.

[0021] Furthermore, the mutant Z-domain protein is mixed with the monoclonal antibody drug at a molar ratio of 40:1.

[0022] Furthermore, the pure protein solution is prepared according to the following method:

[0023] The recombinant *E. coli* (preferably *E. coli* DH10B containing the mutant Z-domain protein and pSupAR-chFSYRS plasmid) was inoculated into LB liquid medium and cultured overnight at 37°C. Then, 20% arabinose aqueous solution and 100mM non-natural amino acid (preferably FSY) aqueous solution were added sequentially at 1% (v / v) to the culture medium, and the culture was induced at 30°C for 20 h. After induction, the cells were collected by centrifugation at 8000 rpm for 10 min, resuspended in lysis buffer (3M sodium chloride, 3mM dithiothreitol (DTT), 500mM Tris-HCl, pH 8.0), and lysed using an ultrasonic disruptor (300W, 20 min, ultrasonic on for 1 s, off for 3 s). The lysed suspension was centrifuged at 10000 rpm for 10 min, and the supernatant was added to Ni-NTA. The protein was incubated on a shaker for 1 hour (the volume added was calculated based on a protein loading of 50 mg / mL for Ni-NTA). Protein purification was then performed using an affinity chromatography column, with gradient elution using lysis buffer and elution buffers at concentrations of 20 mM, 60 mM, 100 mM, 300 mM, and 1 M (3 M sodium chloride, 500 mM Tris-HCl, pH 8.0). All eluent collected from the 100 mM, 300 mM, and 1 M gradient elutions was dialyzed overnight at 4°C with stirring using a dialysis bag (molecular weight cutoff of 3.5 kD). The retentate was then concentrated using an ultrafiltration tube (molecular weight cutoff of 10 kD), and the retentate was collected as the purified protein solution.

[0024] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0025] This invention introduces the non-natural amino acid fluorosulfonic acid-L-tyrosine (FSY) into a specific site of the Z domain using genetic code extension technology. This transforms the non-covalent interaction between the Z domain and IgG monoclonal antibody drugs into a covalent interaction, enhancing the binding force between the two and exhibiting good stability under physiological conditions. This achieves a stable and irreversible binding, significantly improving the stability and uniformity of the coupled molecules.

[0026] The method of this invention can be used as an XDC (antibody-drug conjugate, antibody-drug conjugate probe, antibody-drug conjugate diagnostic reagent, etc.) platform method, and is compatible with various payloads such as drug molecules, imaging molecules, and nucleic acid modifications. (iv) Description of the attached drawings

[0027] Figure 1The image shows the hydrogen nuclear magnetic resonance spectrum of the synthesis of the non-natural amino acid FSY in Example 1.

[0028] Figure 2 This is a Coomassie brilliant blue staining image of the purification process of mutant proteins N11FSY and Y14FSY in step 3 of Example 2. Pel represents the bacterial cells after centrifugation of the induced bacterial culture; Sup represents the supernatant after centrifugation of the lysis buffer; IB represents the precipitated bacterial cells after centrifugation of the lysis buffer; FL represents the flow-through buffer eluted with the lysis buffer; W1-W2 represent the effluent from the low-concentration elution buffers of 20 and 60 mM imidazole; E1-E3 represent the effluent from the high-concentration elution buffers of 100, 300 mM, and 1 M imidazole.

[0029] Figure 3 The image shows a silver staining diagram of the protein from the screening results of covalent cross-linking sites between sfGFP-Z (FSY) and cetuximab in Example 3.

[0030] Figure 4 Silver staining images of proteins from the screening results of Y14FSY and cetuximab covalent cross-linking incubation time (2, 6, 12, 24 h) in Example 4.

[0031] Figure 5 Silver staining image of the protein covalently cross-linked with sfGFP-Z(FSY) and pembrolizumab in Example 5. (V) Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] Unless otherwise specified, all methods described herein are conventional methods. All raw materials, unless otherwise specified, are available from publicly available commercial sources. Quantitative experiments in the following examples were performed in triplicate, and the results were averaged. The room temperature mentioned in this invention refers to 25-30°C.

[0034] Cell culture medium: DMEM complete medium containing 10% fetal bovine serum (FBS) + penicillin (100 μg / mL) + streptomycin (100 μg / mL).

[0035] The LB liquid medium consists of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with water as the solvent. The LB solid medium is the LB liquid medium with 20 g / L agar added.

[0036] Plasmid pSupAR-chFSYRS Source: Journal of the American Chemical Society, 2021, 143: 16377-16382, donated by Professor Chen Peng's research group at Peking University.

[0037] Unless otherwise specified, all other plasmids used were purchased from Nanjing Genscript Biotech Co., Ltd.

[0038] Example 1: Synthesis of the non-natural amino acid fluorosulfonic acid-L-tyrosine (FSY)

[0039] N-(tert-Butoxycarbonyl)-L-tyrosine tert-butyl ester (2 g, 5.93 mmol) was dissolved in 10 mL of acetonitrile, and triethylamine (1.65 mL, 11.9 mmol) was added, followed by activation at room temperature for 10 min. Then, 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-trifluoromethanesulfonate (2.92 g, 8.9 mmol) was added, and the mixture was stirred at room temperature for 6 h. The reaction progress was monitored by TLC (thin-layer chromatography) using ethyl acetate and petroleum ether in a 1:5 volume ratio as the developing solvent. After the reaction was complete, the system was rotary evaporated to dryness, redissolved in 10 mL of ethyl acetate, and extracted sequentially with 20 mL of 10% citric acid aqueous solution, ddH₂O, and saturated NaCl aqueous solution, twice with each solution. The organic phase was dried over anhydrous Na₂SO₄, filtered, and rotary evaporated to remove ethyl acetate. The final product was subjected to silica gel column chromatography (200-300 mesh silica gel, 15 cm column height, 305 mm diameter). The eluent was ethyl acetate and petroleum ether in a volume ratio of 1:15. Gravity elution was performed, and the entire polar eluent was collected and rotary evaporated until no liquid flowed out, yielding 1.26 g of a yellow oily substance with a yield of 63%.

[0040] The above-mentioned yellow oily substance (1.2 g, 2.86 mmol) was dissolved in 10 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature. The reaction progress was monitored by TLC using ethyl acetate and petroleum ether in a 1:8 volume ratio as the developing solvent. After the reaction was complete, the dichloromethane was removed by rotary evaporation. The product was dissolved in 500 μL of methanol, and then added dropwise to 30 mL of cold diethyl ether (0 °C). The mixture was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried under vacuum to obtain a white solid product, fluorosulfonic acid-L-tyrosine (FSY) (970 mg), with a yield of 80%. A 100 mM stock solution was prepared using ddH₂O before use.

[0041] The proton NMR spectrum of FSY is as follows: Figure 1 As shown.

[0042] Example 2: Construction, expression, and purification of sfGFP-Z(FSY) protein

[0043] 1. Construction of recombinant plasmid pBad-sfGFP-Z

[0044] To make the results more visual, C-terminal amide-modified green fluorescent protein (sfGFP) was expressed by fusing a linker peptide (GGGGS) with a Z domain tagged with histidine.

[0045] (1) Using plasmid pBad-IGF2-Z as a template (the nucleotide sequence of IGF2-Z is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO.5), the PCR product was amplified using the primers pZ-F / R in Table 1, in the PCR system in Table 2 and the PCR reaction program in Table 3. The PCR product was digested with Dpn I enzyme (reaction system: 1 μL PCR product + 1 μL Q.Cut Dpn I enzyme + 1 μL 10×Q.CutBuffer, and ultrapure water was brought to a final volume of 10 μL) to obtain fragment Z, the amino acid sequence of which is shown in SEQ ID NO.1.

[0046] (2) Using plasmid pBad-sfGFP (sfGFP nucleotide sequence as shown in SEQ ID NO.6, amino acid sequence as shown in SEQ ID NO.7) as template, amplify using primer pSF-F / R in Table 1, in the PCR system in Table 2 and the PCR reaction program in Table 3. The PCR product is digested with Dpn I enzyme as described in step (1) to obtain linearized vector psfGFP.

[0047] (3) Take linearized vector psfGFP (94 ng) and fragment Z (10 ng), add 2 μL of recombinase Express II and 4 μL of CEbuffer, and bring the volume to 20 μL with ultrapure water. React in a PCR instrument at 37℃ for 30 min to achieve homologous recombination of two linear DNAs.

[0048] (4) Add 1 μL of the recombinant product from step (3) to 30 μL of *E. coli* DH5α competent cells and incubate on ice for 35 min. Then, heat shock the mixture in a 42°C water bath for 90 seconds and immediately cool it on ice for 3 min. Add 600 μL of antibiotic-free LB liquid medium and incubate in a shaker at 37°C for 60 min. Spread the revived bacterial culture onto LB solid medium plates containing 50 μg / mL ampicillin resistance for screening and incubate overnight at 37°C. Pick single colonies and inoculate them into LB liquid medium, amplify them overnight in a shaker at 37°C, and extract plasmids for sequencing to verify the correctness of the recombinant plasmid. The recombinant plasmid pBad-sfGFP-Z was obtained. The amino acid sequence of sfGFP-Z with histidine tag is shown in SEQ ID NO.2 (positions 1-238 represent sfGFP, positions 239-243 represent the linker peptide, positions 244-301 represent the Z domain, and positions 302-307 represent the histidine tag). The nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0049] SEQ ID NO.1

[0050] VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKSEQ ID NO.2

[0051] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGGGGSVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKHHHHHH*

[0052] SEQ ID NO.4 pBad-IGF2-Z gene sequence

[0053] ATGGCAGCTTATAGGCCATCCGAAACCCTTTGCGGTGGCGAGCTGGTGGACACCCTGCAGTTTGTCTGTGGTGATCGTGGTTTTTACTTCAGCCGTCCGGCGAGCCGCGTGAGCCGCCGTAGCAGAGGTATTGTTGAAGAGTGCTGCTTTCGTTCTTGTGATTTGGCTCTGCTGGAAACGTACTGCGCTACCCCGGCGAAGTCCGAGGGTGGCGGCGGCAGCGTTGACAACAAGTTCAACAAAGAACAACAAAACGCGTTCTATGAGATCCTGCACCTGCCGAATTTGAATGAGGAGCAACGTAACGCCTTCATCCAGTCGCTGAAGGACGACCCGTCTCAGAGCGCAAATTTGCTCGCGGAAGCGAAGAAACTGAACGATGCCCAGGCACCGAAACATCATCACCATCATCATTAA

[0054] SEQ ID NO.6 pBad-sfGFP gene sequence

[0055] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCA CAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAACATCATCACCATCATCATTAA

[0056] 2. Mutations of the stop codon TAG were made at different sites of the recombinant plasmid pBad-sfGFP-Z

[0057] Using the recombinant plasmid pBad-sfGFP-Z constructed in step 1 as a template, PCR amplification was performed using the primers in Table 1 (p4-F / R, p5-F / R, p6-F / R, p7-F / R, p10-F / R, p11-F / R, p13-F / R, p14-F / R, p17-F / R, p18-F / R, p24-F / R, p25-F / R, p27-F / R, p28-F / R, p35-F / R), the system in Table 2, and the reaction procedure in Table 3. SEQ ID NO(Z) was then used for PCR amplification. In the amino acid sequence of sfGFP-Z shown in IDNO.2, positions K247, F248, N249, K250, Q253, N254, F256, Y257, L260, H261, E267, E268, R270, N271, and K278 (corresponding to SEQ ID NO.2) are: The nucleotide sequences corresponding to positions 4, 5, 6, 7, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, and 35 of the Z-domain amino acid sequence shown in IDNO.1 were point-mutated to the stop codon TAG. The plasmids of the resulting mutant Z-domain proteins were named as follows: K4TAG, F5TAG, N6TAG, K7TAG, Q10TAG, N11TAG, F13TAG, Y14TAG, L17TAG, H18TAG, E24TAG, E25TAG, R27TAG, N28TAG, and K35TAG.

[0058] The PCR products were digested with Dpn I enzyme using the method and conditions in step 1, and then transformed into E. coli DH5α competent cells for screening and identification, resulting in the 15 target plasmids containing mutant Z domain proteins.

[0059] Table 1 Primers used for plasmid construction.

[0060]

[0061]

[0062] Table 2 PCR reaction system

[0063]

[0064] Table 3 PCR reaction procedure

[0065]

[0066] 3. Expression and purification of mutant protein sfGFP-Z (FSY)

[0067] The 15 plasmids containing mutant Z-domain proteins constructed in step 2 were co-transformed with the pSupAR-chFSYRS plasmid into E. coli DH10B competent cells. The transformation steps were the same as in step 1. FSY was inserted into the 15 sites in the Z-domain of the sfGFP-Z constructed in step 2 that were changed to stop codons.

[0068] After selecting single clones and expanding them overnight in LB liquid medium at 37°C, 20% arabinose aqueous solution and 100mM FSY stock solution were added to the culture medium at a volume concentration of 1% and induced at 30°C for 20h.

[0069] After induction, the cells were collected by centrifugation at 8000 rpm for 10 min. The cells were resuspended in lysis buffer (3M sodium chloride, 3mM dithiothreitol (DTT), 500mM Tris-HCl, pH 8.0) and lysed using an ultrasonic homogenizer (300W, 20 min, sonication on for 1 second, off for 3 seconds). The lysed suspension was centrifuged at 10000 rpm for 10 min, and the supernatant was separated. Ni-NTA beads were added to the supernatant and incubated on a shaker for 1 h (the volume added was calculated based on a Ni-NTA protein loading of 50 mg / mL). Protein purification was performed using an affinity chromatography column, sequentially eluting with lysis buffer and elution buffers at concentrations of 20mM, 60mM, 100mM, 300mM, and 1M (3M sodium chloride, 500mM Tris-HCl, pH 8.0). Purification was monitored by Coomassie staining during SDS-PAGE gel electrophoresis. In 1×PBS (8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and water to a final volume of 1 L), the eluent was eluted at 100 mM, 300 mM, and 1 M gradients using a dialysis bag (molecular weight cutoff of 3.5 kD). All collected eluent was dialyzed overnight at 4°C with stirring. The cutoff was then concentrated using an ultrafiltration tube (molecular weight cutoff of 10 kD). The concentrate was collected to obtain a purified protein solution of a mutant Z-domain protein in sfGFP-Z with FSY inserted at 15 sites where the Z-domain was changed to stop codons. This protein was designated as the sfGFP-Z(FSY) mutant protein. The mutant proteins were named as follows: K4FSY, F5FSY, N6FSY, K7FSY, Q10FSY, N11FSY, F13FSY, Y14FSY, L17FSY, H18FSY, E24FSY, E25FSY, R27FSY, N28FSY, and K35FSY.

[0070] Taking N11FSY and Y14FSY as examples, the purification process is shown in the gel electrophoresis results. Figure 2 As shown, the mutant protein was successfully induced to express in Escherichia coli DH10B with good FSY incorporation efficiency.

[0071] Protein concentration was determined using the Bradford method. The reaction system consisted of 198 μL G250 staining solution + 2 μL of the protein solution to be tested, and the absorbance was measured at 562 nm using a microplate reader. Protein yield was calculated based on the absorbance-concentration standard curve (y = 2.9522x - 0.9981, where y represents the protein concentration in mg / mL and x represents the measured absorbance value) prepared using BSA as the standard protein under the above conditions.

[0072] Example 3: Screening for covalently coupled sites between sfGFP-Z(FSY) mutant protein and cetuximab

[0073] Purified protein solutions of 15 sfGFP-Z(FSY) mutant proteins prepared in Example 2 were incubated with cetuximab (purchased from Merck Healthcare KGaA, Germany, batch number SJ20171039) at a molar ratio of 10:1 (1.2 μg mutant protein, 500 ng cetuximab) at 37°C and pH 9.0 for 12 h, followed by SDS-PAGE gel electrophoresis and silver staining. The silver staining procedure was as follows: the gel after electrophoresis was immersed in a fixative solution (50% methanol + 12% acetic acid + 0.05% formaldehyde aqueous solution) at room temperature for 2 h, washed three times with water, then sensitized with 0.02% NaS₂O₃ solution for 2 min, washed three times with water, and then stained with staining solution (1× silver nitrate solution + 0.05% formaldehyde aqueous solution) for 30 min before development. The conjugation results were observed, and proteins covalently conjugated with cetuximab were screened. The results showed that after incubation with cetuximab, three of the 15 mutant proteins exhibited conjugation: N11FSY, Y14FSY, and E24FSY (e.g., ...). Figure 3 ).

[0074] Example 4: Screening of incubation time for covalent conjugation of mutant protein with cetuximab

[0075] Taking Y14FSY protein as an example, it was incubated with cetuximab at a molar ratio of 40:1 (4.6 μg mutant protein, 500 ng cetuximab) at 37°C and pH 9.0 for 2 h, 6 h, 12 h, and 24 h, respectively. Then, SDS-PAGE gel electrophoresis and silver staining of the protein were performed as in Example 3 to select the optimal incubation time. The highest conjugation efficiency was observed at 12 h, while 24 h incubation showed increased binding of cetuximab heavy chains and other contaminating proteins. Finally, the optimal incubation conditions were determined to be 37°C, pH 9.0, Y14FSY:cetuximab molar ratio = 40:1, and 12 h (e.g., ...). Figure 4 ).

[0076] Example 5: Covalent conjugation of sfGFP-Z(FSY) mutant protein with pembrolizumab

[0077] The mutant proteins N11FSY, Y14FSY, and E24FSY selected in Example 3, which showed covalent conjugation results, and pembrolizumab (purchased from Merck (China) Investment Co., Ltd., batch number S20180019) were incubated at a molar ratio of 40:1 (4.6 μg mutant protein, 500 ng pembrolizumab) at 37°C and pH 9.0 for 12 h. SDS-PAGE gel electrophoresis and silver staining of the proteins were performed using the method described in Example 3. The results showed the same good conjugation effect as the cetuximab group, indicating that the Z domain modified with the non-natural amino acid FSY can serve as a universal platform for conjugating IgG monoclonal antibody drugs, possessing good versatility (e.g., ...). Figure 5 ).

[0078] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for specifically covalently conjugating monoclonal antibody drugs using the Z-domain, characterized in that, The method includes the following steps: 1) introducing the stop codon TAG at a specific site of the amino acid sequence of the Z-domain protein using site-directed mutagenesis; 2) using genetic code expansion technology to introduce non-natural amino acids at the TAG site during protein induction expression to obtain a mutant Z-domain protein containing non-natural amino acids; 3) co-incubating the mutant Z-domain protein with an IgG monoclonal antibody drug at 37°C and pH 9.0 for 2-24 hours to allow the Z-domain to specifically covalently couple with the IgG monoclonal antibody drug.

2. The method as described in claim 1, characterized in that, The amino acid sequence of the Z-domain protein is shown in SEQ ID NO.

1.

3. The method as described in claim 1 or 2, characterized in that, The Z-domain protein-specific sites for introducing non-natural amino acids include sites 11, 14, and 24 of the amino acid sequence shown in SEQ ID NO.

1.

4. The method as described in claim 1, characterized in that, The non-natural amino acid is fluorosulfonic acid-L-tyrosine, with the structure shown below:

5. The method as described in claim 1, characterized in that, The mutant Z-domain protein is a pure protein solution extracted from recombinant E. coli DH10B after the plasmid containing the amino acid at a specific site of the Z-domain protein is mutated to the stop codon TAG and a plasmid carrying an aminoacylated tRNA synthetase / tRNA system capable of recognizing non-natural amino acids is co-transfected with the plasmid. The recombinant E. coli is then induced with arabinose and the expression of non-natural amino acids is added.

6. The method as described in claim 5, characterized in that, The plasmid carrying an aminoacylated tRNA synthetase / tRNA system capable of recognizing non-natural amino acids is the pSupAR-chFSYRS plasmid.

7. The method as described in claim 4, characterized in that, The fluorosulfonic acid-L-tyrosine is prepared according to the following steps: (1) N-(tert-butyloxycarbonyl)-L-tyrosine tert-butyl ester was dissolved in acetonitrile, and triethylamine was added to activate it at room temperature for 10 min. Then 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-trifluoromethanesulfonate was added. The mixture was stirred at room temperature for 6 h. During this period, the reaction progress was monitored by TLC. The developing solvent was ethyl acetate and petroleum ether in a volume ratio of 1:

5. After the reaction was completed, the system was evaporated to dryness by rotary evaporation. After redissolving in ethyl acetate, it was extracted with 10% citric acid aqueous solution, ddH2O and saturated NaCl aqueous solution in sequence. Each solution was extracted twice. The organic phase was dried with anhydrous Na2SO4, filtered and then evaporated by rotary evaporation to remove ethyl acetate. Silica gel column chromatography was performed with ethyl acetate and petroleum ether in a volume ratio of 1:

15. Gravity elution was performed, and all eluent was collected and evaporated by rotary evaporation until no liquid flowed out, resulting in a yellow oily substance. (2) The yellow oily substance was dissolved in dichloromethane, and trifluoroacetic acid was added. The mixture was stirred overnight at room temperature. The reaction progress was monitored by TLC. The developing solvent was ethyl acetate and petroleum ether in a volume ratio of 1:

8. After the reaction was completed, the dichloromethane was removed by rotary evaporation. The product was dissolved in methanol and then added dropwise to cold diethyl ether at 0℃. The mixture was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the precipitate was dried under vacuum to obtain a white solid product, fluorosulfonic acid-L-tyrosine.

8. The method as described in claim 7, characterized in that, In step (1), the molar ratio of N-(tert-butoxycarbonyl)-L-tyrosine tert-butyl ester to triethylamine is 1:1-3; the molar ratio of N-(tert-butoxycarbonyl)-L-tyrosine tert-butyl ester to 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazolium-3-trifluoromethanesulfonate is 1:1-3; the volume of acetonitrile used is 1-5 mL / mmol based on the amount of N-(tert-butoxycarbonyl)-L-tyrosine tert-butyl ester; in step (2), the volume of dichloromethane used is 1-5 mL / mmol based on the amount of yellow oily substance; the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, the volume ratio of dichloromethane to methanol is 1:0.05, and the volume ratio of dichloromethane to cold diethyl ether is 1:

3.

9. The method as described in claim 1, characterized in that, The mutant Z-domain protein was mixed with the monoclonal antibody at a molar ratio of 40:

1.

10. The method as described in claim 5, characterized in that, The pure protein solution was prepared according to the following method: Recombinant *E. coli* was inoculated into LB liquid medium and cultured overnight at 37°C. Then, 20% arabinose aqueous solution and 100 mM non-natural amino acid aqueous solution were added sequentially at 1% (v / v) to the culture medium, and the culture was induced at 30°C for 20 h. After induction, the cells were collected by centrifugation at 8000 rpm for 10 min, resuspended in lysis buffer, and lysed using an ultrasonic homogenizer at 300 W for 20 min (1 second on, 3 seconds off). The lysed bacterial suspension was centrifuged at 10000 rpm for 10 min, and the supernatant was collected. Protein purification was performed using a Ni-NTA affinity chromatography column. Gradient elution was performed sequentially using lysis buffer and elution buffers with imidazole concentrations of 20 mM, 60 mM, 100 mM, 300 mM, and 1 M. All eluent collected from the 100 mM, 300 mM, and 1 M gradient elutions was dialyzed overnight at 4°C using a dialysis bag with a molecular weight cutoff of 3.5 kD. The chopped buffer was then concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kD. The concentrated solution was the purified protein solution. The lysis buffer composition was: 3 M sodium chloride, 3 mM dithiothreitol, 500 mM Tris-HCl, pH 8.

0. The elution buffer composition was: 3 M sodium chloride, 500 mM Tris-HCl, pH 8.0.

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