A claudin 6 antibody comprising a non-natural amino acid modification, and methods of making and using the same

Abstract

The application provides a Claudin 6 antibody containing a non-natural amino acid modification and a preparation method and application thereof, the sequence of the antibody is obtained by replacing original amino acids with non-natural amino acids 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: heavy chain: S114, T115, P148, G156, A157, T159, Q191, V149, L158 or G161; or light chain: T109, A112, K169, S202, S203, L154 or S156. The application introduces non-natural amino acids into specific sites of the antibody and expresses the antibody by using a cell expression system, evaluates the influence of non-natural amino acid insertion at different sites on the expression level, structural stability and binding capacity of the antibody, and provides a new idea for rational design and functional optimization of the antibody.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a Claudin 6 antibody containing non-natural amino acid modifications, its preparation method, and its application. Background Technology

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

[0003] Furthermore, non-natural 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 antibody drug development. This technology has significant application potential in areas such as cancer immunotherapy and the treatment of infectious diseases, and lays a solid foundation for developing safer and more effective novel biotherapeutic drugs.

[0004] Non-natural amino acids refer to an extension of the 20 common amino acids that make up natural eukaryotic cell proteins. Their R-side chains typically carry specific functional groups, such as acetyl, alkynyl, azide, or quinone. These functional groups can be used to specifically modify proteins through chemical reactions such as click chemistry, photocrosslinking, or nucleophilic attack, thereby optimizing or expanding their function. However, the introduction of non-natural amino acids into protein synthesis has long been limited because they lack corresponding genetic codes and necessary translational elements (such as transfer RNA and aminoacyl-tRNA synthetases) within cells.

[0005] With the rapid development of genetic code expansion technology, significant progress has been made in introducing non-natural amino acids during protein expression through the inhibition mechanism of the amber stop codon (UAG). Currently, this strategy has been successfully applied to various expression systems, including E. 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 biotechnology, showing broad application prospects, particularly in protein modification, functional exploration, and drug development.

[0006] The technique of introducing non-natural amino acids into antibodies has been widely applied in the site-specific conjugation of antibody-drug conjugates (ADCs), providing new solutions for improving conjugation efficiency and drug stability. Commonly used non-natural amino acids include p-acetylphenylalanine (pAF), p-azidomethyl-L-phenylalanine (pAMF), and azido-lysine. For example, at the position of alanine (A121) in the heavy chain of trastuzumab (anti-HER2 antibody), the corresponding codon was mutated to an amber stop codon (UAG, A121X), and a modified trastuzumab incorporating pAF was successfully synthesized using orthogonal transfer RNA (tRNA) and aminoacyl-tRNA synthetase (aaRS) derived from Methanococcus jannaschii. Related studies have shown that, in addition to the A121 position of the heavy chain, positions 169 (L169) and 202 (L202) of the light chain of trastuzumab can also serve as insertion sites for non-natural amino acids. After inserting pAF into these sites and conjugating the microtubule disruptor DM1, the conjugate product exhibited significant cytotoxicity in in vitro breast cancer cell experiments. These findings demonstrate that by introducing non-natural amino acids into specific sites on antibodies, not only can precise chemical modification be achieved, but the biological activity of antibody-drug conjugates can also be effectively preserved, laying a solid technical foundation for the development of efficient and stable ADCs.

[0007] Antibodies are a class of highly specific and diverse immunoglobulins, mainly classified into five types: IgG, IgA, IgM, IgE, and IgD. Among them, IgG is widely studied and applied due to its excellent stability and functionality. IgG molecules typically consist of two light chains and two heavy chains linked by disulfide bonds, with a total molecular weight of approximately 150 kDa, exhibiting a typical Y-shaped structure. Its structure includes the Fab region (fragment of antigen binding), the hinge region, and the Fc region (fragment crystallizable), which are respectively involved in antigen binding, molecular flexibility regulation, and the mediation of effector functions.

[0008] Antibodies in IgG form have a molecular weight of approximately 150 kDa and contain over 1300 amino acids. Current research indicates that non-natural amino acid substitution sites on antibodies are commonly found at the linker sequence between the variable region and constant region I of the heavy chain. For example, alanine at position 121 is a common substitution site on the heavy chain of trastuzumab. However, systematic research is still lacking on which specific sites in the antibody molecule can be replaced with non-natural amino acids to ensure successful antibody expression, maintain the antibody's "Y"-shaped structure, and retain its activity (i.e., its binding ability to the antigen is not significantly weakened). This lack of research in this area leaves researchers without valuable reference materials and clear guidelines for designing and optimizing antibodies modified with non-natural amino acids.

[0009] Therefore, this study designed and screened sites that can be used for the introduction of non-natural amino acids, and evaluated the structure and function of antibodies. This study has important guiding and application value for the modification of antibody physicochemical properties and the development of antibody conjugates. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a Claudin6 antibody containing non-natural amino acid modifications, its preparation method, and its applications. This invention focuses on the in vitro expression technology of antibodies containing non-natural amino acids. By introducing non-natural amino acids at specific sites on the antibody and using the Expi293F cell expression system, the effects of non-natural amino acid insertions at different sites on the antibody's expression level, structural stability, and other physicochemical properties were systematically evaluated, providing new ideas and technical support for the rational design and functional optimization of antibodies.

[0011] To achieve this objective, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a Claudin 6 antibody, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2; and the amino acid sequence of the light chain variable region is 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 variable regions of the light chain of the antibody include 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 heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:1; and the light chain amino acid sequence is shown in SEQ ID NO:6.

[0016] Secondly, the present invention provides a Claudin 6 antibody comprising non-natural amino acid modifications, wherein the antibody is obtained by replacing the original amino acids with non-natural amino acids based on the Claudin 6 antibody described in the first aspect; the insertion site of the non-natural amino acid is selected from any one or a combination of at least two of the following:

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

[0018] This invention focuses on antibodies targeting Claudin 6. Based on the antibody's three-dimensional structural information, 17 sites were designed and screened for the introduction of non-natural amino acids, including 10 sites on the heavy chain and 7 sites on the light chain. This invention aims to express antibodies containing non-natural amino acids at these sites using a mammalian expression system without affecting the antibody's basic function, and to evaluate their stability. This provides new ideas and methods for the rational design, functional optimization, and site-specific conjugation of antibody molecules with ADCs.

[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 heavy chain amino acid sequence shown in SEQ ID NO:1, with the heavy chain S114 replaced by p-acetylphenylalanine.

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

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

[0023] In one embodiment of the present invention, the antibody is based on the heavy chain amino acid sequence shown in SEQ ID NO:1, with heavy chain G156 replaced by 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, with p-acetylphenylalanine replacing the heavy chain A157.

[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, with the heavy chain T159 replaced by 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, with the heavy chain Q191 replaced by 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, with heavy chain V149 replaced by 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, with p-acetylphenylalanine replacing the L158 of the heavy chain.

[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, with the heavy chain G161 replaced by 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, with the light chain T109 replaced by 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, with the light chain A112 replaced by 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, with the light chain K169 replaced by 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, with the light chain S202 replaced by 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, with the light chain S203 replaced by 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, with the light chain L154 replaced by 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, with the light chain S156 replaced by p-acetylphenylalanine.

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

[0038] Fourthly, the present invention provides an expression vector containing the nucleic acid molecule described in the third aspect, and after the expression vector is transfected into a host cell, the host cell expresses the Claudin 6 antibody described in the first aspect, or the host cell expresses the Claudin 6 antibody containing non-natural amino acid modifications described in the second aspect.

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

[0040] Fifthly, the present invention provides a host cell containing 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 an Expi293F cell.

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

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

[0044] Eighthly, the present invention provides the use of any one or a combination of at least two of the following: the Claudin 6 antibody of the first aspect, the Claudin 6 antibody comprising non-natural amino acid modifications of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the host cell of the fifth aspect, or the Claudin 6 antibody-drug conjugate of the seventh aspect, in the preparation of a medicament for cancer immunotherapy or treatment of infectious diseases.

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

[0046] (1) The Claudin 6 antibody screened in this invention has the advantages of good antigen binding activity and strong affinity.

[0047] (2) This invention successfully expressed 17 Claudin6 antibody variants containing non-natural amino acids using the Expi293F expression system. These variants introduced non-natural amino acids at different sites on the light and heavy chains, and were systematically compared with the original Claudin6 antibody. Mass spectrometry analysis confirmed the successful introduction of the target non-natural amino acid p-aminophenylalanine (pAF). The study found that specific sites on the heavy chain S114 / T115 / P148 / G156 / A157 / T159 / Q191 / V149 / L158 / G161 and the light chain T109 / A112 / K169 / S202 / S203 / L154 / S156 could effectively accommodate the introduction of non-natural amino acids without affecting structural integrity. This result indicates that these sites have good engineering potential, providing a strong basis for further optimization and development of antibody molecules containing non-natural amino acids. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the design of non-natural amino acid insertion sites on antibodies.

[0049] Figure 2 The structural formula of the non-natural amino acid pAF is given.

[0050] Figure 3 The SDS-PAGE results are for 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 for 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 for 3-H (P148X), 4-H (G156X), 5-H (A157X), 14-L (S202X), and 15-L (S203X).

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

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

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

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

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

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

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

[0060] Figure 13 The mass spectrometry results are for WT and 2-H(T115X).

[0061] Figure 14 The mass spectrometry results are for 1-H (S114X) and 3-H (P148X).

[0062] Figure 15 The mass spectrometry results are for 4-H (G156X) and 6-H (T159X).

[0063] Figure 16 The mass spectrometry results are for 5-H (A157X) and 7-H (Q191X).

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

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

[0066] Figure 19 The mass spectrometry results are for 12-L (A112X) and 14-L (S202X).

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

[0068] Figure 21 The mass spectrometry results are for 16-L (L154X) and 17-L (S156X).

[0069] Figure 22 The results show the cell binding assay results of 18-WT antibody and Claudin 6 cell line.

[0070] Figure 23 The results of cell binding experiments for 18-WT, 1-H(S114X), 2-H(T115X), 3-H(P148X), 4-H(G156X), 5-H(A157X), and Claudin 6 cell lines are presented.

[0071] Figure 24 The results of cell binding experiments for 18-WT, 6-H (T159X), 7-H (Q191X), 8-H (V149X), 9-H (L158), 10-H (G161X), and Claudin 6 cell lines are presented.

[0072] Figure 25 The results of cell binding experiments for 18-WT, 11-L(109X), 12-L(A112X), 13-L(K169X), 14-L(S202X), 15-L(S203X), and Claudin 6 cell lines are presented.

[0073] Figure 26 The results are for cell binding assays of 18-WT, 16-L (154X), 17-L (S156X), and Claudin 6. Detailed Implementation

[0074] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0075] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0076] Example 1

[0077] Screening for high-affinity Claudin 6 antibodies involves the following steps:

[0078] (1) Based on the protein 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 ELISA. The antiserum titer was qualified, indicating that the mice had been induced to produce high-titer antiserum specifically targeting Claudin 6 protein.

[0080] (4) Collect spleen and bone marrow cells from immunized mice, centrifuge at 400g for 5 min, resuspend in 1 mL of red blood cell lysis buffer, lyse on ice for 1 min, terminate lysis with large volume of Macs buffer, centrifuge at 400g for 5 min, resuspend in 1 mL of Macs buffer, add fluorescent antibodies labeled with fluorescent Claudin 6 protein and B cell-specific molecular markers, incubate at 4℃ for 1 h, resuspend in large volume of Macs buffer, centrifuge at 400g for 5 min, discard the supernatant, resuspend cells in buffer, and sort antigen-positive cells using a flow cytometer.

[0081] (5) Mouse positive single cell library construction, the process is as follows: The positive mouse cells obtained by sorting are constructed using a single cell library construction platform. Oil droplets containing a single positive mouse cell are generated by water-in-oil technology. Cell lysis, mRNA capture and reverse transcription are completed in the oil droplets. The reverse transcription product carrying the cell tag barcode is pre-amplified with cDNA and the antibody VDJ gene is amplified. After fragmentation, adapters and library tags are ligated to obtain the antibody library. The naturally paired antibodies of light and heavy chains are obtained by next-generation sequencing and data analysis.

[0082] The sequence of the Claudin 6 antibody obtained through screening is shown below:

[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 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4, and the sequence of CDR3 is PAY.

[0085] The variable regions of the light chain of the antibody include 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.

[0086] 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.

[0087] Example 2

[0088] This embodiment utilizes alphafold mapping to construct the three-dimensional structure of the Claudin 6 antibody. The three-dimensional structure of Claudin 6 is analyzed, and based on relevant literature, non-natural amino acid insertion sites on the Claudin 6 antibody are designed and selected without affecting antibody function. The design of non-natural amino acid insertion sites on the antibody is as follows: Figure 1 As shown in the figure, the right side displays the homologous structure of the overall three-dimensional antibody structure simulated using alphafold mapping. Light chains are represented in green, and heavy chains in cyan. The left side shows the insertion sites of non-natural amino acids on both the light and heavy chains. There are 10 insertion sites on the heavy chain and 7 on the light chain.

[0089] Example 3

[0090] 1. Constructing plasmids

[0091] The light and heavy chain nucleic acid sequences of the fully synthesized antibody were mutated to TAG at the codons of the non-natural amino acid introduction sites, and then subcloned into the pcDNA3.1 vector respectively.

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

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

[0094] 3. Collect and purify the supernatant.

[0095] Transfer cells to a 250 mL centrifuge flask and centrifuge at 5000 rpm for 20 min at 4 °C. Collect the supernatant. Filter the supernatant through 0.45 μm and 0.22 μm filters, respectively. Add the filtered supernatant to a purification column containing 2 mL of protein A packing material; repeat the process twice. Add 50 mL of 1×PBS to the purification column to wash away contaminating proteins. Add 10 mL of 0.1 M pH 3.0 sodium citrate to the purification column to elute proteins, and collect the eluent in a 50 mL centrifuge tube containing 5 mL of 1 M pH 8.0 Tris-HCl. Concentrate the protein using a 50 kDa 15 mL ultrafiltration tube.

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

[0097] Table 1

[0098] 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 the heavy chain of the antibody, and "L" represents the light chain. "X" indicates a non-natural amino acid, which can be p-acetylphenylalanine, azidomethyl-L-phenylalanine, etc. This study uses p-acetylphenylalanine (pAF) as an example. For example, 1-H (S114X) indicates that the serine at position 114 of the antibody heavy chain is replaced with pAF, and 11-L (T109X) indicates that the threonine at position 109 of the antibody light chain is replaced with pAF.

[0100] 4. Protein properties and function detection

[0101] (1) SDS-PAGE detection

[0102] Approximately 2 × 3 μg of each sample was taken and added to both reducing (R) and non-reducing 4 × loading buffer. Samples with added reducing loading buffer were heated in a 95°C metal bath for 5 min.

[0103] Antibody protein SDS-PAGE results as follows Figure 3-6 As shown. Figure 3 These are 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 These are 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 These are the SDS-PAGE results for 3-H (P148X), 4-H (G156X), 5-H (A157X), 14-L (S202X), and 15-L (S203X). Figure 6 These are the SDS-PAGE results for 1-H(S114X) and 2-H(T115X).

[0104] Electrophoresis results showed that 14-L(S202X) and 15-L(S203X) contained non-target bands in addition to the target band. From the results of the non-reduced samples, the molecular weight of the extraneous bands was smaller than that of the target band. From the results of the reduced samples, the molecular weight of the extraneous bands was smaller than that of the target light chain. We speculate that the 14-L(S202X) and 15-L(S203X) experimental groups contained light chains that failed to insert into pAF, and that these light chains combined with the heavy chains to assemble into a "Y" structure. The gel images showed that the remaining reducing and non-reducing electrophoretic bands of the antibody mutants were similar to those of the original Claudin 6 antibody (WT, which does not contain non-natural amino acids) obtained in Example 1.

[0105] (2) HPLC-SEC detection

[0106] 30 μL of sample was taken from each experimental group for HPLC-SEC analysis. Equipment: High-performance liquid chromatography (Shimadzu, LC-2050C). 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 Procedure: First, place the D pump in filtered, degassed ddH₂O and flush for 20 min at a flow rate of 1 mL / min, then adjust the flow rate to 0. Next, 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, flush for 20 min, and then adjust the flow rate to 0. Finally, adjust the flow rate to 0.5 mL / min, install the column, and load the sample after baseline leveling. Select the above program and run the sample. The obtained data were processed and analyzed using GraphPad Prism 8 software.

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

[0109] The original Claudin 6 antibody (WT, without non-natural amino acids) obtained in Example 1 showed a narrow, sharp peak at 7.58 min. The elution times of the 17 antibodies containing non-natural amino acids ranged from 7.50 min to 7.62 min, and their peak shapes were similar to those of the WT antibody, also narrow and sharp. This result indicates that the overall conformation of these 17 antibodies containing non-natural amino acids is highly similar to that of the WT antibody, suggesting that the "Y"-shaped structure of the mutant was not significantly affected. Therefore, replacing the amino acids at these 17 sites with pAF did not significantly change the spatial conformation or structural stability of the antibodies.

[0110] (3) Antibody mass spectrometry detection

[0111] Instruments: Ultra-high performance liquid chromatograph (UHPLC), Thermo Fisher, Vanquish; High resolution mass spectrometer (HDMS), Thermo Fisher, QExactive; 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 removal treatment: Take a portion of the test sample and react with PNGase F enzyme at 37℃ for 2 hours. Reduction: Take a portion of the test sample and reduce with 20mM DTT for 1 hour.

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

[0114] Table 2

[0115]

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

[0117] The theoretical molecular weights of the light and heavy chains of the WT antibody are 23528.09D and 48485.91D, respectively. Sample 1-H(S114X) replaces serine at position 114 on the heavy chain with pAF. Its light chain molecular weight is the same as WT, theoretically 23534.06D; the theoretical molecular weight of its heavy chain is 48588.05D, an increase of 102.14D compared to WT. Mass spectrometry results after desugaring and reduction show that the actual molecular weight difference between the heavy chain of 1-H(S114X) and WT is 102.37D, which matches the theoretical difference. Therefore, 1-H(S114X) antibody successfully replaced S114 with pAF. The same applies to the other samples. (Statistical results are shown in Table 3.)

[0118] Table 3

[0119]

[0120]

[0121] (4) Cell binding experiment: The binding experiment between Claudin 6 antibody (18_WT) and Claudin 6 cell line.

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

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

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

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

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

[0127] Preparation of Claudin 6 cell line: Replace the culture medium of Claudin 6 cell lines with a viability of not less than 95% with MACS medium (PBS, 2% FBS and 2mM EDTA), and adjust the cell concentration to 4×10⁶ cells / year. 6 / mL. Add 50μL of cells to each well of a 96-well plate.

[0128] Antibody dilution: Each antibody was initially diluted at a concentration of 800 nM using Macs solutions in a 4-fold serial dilution.

[0129] FACS assay: Add 50 μL of diluted antibody to each well of a 96-well plate containing Claudin 6 cell line, mix thoroughly, and incubate at 4°C in the dark for 1 hour. Add 200 μL of Macs solution to each well, centrifuge at 400g for 5 min, discard the supernatant, and repeat three times. Add 1 μg / mL of secondary antibody to each well and incubate at 4°C in the dark for 30 min. Then add 200 μL of Macs solution to each well, centrifuge at 400g for 5 min, discard the supernatant, and repeat three times. Resuspend the cells in 200 μL of Macs solution and perform flow cytometry. Data were processed and analyzed using GraphPad Prism 8.

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

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

[0132] As shown in the figure, after adding non-natural amino acid pAF to each of the 17 sites on the antibody, the antibody still bound to the Claudin 6 overexpressing cell line. This indicates that replacing these 17 sites (see Table 1) with non-natural amino acids does not affect the antibody's function, i.e., it does not affect the binding between the antibody and the antigen. Moreover, the binding ability of 18-WT to the Claudin 6 cell line is stronger than that of the Yangshen antibody PC-direct labeling antibody; the binding ability of 1-H (S114X), 8-H (V149X) to the Claudin 6 cell line is comparable to that of 18-WT; the antibody mutants with 6-H (T159X), 10-H (G161X), 14-L (S202X), and 15-L (S203X) sites replaced with non-natural amino acids have a stronger binding ability to the Claudin 6 cell line than 18-WT.

[0133] Final Results Analysis: This study successfully replaced 10 amino acids at sites S114, T115, P148, G156, A157, T159, Q191, V149, L158, and G161 on the heavy chain of the Claudin 6 antibody with the non-natural amino acid pAF. Simultaneously, it also successfully replaced seven amino acids at sites T109, A112, K169, S202, S203, L154, and S156 on the light chain of the Claudin 6 antibody with the non-natural amino acid pAF. All 17 antibody mutants maintained the typical "Y"-shaped antibody structure and were able to specifically bind to Claudin 6 overexpressing cell lines. Among them, the heavy chain S114X and V149X antibody mutants, as well as the light chain S202X and S203X mutants, showed higher binding affinity to Claudin 6 overexpressing cell lines compared to the original Claudin 6 antibody (WT). The heavy chain T159X and G161X mutants exhibited binding affinity to Claudin 6-overexpressing cell lines comparable to WT antibodies. Furthermore, the application of these sites can be further expanded; in addition to introducing p-acetylphenylalanine (pAF), it can also be replaced with azide-methyl-L-phenylalanine (pAMF), providing broader possibilities for antibody functionalization modifications.

[0134] In summary, this invention provides a Claudin 6 antibody and its variants, which introduce non-natural amino acids at different sites on the light and heavy chains. The insertion of non-natural amino acids provides a flexible and efficient strategy for the subsequent development of antibody drugs. The aforementioned insertion sites have good engineering potential, providing a strong basis for further optimization and development of antibody molecules containing non-natural amino acids.

[0135] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and 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 variable regions of the light chain of the antibody include 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 by 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 substitution sites of the non-natural amino acids are selected from any of the following: Heavy chains: S114, T159, V149 or G161; or light chains: S202 or S203; The substituted non-natural amino acid is p-acetylphenylalanine.

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

5. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 4, and after transfection 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 of claim 3 containing non-natural amino acid modifications.

6. A host cell, characterized in that, The host cell contains at least one copy of the expression vector of claim 5, or at least one copy of the nucleic acid molecule of claim 4.

7. A method of preparing the Claudin 6 antibody comprising a non-natural amino acid modification of claim 3, characterized in that, The preparation method includes: synthesizing nucleic acid sequences encoding the light and heavy chains of the antibody; mutating the codons at the non-natural amino acid introduction sites to TAG; cloning the nucleic acid sequence into a vector to obtain plasmids expressing the antibody light and heavy chains; introducing the plasmids expressing the antibody light and heavy chains, pcDNA3.1-tRNA plasmids, and pcDNA3.1-Tet-tRNA ligase plasmids into cells for in vitro expression to obtain Claudin 6 antibodies containing non-natural amino acid modifications.

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