Claudin 6 antibody modified by non-natural amino acid as well as preparation method and application of Claudin 6 antibody

Abstract

The invention provides a Claudin 6 antibody containing non-natural amino acid modification and a preparation method and application thereof, the sequence of the antibody is obtained by replacing original amino acid with non-natural amino acid on the basis of SEQ ID NO: 1 and 6; the replacement site of the non-natural amino acid is selected from any one or a combination of at least two of the following components: a heavy chain: S114, T115, P148, G156, A157, T159, Q191, V149, L158 or G161; or a light chain: T109, A112, K169, S202, S203, L154 or S156. According to the invention, non-natural amino acids are introduced into specific sites of the antibody, and the antibody is expressed by adopting a cell expression system, so that the influence of insertion of non-natural amino acids at different sites on the expression level and structural stability of the antibody and the binding capacity of the antibody are evaluated, and a new thought is provided for rational design and function optimization of the antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a Claudin 6 antibody modified with unnatural amino acids, a preparation method thereof, and applications thereof. Background Art

[0002] In the technology of Antibody-Drug Conjugates (ADC), by introducing unnatural amino acids into antibodies, precise site-specific conjugation of antibodies and drugs is achieved, which not only significantly improves the homogeneity of ADC, but also optimizes its physicochemical properties and pharmacokinetic characteristics. This technological breakthrough helps to enhance the therapeutic effect of antibody-drug conjugates, while significantly reducing its non-specific toxicity and side effects.

[0003] In addition, the unnatural amino acid insertion technology can be widely applied to the engineering design of monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates, providing a flexible and efficient strategy for the development of antibody drugs. This technology has important application potential in the fields of cancer immunotherapy and infectious disease treatment, etc., and lays a solid foundation for the development of safer and more effective new biotherapeutic drugs.

[0004] Unnatural amino acids refer to extended amino acids beyond the range of 20 common amino acids that make up natural eukaryotic cell proteins. Their R side chains usually carry specific functional groups, such as acetyl groups, alkynyl groups, azide groups, or quinones, etc. These functional groups can specifically modify proteins through chemical reactions such as click chemistry, photocrosslinking, or nucleophilic attack, so as to achieve function optimization or expansion. However, due to the lack of corresponding genetic codes and necessary translation elements (such as transfer RNAs and aminoacyl-tRNA synthetases) for unnatural amino acids in cells, their introduction in protein synthesis has been restricted for a long time.

[0005] With the rapid development of genetic code expansion technology, significant progress has been made in the technology of introducing unnatural amino acids during protein expression through the suppression mechanism of the amber stop codon (UAG). Currently, this strategy has been successfully applied in various expression systems such as Escherichia coli, yeast, and mammalian cells. This technology not only significantly expands the chemical space of proteins, but also provides a powerful tool for biological research and the biotechnology field, especially showing broad application prospects in aspects such as protein modification, function exploration, and drug development.

[0006] The technology of introducing unnatural amino acids into antibodies has been widely applied in the field of site-specific conjugation of antibody-drug conjugates (ADCs), providing new solutions for improving conjugation efficiency and drug stability. Commonly used unnatural amino acids include p-Acetylphenylalanine (pAF), p-Azidomethyl-L-phenylalanine (pAMF), and Azido-lysine. For example, at the 121st alanine (A121) position of the heavy chain of Trastuzumab (anti-HER2 antibody), its corresponding codon was mutated to the amber stop codon (UAG, A121X), and using the orthogonal transfer RNA (tRNA) and aminoacyl-tRNA synthetase (aaRS) derived from Methanococcus jannaschii, a modified Trastuzumab incorporating pAF was successfully synthesized. Related studies have shown that in addition to the 121st position of the heavy chain, the 169th (L169) and 202nd (L202) positions of the light chain of Trastuzumab can also serve as insertion sites for unnatural amino acids. After inserting pAF at these sites and conjugating with the microtubule-disrupting agent DM1, the conjugated products showed significant cytotoxicity in in vitro breast cancer cell experiments. These research results indicate that by introducing unnatural amino acids at specific sites of antibodies, not only can precise chemical modification be achieved, but also the biological activity of antibody-drug conjugates can be effectively retained, laying a solid technical foundation for the development of highly efficient and stable ADCs.

[0007] Antibodies are a class of immunoglobulins with high specificity and diversity, mainly divided into five types: IgG, IgA, IgM, IgE, and IgD. Among them, IgG has been widely studied and applied due to its excellent stability and functionality. An IgG molecule is usually composed of two light chains and two heavy chains connected by disulfide bonds, with a total molecular weight of approximately 150 kDa, presenting a typical Y-shaped structure. Its structure includes the Fab region (fragment of antigen binding, Fab), the Hinge Region, and the Fc region (fragment crystallizable, Fc), which are involved in antigen binding, molecular flexibility regulation, and the mediation of effector functions, respectively.

[0008] Antibodies in IgG form have a molecular weight of approximately 150 kD and consist of more than 1,300 amino acids. Existing studies have shown that the common site for replacing non-natural amino acids on antibodies is the linker sequence between the heavy-chain variable region and constant region I. For example, on the heavy chain of trastuzumab, alanine at position 121 is a common replacement site. However, which specific sites in the antibody molecule can be replaced with non-natural amino acids to ensure smooth antibody expression, maintain the "Y"-shaped structure of the antibody, and still have activity after replacement, that is, the binding ability to the antigen is not significantly weakened. Currently, there is still a lack of systematic research in this area. The lack of research in this field makes it lack valuable reference materials and clear guiding principles for researchers to design and optimize non-natural amino acid-modified antibodies.

[0009] Therefore, this study designed and screened sites for introducing non-natural amino acids, and evaluated the structure and function of the antibody. This study has important guiding and application values for the modification of antibody physical and chemical properties and the development of antibody conjugates. Summary of the Invention

[0010] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a Claudin 6 antibody modified with non-natural amino acids, its preparation method and application. The present invention focuses on the in vitro expression technology of antibodies containing non-natural amino acids. By introducing non-natural amino acids at specific sites of the antibody and using the Expi293F cell expression system, the effects of non-natural amino acid insertion at different sites on the physical and chemical properties such as antibody expression level and structural stability are systematically evaluated, providing new ideas and technical support for the rational design and function optimization of antibodies.

[0011] To achieve the purpose of this invention, the following technical solutions are adopted:

[0012] In the first aspect, the present invention provides a Claudin 6 antibody, the amino acid sequence of the heavy-chain variable region of the antibody is as shown in SEQ ID NO:2; the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:7;

[0013] The heavy-chain variable region of the antibody includes CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4, and the sequence of CDR3 is PAY;

[0014] The light-chain variable region of the antibody includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:5.

[0015] Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 1; the amino acid sequence of the light chain is as shown in SEQ ID NO: 6.

[0016] In a second aspect, the present invention provides a Claudin 6 antibody comprising non-natural amino acid modification, which is obtained by replacing the original amino acids with non-natural amino acids on the basis of the Claudin 6 antibody described in the first aspect; the insertion sites of the non-natural amino acids are selected from any one or a combination of at least two of the following:

[0017] Heavy chain: S114, T115, P148, G156, A157, T159, Q191, V149, L158 or G161; or light chain: T109, A112, K169, S202, S203, L154 or S156.

[0018] The present invention takes the antibody targeting Claudin 6 as the research object, combines the three-dimensional structure information of the antibody, designs and screens 17 sites for the introduction of non-natural amino acids, including 10 sites designed on the heavy chain and 7 sites designed on the light chain. The present invention attempts to express the antibodies containing non-natural amino acids at these sites using a mammalian expression system and evaluate the stability on the premise of not affecting the basic functions of the antibodies, providing new ideas and methods for the rational design, function optimization of antibody molecules and site-specific conjugation of ADC.

[0019] Preferably, the inserted non-natural amino acid is p-acetylphenylalanine or azidomethyl-L-phenylalanine.

[0020] In one embodiment of the present invention, the antibody is based on the amino acid sequence of the heavy chain shown in SEQ ID NO: 1, and S114 of the heavy chain is replaced with p-acetylphenylalanine.

[0021] In one embodiment of the present invention, the antibody is based on the amino acid sequence of the heavy chain shown in SEQ ID NO: 1, and T115 of the heavy chain is replaced with p-acetylphenylalanine.

[0022] In one embodiment of the present invention, the antibody is based on the amino acid sequence of the heavy chain shown in SEQ ID NO: 1, and P148 of the heavy chain is replaced with p-acetylphenylalanine.

[0023] In one embodiment of the present invention, the antibody is based on the amino acid sequence of the heavy chain shown in SEQ ID NO: 1, and G156 of the heavy chain is replaced with p-acetylphenylalanine.

[0024] In one embodiment of the present invention, the antibody is based on the heavy chain amino acid sequence shown in SEQ ID NO:1, and the heavy chain A157 is replaced with p-acetylphenylalanine.

[0025] In one embodiment of the present invention, the antibody is based on the heavy chain amino acid sequence shown in SEQ ID NO:1, and the heavy chain T159 is replaced with p-acetylphenylalanine.

[0026] In one embodiment of the present invention, the antibody is based on the heavy chain amino acid sequence shown in SEQ ID NO:1, and the heavy chain Q191 is replaced with p-acetylphenylalanine.

[0027] In one embodiment of the present invention, the antibody is based on the heavy chain amino acid sequence shown in SEQ ID NO:1, and the heavy chain V149 is replaced with p-acetylphenylalanine.

[0028] In one embodiment of the present invention, the antibody is based on the amino acid sequence of the heavy chain shown in SEQ ID NO:1, and the heavy chain L158 is replaced with p-acetylphenylalanine.

[0029] In one embodiment of the present invention, the antibody is based on the heavy chain amino acid sequence shown in SEQ ID NO:1, and the heavy chain G161 is replaced with p-acetylphenylalanine.

[0030] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO:6, and the light chain T109 is replaced with p-acetylphenylalanine.

[0031] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO:6, and the light chain A112 is replaced with p-acetylphenylalanine.

[0032] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO:6, and the light chain K169 is replaced with p-acetylphenylalanine.

[0033] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO:6, and the light chain S202 is replaced with p-acetylphenylalanine.

[0034] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO:6, and the light chain S203 is replaced with p-acetylphenylalanine.

[0035] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO: 6, and L154 of the light chain is replaced with p-acetylphenylalanine.

[0036] In one embodiment of the present invention, the antibody is based on the light chain amino acid sequence shown in SEQ ID NO: 6, and S156 of the light chain is replaced with p-acetylphenylalanine.

[0037] In a third aspect, the present invention provides a nucleic acid molecule that encodes the Claudin 6 antibody comprising non-natural amino acid modifications described in the first aspect, or encodes the Claudin 6 antibody comprising non-natural amino acid modifications described in the second aspect.

[0038] In a fourth aspect, the present invention provides an expression vector that contains the nucleic acid molecule described in the third aspect, and after being transfected into a host cell, the expression vector enables the host cell to express the Claudin 6 antibody described in the first aspect, or enables the host cell to express the Claudin 6 antibody comprising non-natural amino acid modifications described in the second aspect.

[0039] Preferably, the expression vector is the pcDNA3.1 vector.

[0040] In a fifth aspect, the present invention provides a host cell that contains at least one copy of the expression vector described in the fourth aspect, or at least one copy of the nucleic acid molecule described in the third aspect.

[0041] Preferably, the host cell is Expi293F cell.

[0042] In a sixth aspect, the present invention provides a method for preparing the Claudin 6 antibody comprising non-natural amino acid modifications described in the second aspect. The preparation method includes: synthesizing the nucleic acid sequences encoding the heavy and light chains of the antibody, mutating the codons at the sites for introducing non-natural amino acids into TAG; cloning the nucleic acid sequences onto a vector to obtain a plasmid expressing the heavy and light chains of the antibody; introducing the plasmid expressing the heavy and light chains of the antibody, the pcDNA3.1-tRNA plasmid, and the pcDNA3.1-Tet-tRNA ligase plasmid into cells for in vitro expression to obtain the Claudin 6 antibody comprising non-natural amino acid modifications.

[0043] In a seventh aspect, the present invention provides a Claudin 6 antibody-drug conjugate, and the conjugate includes the Claudin 6 antibody described in the first aspect, or the Claudin 6 antibody comprising non-natural amino acid modifications described in the second aspect.

[0044] In an eighth aspect, the present invention provides the use of any one or a combination of at least two of the Claudin 6 antibodies described in the first aspect, the Claudin 6 antibodies containing unnatural amino acid modifications described in the second aspect, the nucleic acid molecules described in the third aspect, the expression vectors described in the fourth aspect, the host cells described in the fifth aspect, or the Claudin 6 antibody-drug conjugates described in the seventh aspect in the preparation of drugs for cancer immunotherapy or infectious disease treatment.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The Claudin 6 antibodies screened in the present invention have advantages such as good antigen-binding activity and strong affinity.

[0047] (2) The present invention successfully expressed 17 Claudin6 antibody variants containing unnatural amino acids using the Expi293F expression system. These variants introduced unnatural amino acids at different sites on the light and heavy chains and were systematically compared with the original Claudin 6 antibody. The expressed antibodies were characterized by mass spectrometry analysis technology to confirm the successful introduction of the target unnatural amino acid p-aminophenylalanine (pAF). The study found that specific sites located on the heavy chain at S114 / T115 / P148 / G156 / A157 / T159 / Q191 / V149 / L158 / G161, and on the light chain at T109 / A112 / K169 / S202 / S203 / L154 / S156 can effectively accommodate the introduction of unnatural amino acids without affecting the structural integrity. This result indicates that the above sites have good engineering potential, providing a strong basis for further optimizing and developing antibody molecules containing unnatural amino acids. Description of the Drawings

[0048] Figure 1 Schematic diagram for the design of unnatural amino acid insertion sites on the antibody.

[0049] Figure 2 Structural formula of the unnatural amino acid pAF.

[0050] Figure 3 SDS-PAGE results of antibody proteins 6-H (T159X), 7-H (Q191X), 8-H (V149X), 9-H (L158X), 10-H (G161X), and 11-L (T109X).

[0051] Figure 4SDS-PAGE results of antibody proteins 12-L (A112X), 13-L (K169X), 14-L (S202X), 15-L (S203X), 16-L (L154X) and 17-L (S156X).

[0052] Figure 5 SDS-PAGE results of 3-H (P148X), 4-H (G156X), 5-H (A157X), 14-L (S202X) and 15-L (S203X).

[0053] Figure 6 SDS-PAGE results of 1-H (S114X) and 2-H (T115X).

[0054] Figure 7 HPLC-SEC detection results of WT (1 / 4), 1-H (S114X), 3-H (P148X) and 4-H (G156X).

[0055] Figure 8 HPLC-SEC detection results of 2-H (T115X) and 5-H (A157X).

[0056] Figure 9 HPLC-SEC detection results of 6-H (T159X), 7-H (Q191X), 9-H (L158X) and 10-H (G161X).

[0057] Figure 10 HPLC-SEC detection results of 8-H (V149X) and 11-L (T109X).

[0058] Figure 11 HPLC-SEC detection results of 12-L (A112X), 13-L (K169X), 14-L (S202X) and 16-L (L154X).

[0059] Figure 12 HPLC-SEC detection results of 14-L (S202X) and 17-L (S156X).

[0060] Figure 13 Mass spectrometry detection results of WT and 2-H (T115X).

[0061] Figure 14 Mass spectrometry detection results of 1-H (S114X) and 3-H (P148X).

[0062] Figure 15 Mass spectrometry detection results of 4-H (G156X) and 6-H (T159X).

[0063] Figure 16 Mass spectrometry detection results for 5-H (A157X) and 7-H (Q191X).

[0064] Figure 17 Mass spectrometry detection results for 8-H (V149X) and 10-H (G161X).

[0065] Figure 18 Mass spectrometry detection results for 9-H (L158X) and 11-L (T109X).

[0066] Figure 19 Mass spectrometry detection results for 12-L (A112X) and 14-L (S202X).

[0067] Figure 20 Mass spectrometry detection results for 13-L (K169X) and 15-L (S203X).

[0068] Figure 21 Mass spectrometry detection results for 16-L (L154X) and 17-L (S156X).

[0069] Figure 22 Results of the cell binding experiment for 18-WT antibody and Claudin 6 cell line.

[0070] Figure 23 Results of the cell binding experiment for 18-WT, 1-H (S114X), 2-H (T115X), 3-H (P148X), 4-H (G156X), 5-H (A157X) and Claudin 6 cell line.

[0071] Figure 24 Results of the cell binding experiment for 18-WT, 6-H (T159X), 7-H (Q191X), 8-H (V149X), 9_H (L158), 10-H (G161X) and Claudin 6 cell line.

[0072] Figure 25 Results of the cell binding experiment for 18-WT, 11-L (109X), 12-L (A112X), 13-L (K169X), 14-L (S202X), 15-L (S203X) and Claudin 6 cell line.

[0073] Figure 26 Results of the cell binding experiment for 18-WT, 16-L (154X), 17-L (S156X) and Claudin 6. Specific implementation methods

[0074] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0075] For those not specified in the embodiments in terms of specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions. For reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through regular channels.

[0076] Example 1

[0077] Screening for high-affinity Claudin 6 antibodies includes the following process:

[0078] (1) According to the protein sequence and gene sequence information of Claudin 6 (UniProt: P56747), plasmid DNA was designed and prepared for subsequent mouse immunization.

[0079] (2) The mice were immunized three times with the plasmid DNA obtained in the previous step. One week after the third immunization, the serum titer was detected by the ELISA method. The qualified anti-serum titer indicated that the mice had been induced to produce a high-titer anti-serum specifically against the Claudin 6 protein.

[0080] (4) The spleens and bone marrow cells of the immunized mice were collected, centrifuged at 400g for 5 min, resuspended in 1 mL of red blood cell lysis buffer, lysed on ice for 1 min, terminated with a large volume of Macs buffer, centrifuged at 400g for 5 min, resuspended in 1 mL of Macs buffer, added with fluorescently labeled Claudin 6 protein and fluorescent antibodies of B cell-specific molecular markers, incubated at 4 °C for 1 h, resuspended with a large volume of Macs buffer, centrifuged at 400g for 5 min, the supernatant was discarded, the cells were resuspended in the buffer, and antigen-positive cell sorting was performed using a flow cytometer.

[0081] (5) Construction of a mouse positive single-cell library, the process is as follows: Using a single-cell library construction platform to construct a library for the sorted positive mouse cells, generating oil droplets containing a single positive mouse cell through the water-in-oil technology, and completing cell lysis, mRNA capture, and reverse transcription within the oil droplets. The reverse transcription products carrying cell barcode were subjected to cDNA pre-amplification and antibody VDJ gene amplification, ligated with adapters and library tags after fragmentation to obtain an antibody library, and antibodies with natural pairing of heavy and light chains were obtained through next-generation sequencing and data analysis.

[0082] The sequences of the screened Claudin 6 antibodies are shown as follows:

[0083] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.

[0084] The heavy chain variable region of the antibody includes CDR1 shown in SEQ ID NO: 3, CDR2 shown in SEQ ID NO: 4, and the sequence of CDR3 is PAY.

[0085] The light chain variable region of the antibody includes CDR1 shown in SEQ ID NO: 8, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 5.

[0086] The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO: 1; the amino acid sequence of the light chain is shown in SEQ ID NO: 6.

[0087] Example 2

[0088] In this example, alphafold was used to construct the three-dimensional structure of the Claudin 6 antibody, analyze the three-dimensional structure of Claudin 6, and in combination with relevant literature, on the premise of not affecting the function of the antibody, design and select the sites for introducing unnatural amino acids on the Claudin 6 antibody. The design of the sites for inserting unnatural amino acids on the antibody is as Figure 1 shown. Among them, the homologous structure of the overall three-dimensional structure of the antibody simulated by alphafold is shown on the right side of the figure. The light chain is shown in green, and the heavy chain is shown in cyan. On the left are the sites for inserting unnatural amino acids on the heavy and light chains. Among them, there are 10 sites on the heavy chain and 7 sites on the light chain.

[0089] Example 3

[0090] 1. Construct plasmids

[0091] Fully synthesize the nucleic acid sequences of the heavy and light chains of the antibody, mutate the codons at the sites for introducing unnatural amino acids into TAG, and then subclone them onto the pcDNA3.1 vector respectively.

[0092] 2. Express using the Expi293F in vitro expression system

[0093] Take 200 mL of 1.5×10 6, Expi293F cells with a viability of 98% were used. pcDNA3.1-light chain, pcDNA3.1-heavy chain, pcDNA3.1-tRNA, and pcDNA3.1-Tet-tRNAligase plasmids were mixed with PEI (1 mg / mL) at a mass ratio of 1:3 and incubated at room temperature for 20 min, then added dropwise to the cells. After 24 h of transfection, pAF with a final concentration of 1 mM was added to the cells and cultured at 37°C for 5 days. Figure 2 It is the structural formula of the unnatural amino acid pAF.

[0094] 3. Collect the supernatant and purify

[0095] The cells were poured into a 250 mL centrifuge bottle and centrifuged at 5000 rpm at 4°C for 20 min. The supernatant was collected. The supernatant was filtered through 0.45 μm and 0.22 μm filters respectively. The filtered supernatant was added to a purification column containing 2 mL of protein A packing; the operation was repeated twice. 50 mL of 1×PBS was added to the purification column to wash away the miscellaneous proteins. 10 mL of 0.1 M sodium citrate with pH 3.0 was added to the purification column to elute the protein, and the eluate was collected with a 50 mL centrifuge tube containing 5 mL of 1 M Tris-HCl with pH 8.0. The protein was concentrated with a 50 Kd 15 mL ultrafiltration tube.

[0096] Table 1 shows the unnatural amino acid insertion sites and antibody expression information.

[0097] Table 1

[0098] Name Theoretical Molecular Weight (D) Concentration (mg / mL) Yield (μg) 1-H (S114X) 48588.05 0.16 32.20 2-H (T115X) 48574.02 0.18 37.80 3-H (P148X) 48578.01 0.426 65.38 4-H (G156X) 48618.07 0.447 68.11 5-H (A157X) 48604.05 0.598 81.76 6-H (T159X) 48573.98 0.2 32.48 7-H (Q191X) 48546.99 0.28 57.60 8-H (V149X) 48575.99 0.13 18.62 9-H (L158X) 48561.97 0.28 49.70 10-H (G161X) 48618.07 0.26 53.80 11-L (T109X) 23616.16 0.27 62.33 12-L (A112X) 23646.23 0.4 74.48 13-L (K169X) 23589.13 0.66 139.65 14-L (S202X) 23630.23 1.61 284 15-L (S203X) 23630.23 1.36 255.50 16-L (L154X) 23604.15 0.57 136.94 17-L (S156X) 23630.23 0.33 76.00 18-H_WT / 19-L_WT 48485.91 / 23528.09 1.97 1370

[0099] In Table 1 above, "H" represents Heavy chain, that is, the heavy chain of the antibody. "L" represents Light chain, that is, the light chain of the antibody. "X" represents an unnatural amino acid, and the unnatural amino acid can be p-acetylphenylalanine, azidomethyl-L-phenylalanine, etc. In this study, p-acetylphenylalanine (pAF) was taken as an example. For example, 1-H(S114X) means replacing serine at position 114 on the heavy chain of the antibody with pAF, and 11-L(T109X) means replacing threonine at position 109 on the light chain of the antibody with pAF.

[0100] 4. Detection of protein properties and functions

[0101] (1) SDS-PAGE detection

[0102] Take about 2×3 μg of each sample and add reducing (R) and non-reducing 4× loading buffer respectively. Among them, the sample added with reducing loading buffer was heated in a 95°C metal bath for 5 min.

[0103] The SDS-PAGE results of the antibody proteins are as Figures 3 - 6 shown. Figure 3 The SDS-PAGE results of antibody proteins 6-H (T159X), 7-H (Q191X), 8-H (V149X), 9-H (L158X), 10-H (G161X) and 11-L (T109X). Figure 4 The SDS-PAGE results of antibody proteins 12-L (A112X), 13-L (K169X), 14-L (S202X), 15-L (S203X) and 16-L (L154X). Figure 5 The SDS-PAGE results of 3-H (P148X), 4-H (G156X), 5-H (A157X), 14-L (S202X) and 15-L (S203X). Figure 6 The SDS-PAGE results of 1-H (S114X) and 2-H (T115X).

[0104] The electrophoresis results showed that in addition to the target bands, 14-L (S202X) and 15-L (S203X) also contained non-target bands. From the results of the non-reducing samples, it was found that the molecular weight of the impurity bands was smaller than that of the target bands. From the results of the reducing samples, it was found that the molecular weight of the impurity bands was smaller than that of the target light chains. We speculated that the light chains that were not successfully inserted into pAF were contained in the 14-L (S202X) and 15-L (S203X) experimental groups, and these light chains combined with the heavy chains to assemble into a "Y" structure. It could be seen from the gel diagram that the reducing and non-reducing electrophoresis bands of the remaining antibody mutants were similar to those of the original Claudin 6 antibody (abbreviated as WT, without unnatural amino acids) obtained in Example 1.

[0105] (2) HPLC-SEC detection

[0106] Take 30 μL of the sample from each experimental group for HPLC-SEC detection. Equipment: High performance liquid chromatography (Shimadzu, LC-2050C). Chromatographic column: XBridge Protein BEH SEC Column, 3.5 μm, 7.8 mm × 150 mm. Buffer: 1× PBS. Program: Flow rate: 0.5 mL / min; Total duration: 15 min; Temperature: 25°C. Light source: Deuterium lamp; Wavelength: 280 nM.

[0107] Experimental procedures: First, place the D pump in filtered and degassed ddH2O and rinse it at a flow rate of 1 mL / min for 20 min, then adjust the flow rate to 0. Place the D pump in 1×PBS, adjust the flow rate to 1 mL / min, set the column oven temperature to 25 °C, turn on the deuterium lamp, and rinse for 20 min before adjusting the flow rate to 0. Adjust the flow rate to 0.5 mL / min, install the chromatographic column, and after the baseline is flushed flat, load the sample. Select the above program to run the sample. The data obtained is processed and analyzed using GraphPad Prism 8 software.

[0108] The HPLC-SEC detection results of the antibody are as Figures 7 - 12 , Figure 7 the HPLC-SEC detection results of WT(1 / 4), 1-H(S114X), 3-H(P148X), and 4-H(G156X); Figure 8 the HPLC-SEC detection results of 2-H(T115X) and 5-H(A157X); Figure 9 the HPLC-SEC detection results of 6-H(T159X), 7-H(Q191X), 9-H(L158X), and 10-H(G161X); Figure 10 the HPLC-SEC detection results of 8-H(V149X) and 11-L(T109X); Figure 11 the HPLC-SEC detection results of 12-L(A112X), 13-L(K169X), 14-L(S202X), and 16-L(L154X); Figure 12 the HPLC-SEC detection results of 14-L(S202X) and 17-L(S156X).

[0109] Among them, the original Claudin 6 antibody (WT, without unnatural amino acids) obtained in Example 1 has a narrow and sharp peak at 7.58 min. The elution times of the 17 antibodies containing unnatural amino acids are between 7.50 min and 7.62 min, and the peak shapes are similar to that of the WT antibody, also narrow and sharp. This result indicates that the overall conformations of these 17 antibodies containing unnatural amino acids are highly similar to that of the WT antibody, suggesting that the "Y"-shaped structure of the mutants is not significantly affected. Therefore, after replacing the amino acids at these 17 sites with pAF, the spatial conformation and structural stability of the antibody do not change significantly.

[0110] (3) Mass spectrometry detection of the antibody

[0111] Instruments: Ultra-high performance liquid chromatograph, ThermoFisher, Vanquish. High-resolution mass spectrometer, ThermoFisher, QExactive. Chromatographic column: UP3WC4-100 / 021, Yimi Biotechnology. PNGase F: Shanghai Yongtuo. DTT: Sigma-Aldrich. Mobile phase A: 0.1% formic acid aqueous solution. Mobile phase B: 0.1% formic acid acetonitrile solution.

[0112] Sample preparation: Sugar-cutting treatment: Take a part of the test sample and react it with PNGase F enzyme at 37 °C for 2 hours. Reduction: Take a part of the test sample and reduce it with 20 mM DTT for 1 hour.

[0113] The mobile phase gradient is shown in Table 2:

[0114] Table 2

[0115]

[0116] The mass spectrometry detection results of the antibody are as Figures 13 - 21 , Figure 13 the mass spectrometry detection results of WT and 2-H (T115X); Figure 14 the mass spectrometry detection results of 1-H (S114X) and 3-H (P148X); Figure 15 the mass spectrometry detection results of 4-H (G156X) and 6-H (T159X); Figure 16 the mass spectrometry detection results of 5-H (A157X) and 7-H (Q191X); Figure 17 the mass spectrometry detection results of 8-H (V149X) and 10-H (G161X); Figure 18 the mass spectrometry detection results of 9-H (L158X) and 11-L (T109X); Figure 19 the mass spectrometry detection results of 12-L (A112X) and 14-L (S202X); Figure 20 the mass spectrometry detection results of 13-L (K169X) and 15-L (S203X); Figure 21 the mass spectrometry detection results of 16-L (L154X) and 17-L (S156X). Among them, the samples of 14-L (S202X) and 15-L (S203X) contain two kinds of light chains, one is the target light chain, and the other is a light chain about 1400 D smaller than the target light chain. The results are consistent with the electrophoresis results. The measured molecular weights of the remaining 15 amino acids all conform to the theoretical molecular weights.

[0117] The theoretical molecular weights of the light and heavy chains of the WT antibody are 23,528.09 D and 48,485.91 D respectively; Sample 1-H(S114X) replaces serine at position 114 on the heavy chain of the antibody with pAF. The molecular weight of its light chain is the same as that of WT, theoretically 23,534.06 D; the molecular weight of the heavy chain is theoretically 48,588.05 D, which is theoretically 102.14 D higher than that of WT. The mass spectrometry results after deglycosylation and reduction of the sample show that the actual molecular weight difference between the heavy chain of 1-H(S114X) and the heavy chain of WT is 102.37 D, which is consistent with the theoretical molecular weight difference. Therefore, S114 in the 1-H(S114X) antibody was successfully replaced with pAF. The same applies to the other samples. (The statistical results are shown in Table 3)

[0118] Table 3

[0119]

[0120]

[0121] (4) Cell binding experiment Binding experiment of Claudin 6 antibody (18_WT) and Claudin 6 cell line.

[0122] Antigen: Claudin 6 cell line (self-developed).

[0123] Primary antibody: 18 antibodies involved in this study (1 WT, 17 containing unnatural amino acids).

[0124] Directly labeled antibody: Human Claudin-6 Alexa 647-conjugated Antibody (R&D).

[0125] Secondary antibody: Alexa 647 AffiniPure Goat Anti-Mouse IgG (Jackson).

[0126] Flow cytometer: Attune NxT Acoustic Focusing Cytometer (Thermo, AFC2).

[0127] Prepare Claudin 6 cell line: Replace the medium of the Claudin 6 cell line with a viability of not less than 95% with MACS culture medium (PBS, 2% FBS and 2 mM EDTA), and adjust the cell concentration to 4×10 6 / mL. Add 50 μL of cells to each well of a 96-well plate.

[0128] Antibody dilution: The initial concentration of each antibody was 800 nM, and it was serially diluted 4-fold with Macs solution.

[0129] FACS assay: To each well of a 96-well plate containing Claudin 6 cell line, 50 μL of the diluted antibody was added. After mixing well, the plate was incubated at 4 °C in the dark for 1 hour. Then, 200 μL of Macs solution was added to each well, and the plate was centrifuged at 400 g for 5 min. The supernatant was discarded, and this washing step was repeated three times. Next, 1 μg / mL of secondary antibody was added to each well, and the plate was incubated at 4 °C in the dark for 30 min. Then, 200 μL of Macs solution was added to each well, and the plate was centrifuged at 400 g for 5 min. The supernatant was discarded, and this washing step was repeated three times. The cells were resuspended in 200 μL of Macs solution and analyzed by flow cytometry. The data were processed and analyzed using GraphPad Prism 8.

[0130] The experimental results of the antibody-cell binding assay showed that the Yangshen antibody bound to the Claudin 6 overexpressing cell line used in this study but did not reach a plateau, and the binding ability of the 18-WT antibody to the Claudin 6 overexpressing cell line was stronger than that of the PC-direct labeled antibody.

[0131] Binding assay of 18 Claudin 6 antibodies (18-WT and 17 antibodies containing one pAF) to the Claudin 6 cell line. Figures 22 - 26 Showed the results of the binding assay of the 18 antibodies involved in this study to the Claudin 6 overexpressing cell line. Figure 22 Were the results of the cell binding assay of the 18-WT antibody to the Claudin 6 cell line. Figure 23 Were the results of the cell binding assay of 18-WT, 1-H(S114X), 2-H(T115X), 3-H(P148X), 4-H(G156X), and 5-H(A157X) to the Claudin 6 cell line. Figure 24 Were the results of the cell binding assay of 18-WT, 6-H(T159X), 7-H(Q191X), 8-H(V149X), 9_H(L158), and 10-H(G161X) to the Claudin 6 cell line. Figure 25 Were the results of the cell binding assay of 18-WT, 11-L(109X), 12-L(A112X), 13-L(K169X), 14-L(S202X), and 15-L(S203X) to the Claudin 6 cell line. Figure 26 Were the results of the cell binding assay of 18-WT, 16-L(154X), and 17-L(S156X) to Claudin 6.

[0132] As can be seen from the figure, after adding the unnatural amino acid pAF to 17 sites of the antibody respectively, the antibody still binds to the Claudin 6 overexpressing cell line. This indicates that replacing these 17 sites (see Table 1) with unnatural amino acids does not affect the function of the antibody, that is, it does not affect the binding of the antibody to the antigen. Moreover, the binding ability of 18-WT to the Claudin 6 cell line is stronger than that of the positive control antibody PC-direct label antibody, and the binding abilities of 1-H(S114X) and 8-H(V149X) to the Claudin 6 cell line are comparable to that of 18-WT; the binding ability of the antibody mutants with the four sites 6-H(T159X), 10-H(G161X), 14-L(S202X), and 15-L(S203X) replaced with unnatural amino acids to the Claudin 6 cell line is stronger than that of 18-WT.

[0133] Final result analysis: In this study, the amino acids at 10 sites, namely S114, T115, P148, G156, A157, T159, Q191, V149, L158, and G161 on the heavy chain of the Claudin 6 antibody, were successfully replaced with the unnatural amino acid pAF; at the same time, the amino acids at 7 sites, namely T109, A112, K169, S202, S203, L154, and S156 on the light chain of the Claudin 6 antibody, were also successfully replaced with the unnatural amino acid pAF. These 17 antibody mutants all maintain the typical "Y"-shaped antibody structure and can specifically bind to the Claudin 6 overexpressing cell line. Among them, the heavy chain S114X and V149X antibody mutants, and the light chain S202X and S203X mutants show higher binding abilities to the Claudin 6 overexpressing cell line compared with the original Claudin 6 antibody (WT). The binding abilities of the heavy chain T159X and G161X mutants to the Claudin 6 overexpressing cell line are comparable to that of the WT antibody. In addition, the application of these sites can be further expanded. Besides introducing p-acetylphenylalanine (pAF), they can also be replaced with azidomethyl-L-phenylalanine (pAMF), providing more extensive possibilities for antibody functional modification.

[0134] In summary, the present invention provides a Claudin 6 antibody and its variants, which introduce unnatural amino acids at different sites on the light chain and heavy chain respectively. The insertion of unnatural amino acids provides a flexible and efficient strategy for the subsequent development of antibody drugs; the above insertion sites have good engineering potential, providing a strong basis for further optimizing and developing antibody molecules containing unnatural amino acids.

[0135] The applicant declares that the above description is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A Claudin 6 antibody, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7; The heavy chain variable region of the antibody includes CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4, and the sequence of CDR3 is PAY; The light chain variable region of the antibody includes CDR1 as shown in SEQ ID NO:8, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:

5.

2. The Claudin 6 antibody according to claim 1, characterized in that The heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 1; the light chain amino acid sequence is shown in SEQ ID NO:

6.

3. A Claudin 6 antibody comprising a non-natural amino acid modification, characterized in that: The antibody is obtained by replacing the original amino acids with non-natural amino acids based on the Claudin 6 antibody of claim 2; the insertion site of the non-natural amino acid is selected from any one or a combination of at least two of the following: Heavy chain: S114, T115, P148, G156, A157, T159, Q191, V149, L158 or G161; or light chain: T109, A112, K169, S202, S203, L154 or S156.

4. The Claudin 6 antibody modified with a non-natural amino acid according to claim 3, characterized in that: The inserted non-natural amino acid is p-acetylphenylalanine or azidomethyl-L-phenylalanine.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Claudin 6 antibody of claim 1 or 2, or encodes the Claudin 6 antibody comprising a non-natural amino acid modification of claim 3 or 4.

6. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule of claim 5, and after being transfected into a host cell, the expression vector causes the host cell to express the Claudin 6 antibody of claim 1 or 2, or causes the host cell to express the Claudin 6 antibody modified with a non-natural amino acid of claim 3 or 4.

7. A host cell, characterized in that The host cell contains at least one copy of the expression vector of claim 6, or at least one copy of the nucleic acid molecule of claim 5; Preferably, the host cell is an Expi293F cell.

8. A method for preparing a Claudin 6 antibody modified with a non-natural amino acid according to claim 3 or 4, characterized in that: The preparation method comprises: synthesizing a nucleic acid sequence encoding the light and heavy chains of an antibody, mutating the codon of a non-natural amino acid introduction site into TAG; cloning the nucleic acid sequence into a vector to obtain a plasmid expressing the light and heavy chains of the antibody; introducing the plasmid expressing the light and heavy chains of the antibody, a pcDNA3.1-tRNA plasmid and a pcDNA3.1-Tet-tRNA ligase plasmid into cells for in vitro expression, and obtaining a Claudin 6 antibody modified with a non-natural amino acid.

9. A Claudin 6 antibody-drug conjugate, characterized in that: The conjugate comprises the Claudin 6 antibody according to claim 1 or 2, or the Claudin 6 antibody modified with a non-natural amino acid according to claim 3 or 4.

10. Use of any one or a combination of at least two of the Claudin 6 antibody of claim 1 or 2, the Claudin 6 antibody modified with a non-natural amino acid of claim 3 or 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7 or the Claudin 6 antibody-drug conjugate of claim 9 in the preparation of a drug for cancer immunotherapy or infectious disease treatment.

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