Variants of Antibody Variable Regions and Their Use

Abstract

The present invention provides variants of antibodies and their use. Specifically, based on the Kabat numbering system, the variants of the antibodies have cysteine residues engineered at any one or more positions selected from the following: positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 in the heavy chain variable region or any combination thereof, or positions 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 in the light chain variable region or any combination thereof. The mercapto groups of the engineered cysteines can maintain partial activity during and after antibody expression. The mercapto groups that maintain activity may be directly used for reaction with other active groups without undergoing a reduction treatment. The cysteine-engineered antibodies of the present invention can obtain active groups for drug binding, which is advantageous for simplifying the production process of antibody-drug conjugates, improving the uniformity of antibody-drug conjugates, enhancing drug efficacy, and reducing toxic side effects.

Description

Technical Field

[0001] The present invention relates to the field of antibodies. Specifically, it relates to cysteine mutants of antibodies, compositions and / or conjugates containing the same, and their use.

Background Art

[0002] An antibody-drug conjugate (ADC) is an antibody product to which a drug molecule obtained by linking an antibody and a drug by biological or chemical means is linked. Antibody-drug conjugates reduce the killing effect of drugs on normal cells due to the specificity of monoclonal antibodies and are mainly applied to the treatment of tumors. The production of antibody-drug conjugates mainly performs a binding reaction by the amino group residue of lysine or the mercapto group residue of cysteine. Whether it is a binding by lysine residues or a binding by cysteine residues, there are several drawbacks in the conventional non-site-specific binding methods, and site-specific binding by site-specific binding technology can enhance the uniformity of antibody-drug conjugates [1] 。

[0003] An antibody molecule contains four polypeptide chains. Among them, the two chains with relatively large molecular weights are called heavy chains (HC), and the two chains with relatively small molecular weights are called light chains (LC). The amino acid compositions of the two H chains and the two L chains in the same antibody molecule are exactly the same. Analyzing the amino acid sequences of the heavy and light chains of different antibodies, it was found that the amino acid sequences near the N-terminus of the heavy and light chains vary greatly, and the amino acid sequences of other parts are relatively constant. Therefore, the region where the amino acid sequences near the N-terminus of the light and heavy chains of the antibody change greatly is called the variable region (V), which occupies 1 / 4 and 1 / 2 of the heavy and light chains respectively. The region where the amino acid sequence near the C-terminus is relatively stable is called the constant region (C), which occupies 3 / 4 and 1 / 2 of the heavy and light chains respectively.

[0004] A single-chain antibody (single chain antibody fragment, scFv) is an antibody in which the heavy-chain variable region and the light-chain variable region of the antibody are linked by an amino acid short peptide (linker). In a single-chain antibody (scFv), the heavy and light chains of the antibody variable region are separated by short amino acids and linked by disulfide bonds [2] . ScFv has a small molecular weight, is suitable for expression in yeast and bacteria, is useful for rapid mass production, and is a candidate for ideal high-throughput selection technologies such as phage display, cell display, yeast display, and ribosome display [3] . Single-chain antibody-drug conjugates have great potential. Although scFv has a short half-life, its tumor penetration ability is greater than that of the Fab fragment. Therefore, single-chain antibody-drug conjugates are effective and have low side effects [4] . The data indicate that single-chain antibody-drug conjugates are more resistant than IgG antibody-drug conjugates [5,6] .

[0005] Site-specific binding technology is applied not only to complete IgG antibody molecules but also to scFv and bispecific antibody-drug conjugates based on the scFv format. The reactive amino acid in site-specific binding technology is most commonly cysteine. By means of site-specific mutagenesis technology, an antibody-drug conjugate with a controllable drug-antibody binding ratio can be developed. By selecting an appropriate site for cysteine mutation, the mercapto group on the cysteine obtained by the mutation can be kept in a free state during expression and purification and directly applied to the downstream binding reaction. Due to the universal application of cysteine, maleimide is the most popular linker group [7] , and then the antibody is conjugated to another functional molecule (antibody fragment, polypeptide or small molecule drug) via a linker containing maleimide

[0006] Most of the mercapto groups on cysteines introduced into antibodies by cysteine engineering are oxidized during cell culture, such as reacting with the mercapto groups of free cysteine in the cell culture medium, to form disulfide bonds. The oxidized mercapto groups have activity after being reduced and are used in subsequent binding reactions. However, the reduction reaction simultaneously opens the disulfide bonds in the antibody, and these opened disulfide bonds in the antibody need to be oxidized again to restore the disulfide bond structure in the antibody. These series of reprocessing steps after antibody expression complicate the production process of antibody-drug conjugates and also pose challenges to quality control.

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention mainly provides cysteine-engineered antibodies. According to the Kabat numbering system, the antibody has cysteine residues engineered at any one or more positions selected from the following: positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 in the heavy chain variable region or any combination thereof, and their corresponding IMGT numbers are 13, 39, 40, 43, 49, 52, 56, 67, 68, 75, 77, 86, 87, 122 respectively; or positions 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 in the light chain variable region or any combination thereof, and their corresponding IMGT numbers are 19, 21, 50, 52, 53, 54, 76, 87, 91, 94, 103 respectively. The mercapto groups of the engineered cysteines can maintain partial activity during and after antibody expression. The mercapto groups with maintained activity may be directly used in reactions with other active groups without undergoing a reduction treatment.

Means for Solving the Problems

[0008] Specifically, the present invention provides the following aspects: 1. A cysteine - engineered antibody having cysteine residues engineered at one or more positions selected from the following, based on the Kabat numbering system: Position 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 in the heavy - chain variable region, or any combination thereof, or Position 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 in the light - chain variable region, or any combination thereof.

[0009] 2. The cysteine - engineered antibody according to item 1, having cysteine residues engineered at one or more positions selected from the following, based on the Kabat numbering system: Position 12, 35, 61, 51, 69, 78 in the heavy - chain variable region, or any combination thereof, or Position 46, 87 in the light - chain variable region, or any combination thereof.

[0010] 3. The cysteine - engineered antibody according to item 1, having cysteine residues engineered at one or more positions selected from the following, based on the Kabat numbering system: Position 12, 35, 61, 51 in the heavy - chain variable region, or any combination thereof, or Position 46, 87 in the light - chain variable region, or any combination thereof.

[0011] 4. The cysteine - engineered antibody according to item 1, obtained based on the following wild - type antibodies: An antibody comprising the heavy - chain variable region shown in SEQ ID NO:4 and the light - chain variable region shown in SEQ ID NO:5, An antibody comprising the heavy - chain variable region shown in SEQ ID NO:75 and the light - chain variable region shown in SEQ ID NO:92, An antibody comprising the heavy - chain variable region shown in SEQ ID NO:81 and the light - chain variable region shown in SEQ ID NO:95, An antibody comprising a heavy chain variable region shown in SEQ ID NO:85 and a light chain variable region shown in SEQ ID NO:96, An antibody comprising a heavy chain variable region shown in SEQ ID NO:89 and a light chain variable region shown in SEQ ID NO:97, An antibody comprising a heavy chain shown in SEQ ID NO:100 and a light chain shown in SEQ ID NO:101, An antibody comprising a heavy chain variable region shown in SEQ ID NO:108 and a light chain variable region shown in SEQ ID NO:109, An antibody comprising a heavy chain variable region shown in SEQ ID NO:113 and a light chain variable region shown in SEQ ID NO:114, and / or An antibody comprising a heavy chain variable region shown in SEQ ID NO:130 and a light chain variable region shown in SEQ ID NO:133.

[0012] 5. The light chain is of the λ type or κ type, Optionally, an anti-HER2 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-VEGFR-2 antibody, an anti-EGFR antibody, and / or an anti-c-Met antibody, Optionally, the cysteine-engineered antibody according to item 1, which is a single-chain antibody, an IgG antibody, or a bispecific antibody, for example, a bispecific antibody in the form of BiTE / DART / Diabody.

[0013] 6. The cysteine-engineered antibody according to item 1, wherein when the antibody is a single-chain antibody, it further comprises a linker chain, preferably further comprises the linker chain shown in SEQ ID NO:6.

[0014] 7. A conjugate comprising the cysteine-engineered antibody according to any one of items 1 to 6 and a conjugate moiety.

[0015] 8. The conjugate part is selected from polyethylene glycol, cytotoxic agent, active peptide, nanobody, single domain antibody, Fab fragment, Fab' fragment, scFv, small molecule drug (such as topoisomerase inhibitor, tubulysin A, DM1, PBD, MMAE or MMAF, etc.), chemotherapeutic agent or radiotherapy agent, The conjugate part is conjugated to the cysteine-engineered antibody via a linker, Preferably, the linker contains an electrophilic group (preferably a maleimide group or a haloacetamide group), and optionally, the linker is mc-VC-PAB. The conjugate according to item 7.

[0016] 9. The polyethylene glycol is maleimide monomethoxypolyethylene glycol, preferably mPEG2000-Mal, mPEG5000-Mal, mPEG10000-Mal. The conjugate according to item 8.

[0017] 10. A pharmaceutical composition comprising the cysteine-engineered antibody according to any one of items 1 to 6 or the conjugate according to any one of items 7 to 9, and optionally a pharmaceutically acceptable carrier.

[0018] 11. Use of the cysteine-engineered antibody according to any one of items 1 to 6, the conjugate according to any one of items 7 to 9, or the pharmaceutical composition according to item 10 in the manufacture of a drug or kit for treating cancer (such as HER2-positive cancer, preferably breast cancer).

[0019] 12. A kit comprising the cysteine-engineered antibody according to any one of items 1 to 6, the conjugate according to any one of items 7 to 9, or the pharmaceutical composition according to item 10.

Advantages of the Invention

[0020] 1. By utilizing cysteine mutations, an active group capable of drug binding can be obtained, which is widely used and easy to bind.

[0021] 2. Compared with non-site-specific mutations, site-specific mutations can improve the uniformity of antibody-drug conjugates.

[0022] 3. The selected cysteine mutation site can maintain the activity of a part of the mercapto group after the expression of the antibody or antibody fragment, and can be used in the binding reaction without the need for treatment.

[0023] 4. The site-specific cysteine mutations in this specification do not change the activity of the antibody, and after drug binding, can improve the drug efficacy and reduce the toxic side effects.

[0024] 5. The mutation sites in the present invention are suitable for most antibodies and have a certain versatility.

[0025] 6. The present invention can construct bispecific antibody-drug conjugates by utilizing site mutations of different antibodies.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0028] The restricted endonuclease used in the examples was purchased from Thermo Fisher Scientific (China) Co., Ltd., and the reagents or materials used in the following examples are all commonly available in the art if the source is not explicitly mentioned.

[0029] Definition "Single-chain Fv" is also abbreviated as "sFv" or "scFv", and is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker chain between the VH domain and the VL domain that enables the sFv to form a structure desirable for antigen binding.

[0030] The polypeptide linker chain has various forms classified into flexible linker chains, rigid linker chains, and cleavable linker chains. The most common flexible linker chain is (GGGGS)n, and other common flexible linker chains are KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8 composed of pure glycine, or a slightly shorter (Gly)6. Rigid linker chains are (EAAAK)n, (XP)n, where X can specify any amino acid, and it is recommended to select alanine (Ala), lysine (Lys), or glutamic acid (Glu). Cleavable linker chain sequences are LEAGCKNFFPR↓SFTSCGSLE, etc. The linker chain used herein is the most common flexible linker chain, and its sequence is GGGGSGGGGSGGGGS.

[0031] In the cysteine-engineered antibodies of the present invention, since the selected mutation sites are all located in the framework regions of the antibody variable regions, they can be applied to cysteine mutations at the corresponding sites of any antibody including, but not limited to, anti-HER2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-VEGFR-2 antibodies, anti-EGFR antibodies, anti-c-Met antibodies, etc.

[0032] Here, HER2 (human epidermal growth factor receptor-2, ErbB2 / P185) is the second member of the human epidermal growth factor receptor (EGFR) family, encoded by the proto-oncogene erbB2 / Her2. The coding gene is located on human chromosome 17q21, with a molecular weight of 185 kDa, and it is a tyrosine kinase receptor membrane glycoprotein. HER2 forms heterodimers with HER1, HER3, or HER4, thereby causing activation of downstream signaling pathways such as MAPK and PI3K, over-conducting growth signals, and causing malignant proliferation of cells. Among various human tumors, HER2 proto-oncogene amplification or protein overexpression phenomena have been discovered in all of them, including 25 - 30% of breast cancers, 35 - 45% of pancreatic cancers, 90% of colorectal cancers, 16 - 57% of non-small cell lung cancers, 9 - 38% of gastric cancers, etc. Clinically, they are called HER2-positive tumors, which mainly have a high degree of tumor malignancy, are prone to progression and metastasis, are not sensitive to radiotherapy and chemotherapy, are prone to recurrence, and have a short survival period for patients.

[0033] In 1975, Kohler and Milstein invented the hybridoma technology for preparing monoclonal antibodies (mAb). Subsequently, mAb drugs have developed rapidly and been applied clinically. In recent years, antibody drugs targeting HER2 have also become a new hot spot in the treatment of HER2-positive tumors. Trastuzumab (Herceptin) is a humanized monoclonal antibody drug against HER2 developed by Genentech. The US FDA approved its sale in 1998. Currently, the combination chemotherapy drugs of Trastuzumab (such as paclitaxel) have already become the first-line treatment regimens for HER2-overexpressing advanced metastatic breast cancer and advanced gastric cancer. In the treatment of HER2-positive metastatic breast cancer, the clinical effective rate reaches 38%. The European EMEA has approved the combination chemotherapy drugs of Trastuzumab as the first-choice treatment regimen for treating HER2-positive advanced gastric cancer.

[0034] As used herein, the term "linker" refers to a chemical moiety that includes a covalent bond or a chain of atoms that covalently attaches an antibody to a conjugate moiety (such as a drug moiety, etc.). In various embodiments, the linker is designated as L. The "linker" (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties (D) and an antibody unit (Ab) to form an antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs) can be conveniently prepared using a linker having a reactive functional group for the conjugation of the drug and the antibody. The cysteine thiol of a cysteine-engineered antibody (CYSMAB) can form a bond with an electrophilic functional group of a linker reagent, a drug moiety, or a drug-linker intermediate.

[0035] In one aspect, the linker has a reactive site having an electrophilic group that reacts with a nucleophilic cysteine present on the antibody. The cysteine thiol of the antibody reacts with the electrophilic group on the linker to form a covalent bond with the linker. Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups. Common linkers are mainly divided into two types: non-cleavable and cleavable. Non-cleavable linkers are mainly thioether linkers, and cleavable linkers are classified into acid-cleavable, reducible, enzyme-cleavable, etc. Among them, the most commonly used cleavable linker includes Val-Cit dipeptide, etc. In this specification, mc-VC-PAB, which is also a commonly used linker for ADCs and can be purchased from MedChemExpress, is adopted as the linker.

[0036] As used herein, the drug moiety D used in the ADC may be a topoisomerase inhibitor (such as etoposide, teniposide, doxorubicin, etc.), tubulysin A, DM1, MMAE, etc.

[0037] Here, tubulysin A (TubA) is a product of myxobacteria and has anti-angiogenic, anti-mitotic, and anti-proliferative effects in vitro. The structure is shown in the following figure:

[0038] [Chemistry]

[0039] Here, DM1 is a derivative of the antimicrotubule drug maytansine, a tubulin inhibitor that binds to the ends of microtubules to suppress microtubule dynamics, a maytansine alkaloid capable of binding to an antibody, and can be used to overcome the systemic toxicity associated with maytansine and enhance tumor-specific delivery. The structure is shown in the following figure:

[0040] [Chemistry]

[0041] Here, MMAE is a synthetic derivative of auristatin 10, which exerts an effective mitotic inhibitory effect by suppressing tubulin polymerization. The structure is shown in the following figure:

[0042] [Chemistry]

[0043] Here, PBD, also called pyrrolobenzodiazepine, can form effective cytotoxic DNA interstrand cross-links by binding to the minor groove of DNA, thereby blocking cell division and killing cancer cells.

[0044] Here, MMAF (Monomethylauristatin F), a tubulin polymerization inhibitor effective as an antitumor drug, is commercially available.

[0045] As used herein, the term "bioactive peptide," also called a biologically active peptide, means a peptide compound that has a beneficial or physiological effect on the life activities of an organism.

[0046] The terms "subject", "patient", "individual" and similar terms are used interchangeably and, unless otherwise specified, mean mammals such as humans and non-human primates, and rabbits, rats, mice, goats, pigs and other mammalian species. The terms do not necessarily indicate that the subject has been diagnosed with a particular disease. The term "patient" means a subject under medical supervision. A patient may be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment program, etc. The term "cancer patient" or "AML patient" can refer to an individual diagnosed with cancer who is currently receiving treatment or is at risk of recurrence, such as after tumor removal surgery. In some embodiments, a cancer patient is diagnosed with cancer and is a candidate for treatment. A cancer patient may include an individual who has not received treatment, an individual who is currently receiving treatment, an individual who has undergone surgery, and an individual who has discontinued treatment.

[0047] In the context of cancer treatment, a subject in need of treatment can refer to an individual who has cancer or a pre-cancerous condition, already has cancer and is at risk of recurrence, is suspected of having cancer, or is receiving standard cancer treatments such as radiation therapy or chemotherapy.

[0048] The terms "cancer", "tumor" and similar terms include precancerous cells, neoplastic cells and cancerous cells and may refer to solid tumors or non-solid cancers. Cancer includes benign and malignant neoplasms (abnormal growths).

[0049] The term "cancer" can refer to leukemia, carcinoma, sarcoma, adenocarcinoma, lymphoma, solid tumor, lymphatic cancer, etc. Examples of different types of cancer include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, small cell lymphoma, large cell lymphoma, monocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, lung cancer (e.g., non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (i.e., hepatocarcinoma), kidney cancer (i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, thoracic cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, anal cancer, pancreatic cancer, cholangiocarcinoma, gastrointestinal cancer tumors, esophageal cancer, gallbladder cancer, appendiceal cancer, small intestine cancer, stomach (gastric) cancer, central nervous system cancer, skin cancer, choriocarcinoma, urothelial cancer, head and neck cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, including but not limited to these.

Examples

[0050] Example 1. Screening for mutant sites of anti-HER2 trastuzumab single-chain antibody Based on the 4X4X model in the protein database PDB, four parameters of the antibody, namely residue solvent accessibility, sidechain solvent accessibility, percent solvent accessibility, and percent sidechain solvent accessibility, were predicted using Discovery Studio software. Considering that the nutrient and mutant cysteine reactions in the cell culture process affect subsequent binding, sites that are too exposed cannot be selected. The inventor comprehensively considered and finally selected sites in the non-CDR region where all of the above four parameters are ≤ 20 for mutation screening, that is, positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 in the heavy chain variable region, or positions 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 in the light chain variable region.

[0051] Example 2. Screening and expression of anti-HER2 trastuzumab single-chain antibody cysteine mutant cell lines (1) Construction of anti-HER2 trastuzumab single-chain antibody and mutant expression vectors Use the DNA encoding the antibody described in this specification (the corresponding wild-type sequence is from the USP Medicines Compendium). The specific nucleotide sequences of the heavy-chain variable region, light-chain variable region, and linker chain are shown in SEQ ID NOs: 1-3, and the amino acid sequences are shown in SEQ ID NOs: 4-6. Then, based on the Kabat numbering, substitute the amino acid at position 12 of the heavy chain with cysteine to obtain the variant HC12C of the present invention, substitute the amino acid at position 35 of the heavy chain with cysteine to obtain the variant HC35C of the present invention, substitute the amino acid at position 61 of the heavy chain with cysteine to obtain the variant HC61C of the present invention, substitute the amino acid at position 21 of the light chain with cysteine to obtain the variant LC21C of the present invention, substitute the amino acid at position 47 of the light chain with cysteine to obtain the variant LC47C of the present invention, substitute the amino acid at position 48 of the light chain with cysteine to obtain the variant LC48C of the present invention, substitute the amino acid at position 71 of the light chain with cysteine to obtain the variant LC71C of the present invention, substitute the amino acid at position 75 of the light chain with cysteine to obtain the variant LC75C of the present invention, substitute the amino acid at position 34 of the heavy chain with cysteine to obtain the variant HC34C of the present invention, substitute the amino acid at position 38 of the heavy chain with cysteine to obtain the variant HC38C of the present invention, substitute the amino acid at position 44 of the heavy chain with cysteine to obtain the variant HC44C of the present invention, substitute the amino acid at position 47 of the heavy chain with cysteine to obtain the variant HC47C of the present invention, substitute the amino acid at position 51 of the heavy chain with cysteine to obtain the variant HC51C of the present invention, substitute the amino acid at position 60 of the heavy chain with cysteine to obtain the variant HC60C of the present invention, substitute the amino acid at position 61 of the heavy chain with cysteine to obtain the variant HC61C of the present invention, substitute the amino acid at position 67 of the heavy chain with cysteine to obtain the variant HC67C of the present invention, substitute the amino acid at position 69 of the heavy chain with cysteine to obtain the variant HC69C of the present invention, substitute the amino acid at position 78 of the heavy chain with cysteine to obtain the variant HC78C of the present invention, substitute the amino acid at position 79 of the heavy chain with cysteine to obtain the variant HC79C of the present invention, substitute the amino acid at position 114 of the heavy chain with cysteine to obtain the variant HC114C of the present invention, substitute the amino acid at position 19 of the light chain with cysteine to obtain the variant LC19C of the present invention, substitute the amino acid at position 44 of the light chain with cysteine to obtain the variant LC44C of the present invention, substitute the amino acid at position 46 of the light chain with cysteine to obtain the variant LC46C of the present invention,The amino acid at position 62 of the light chain was replaced with cysteine to obtain the variant LC62C of the present invention, the amino acid at position 78 of the light chain was replaced with cysteine to obtain the variant LC78C of the present invention, and the amino acid at position 87 of the light chain was replaced with cysteine to obtain the variant LC87C of the present invention. The above-mentioned antibody DNA was chemically synthesized (Suzhou GenScript Biotechnology Co., Ltd.).

[0052] Then, the gene of the single-chain antibody was double-digested with HindIII and XhoI, and the single-chain antibody gene was ligated with the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated with HindIII and XhoI using T4 ligase. After successful ligation, the expression vectors of the anti-HER2 single-chain antibody and its mutants were successfully constructed.

[0053] [Table 1] TIFF2025521609000005.tif197170

[0054] (2) Screening and purification of the expression vectors of the anti-HER2 trastuzumab single-chain antibody and mutants The successfully constructed expression vector was introduced into the DH5α competent Escherichia coli strain (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.). The transformed DH5α strain was double-digested with two enzymes, HindIII and XhoI, and sequenced for verification, and positive clones were selected. The obtained Escherichia coli was lysed using a plasmid extraction kit (purchased from Kangwei Century), and the plasmid was extracted to obtain a purified expression vector.

[0055] (3) Expression of the anti-HER2 trastuzumab single-chain antibody and mutants in CHO cells The purified expression vector was transfected into CHO-K1 cells (purchased from ATCC) by electroporation, seeded in 96-well plates, grown for 15 days, monoclonal cells were selected, and the cell antibody yield was measured by Western blot. The first 20% were selected and transferred to 24-well plates. After growing for 7 days, the cell antibody yield was measured again. The first 5-6 strains were selected and transferred to shake flask culture. After stabilization in serum-free culture, they were continuously cultured for 7 days and then harvested to achieve the expression of anti-HER2 single-chain antibody and its variants in CHO-K1 cells. Purification and subsequent experiments were performed on the antibody.

[0056] Example 3. Purification by Ni-affinity chromatography column The cells and medium were transferred to a 50 mL centrifuge tube, placed in a high-speed refrigerated centrifuge, centrifuged at 8000 r for 15 min, the supernatant was retained, and the cell debris was discarded. After diluting the medium with 3 volumes of equilibration buffer, it was filtered once through a 0.22 μm microporous filtration membrane, and the filtrate was collected for use. The pump, sample injector, and purification column were washed with 10 column volumes of ultrapure water and then with PBS. Sampling was awaited and then carried out. Elution was performed with an eluent containing 250 mM imidazole, and the purified antibody protein solution was taken into an EP tube and stored at 2-8 °C. The SDS-PAGE purity detection results are shown in Figure 1. As can be seen from the electrophoresis diagram, the antibody band was single and the purity met the requirements of subsequent experiments.

[0057] Example 4. Detection of the occupancy rate of free mercapto groups on the antibody The present invention selects cell lines more suitable for further binding by detecting the content of free mercapto groups in antibody products using the Ellman's method (Thermo Fisher Scientific (China) Co., Ltd.). A 0.1 M Na2HPO4 solution, a DTNB (10 mM) solution, and a cysteine standard solution were formulated, and their final concentrations were set to 0.0059 m to 0.375 m. At the same time, a measurement antibody sample with a concentration of 5 mg / mL was prepared. To 1.5 mL EP tubes containing the cysteine standard solution and the measurement sample respectively, 250 μL of 0.1 M Na2HPO4 and 5 μL of the DTNB solution were added, vortexed to mix uniformly, and then incubated at room temperature in the dark for 5 min. The OD value was detected at a wavelength of 412 nm. A standard curve was created from the OD values, and the content of mercapto groups in the sample was calculated.

[0058]

Table 2

[0059] As can be seen from Table 1, the wild type contains almost no free mercapto groups, while the cysteine-engineered mutants have free mercapto groups available for binding.

[0060] Example 5. Measurement of the affinity between an antibody and an antigen Using the biolayer interferometry (BLI) technique, the affinity between the wild-type antibody, the cysteine-engineered antibody mutant and the antigen HER-FC was measured using a Pro A probe (ForteBio).

[0061] HER-FC (Beijing Protein Innovation Co., Ltd.) was dissolved in PBS to a working concentration of 10 μg / mL, and the antibody was dissolved in PBS2 and serially diluted to concentrations of 18.75 nM, 37.5 nM, 75 nM, 150 nM, and 300 nM respectively. The working volume was 200 μL in all cases.

[0062] The data map was fitted by the OCTET system and data processing software, and the software was used to calculate the intermolecular interaction force between an antibody or the like and HER2, and the results expressed by the KD value are shown in Table 2.

[0063]

Table 3

[0064] From the data in the table, it was found that there was almost no difference in the affinity of the wild-type antibody and the mutant for the antigen, and the selected mutation site did not change the effect of the antibody.

[0065] Example 6. Measurement of antibody-antigen binding The binding force between the wild-type antibody, the cysteine-engineered antibody mutant and the antigen HER-Fc (Beijing Sino Biological Inc.) was measured using the enzyme-linked immunosorbent assay (ELISA). The results are shown in Figure 3.

[0066]

Table 4

[0067] From the data in the table, it was found that there was almost no difference in the binding force of the wild-type antibody and the mutant for the antigen, and the selected mutation site did not change the effect of the antibody.

[0068] Example 7. Screening of anti-HER2 trastuzumab single-chain antibody two-site cysteine mutant cell lines Based on Examples 1 to 6, the present invention further performed two-site cysteine mutations shown in the following table on the anti-HER2 trastuzumab single-chain antibody.

[0069]

Table 5

[0070] (1) Construction of anti-HER2 trastuzumab single-chain antibody and two-site mutant expression vectors DNA encoding the antibody described in this specification (USP Medicines Compendium from its corresponding wild-type sequence) is used. The specific nucleotide sequences of the heavy-chain variable region, light-chain variable region, and linker chain are shown in SEQ ID NOs: 1-3, and the amino acid sequences are shown in SEQ ID NOs: 4-6. Furthermore, based on the Kabat numbering, the amino acid at position 12 and the amino acid at position 35 of the heavy chain are substituted with cysteine to obtain variant 12C-35C of the present invention; the amino acid at position 12 and the amino acid at position 61 of the heavy chain are substituted with cysteine to obtain variant 12C-61C of the present invention; the amino acid at position 35 and the amino acid at position 61 of the heavy chain are substituted with cysteine to obtain variant 35C-61C of the present invention; the amino acid at position 51 and the amino acid at position 61 of the heavy chain are substituted with cysteine to obtain variant 51C-61C of the present invention; the amino acid at position 51 of the heavy chain and the amino acid at position 46 of the light chain are substituted with cysteine to obtain variant 51C-46C of the present invention; the amino acid at position 61 of the heavy chain and the amino acid at position 46 of the light chain are substituted with cysteine to obtain variant 61C-46C of the present invention; the amino acid at position 61 of the heavy chain and the amino acid at position 87 of the light chain are substituted with cysteine to obtain variant 61C-87C of the present invention; the amino acid at position 21 and the amino acid at position 47 of the light chain are substituted with cysteine to obtain variant 21C-47C of the present invention; the amino acid at position 21 and the amino acid at position 48 of the light chain are substituted with cysteine to obtain variant 21C-48C of the present invention; the amino acid at position 21 and the amino acid at position 75 of the light chain are substituted with cysteine to obtain variant 21C-75C of the present invention; the amino acid at position 46 and the amino acid at position 87 of the light chain are substituted with cysteine to obtain variant 46C-87C of the present invention. The DNA of the above antibody was chemically synthesized (Suzhou GenScript Biotechnology Co., Ltd.), and the sequence listing is shown below.

[0071] Then, the gene of the single-chain antibody was double-digested with HindIII and XhoI, and the single-chain antibody gene was ligated with the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated with HindIII and XhoI using T4 ligase. After successful ligation, the expression vectors of the anti-HER2 single-chain antibody and its two-site mutants were successfully constructed.

[0072]

Table 6

[0073] (2) Screening and purification of the anti-HER2 trastuzumab single-chain antibody and two-site variant expression vector The successfully constructed expression vector was introduced into the DH5α competent Escherichia coli strain. The transformed DH5α strain was subjected to double enzyme digestion using two enzymes, HindIII and XhoI, and sequencing was performed for verification, and positive clones were selected. The obtained Escherichia coli was lysed using a plasmid extraction kit (purchased from Kangwei Century), and the plasmid was extracted to obtain a purified expression vector.

[0074] (3) Expression of the anti-HER2 trastuzumab single-chain antibody and two-site variant in CHO cells The purified expression vector was transfected into CHO-K1 cells (purchased from ATCC) by electroporation, plated in a 96-well plate, grown for 15 days, monoclonal cells were selected, and the cell antibody yield was measured by Western blot. The first 20% were selected and transferred to a 24-well plate. After growing for 7 days, the cell antibody yield was measured. The first 5-6 strains were selected and transferred to shake flask culture. After stabilization in serum-free culture, they were continuously cultured for 7 days and then harvested to achieve the expression of the anti-HER2 single-chain antibody in CHO-K1 cells, and purification and subsequent experiments were performed on the antibody.

[0075] The two-site variant of the anti-HER2 trastuzumab single-chain bispecific antibody also has a technical effect similar to that of the single-site variant described in Examples 1 to 6. For its binding effect, reference may be made to the experimental results of Example 11 "Binding and Detection of the Antibody to vcMMAE" and Example 13 "Detection of Cell Proliferation Inhibitory Effect".

[0076] Example 8. Screening of other single-chain antibody cysteine mutant cell lines Examples 1 to 7 verified each mutation site using the anti-HER2 trastuzumab single-chain antibody as an example. The present invention performed mutations at corresponding sites of other antibodies and verified that the above mutation sites are versatile for various antibodies. Specifically, the present invention performed cysteine mutations on the amino acids at positions 46 and 87 of the light chain of the pertuzumab single-chain antibody, and named the mutants Per-46C and Per-87C respectively. A cysteine mutation was performed on the amino acid at position 46 of the light chain of the rituximab single-chain antibody, and the mutant was named Rit-46C. A cysteine mutation was performed on the amino acid at position 46 of the light chain of the muromonab single-chain antibody, and the mutant was named Mur-46C. A cysteine mutation was performed on the amino acid at position 87 of the light chain of the ramucirumab single-chain antibody, and the mutant was named Ram-87C. The sequence listing is shown below.

[0077]

Table 7

[0078] Mutants at corresponding sites of the trastuzumab single-chain antibody, rituximab single-chain antibody, muromonab single-chain antibody, and ramucirumab single-chain antibody also have effects similar to those of the anti-HER2 trastuzumab single-chain antibody. For the binding effect, reference may be made to the experimental results of "Binding and Detection of Other Antibody Single-Chain Antibodies to mPEG2000-MAL in Example 12". Therefore, the above mutation sites screened in the present invention are versatile for various antibodies.

[0079] Example 9. Screening of Anti-HER2 Single-Chain Bispecific Antibody Two-Site Cysteine Mutation Cell Lines Based on the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody, a bispecific antibody in the BiTE form named BiTE-WT was constructed. Moreover, cysteine mutations were performed at position 46 or 87 of the light chain on the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody, respectively, to construct a cysteine-mutated bispecific antibody in the BiTE form. For the cysteine-mutated bispecific antibody in the BiTE form, with the trastuzumab antibody VL mutation site in front and the trastuzumab VL mutation site behind, they were named BiTE-46-46, BiTE-46-87, BiTE-87-46, and BiTE-87-87, respectively.

[0080] The ligation form of BiTE is Herceptin VL - GGGGSGGGGSGGGGS - Herceptin VH - GGGGS - Perjeta VH - GGGGSGGGGSGGGGS - Perjeta VL, and the sequence is shown in the following table.

[0081]

Table 8

[0082] The anti-HER2 single-chain bispecific antibody two-site mutant also has a technical effect similar to that of the single-specific antibody single-site mutant described in Examples 1 to 6. For its binding effect, reference may be made to the experimental results of Example 11 "Binding and Detection of Antibody to vcMMAE" and Example 13 "Detection of Cell Proliferation Inhibitory Effect".

[0083] Example 10. Binding and Detection of Anti-HER2 Trastuzumab Single-Chain Antibody to mPEG2000-MAL This example used the single-site mutant of the purified anti-HER2 trastuzumab single-chain antibody obtained in Example 3, and experiments were carried out with mPEG2000-MAL as the conjugate part. Here, the binding reaction conditions between the antibody and the conjugate part were: antibody: conjugate part (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature 37 °C, reaction time 3 h, reaction buffer PBS (pH 7.2). For the SDS-PAGE electrophoresis diagrams of the wild-type or mutant single-chain antibody binding to mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01), refer to Figures 2 to 8 respectively.

[0084] This specification detected the binding of the mutant single-chain antibody to mPEG2000-MAL in the SDS-PAGE experiment. The protein band after binding migrated upward, indicating an increase in molecular weight. As a result, it was shown that the binding of each mutant to mPEG2000-MAL was successful. From the binding results of the antibody to mPEG2000-MAL, it was found that the mutant single-chain antibody in this specification has a reactive mercapto group that can react with a drug linker having a functional group that reacts with a mercapto group (for example, a maleimide functional group), and thus it can be reasonably expected that the antibody and the drug molecule can also be linked.

[0085] Example 11. Binding and Detection of Antibody to vcMMAE In this example, one sample each of the purified single-site mutant (LC87) of the anti-HER2 trastuzumab single-chain antibody obtained in Example 3, the two-site mutant (HC61C-LC87C) of the anti-HER2 trastuzumab single-chain antibody obtained in Example 7, and the two-site mutant bispecific antibody (BiTE-87-87) obtained after purification in Example 9 were selected, and experiments were carried out using the drug molecule vcMMAE (purchased from Nanjing Adooq Bioscience Co., Ltd., product identification number A14362) as the conjugate part. Here, the binding reaction conditions between the antibody and the conjugate part were antibody:conjugate part (vcMMAE) = 1:5 (molar ratio), reaction temperature 37 °C, reaction time 3 h, and reaction buffer PBS (pH 7.2). For the typical mass spectra before and after the binding of the single-site mutant single-chain antibody to vcMMAE, refer to Figures 9 and 10. Figure 9 is the result diagram of the molecular weight of the single-chain antibody before binding. The molecular weight of the single-chain antibody is 26521.3 Da. Figure 10 is the result diagram of the molecular weight of the antibody after binding to vcMMAE. The molecular weight of the antibody after binding to vcMMAE is 27838.2 Da. The difference was calculated as the molecular weight of vcMMAE, i.e., 1316.9 Da, which proved that each mutant single-chain antibody successfully bound to one molecule of vcMMAE. For the typical mass spectra before and after the binding of the two-site mutant, refer to Figures 11 and 12. Figure 11 is the result diagram of the molecular weight of the single-chain antibody before binding. The molecular weight of the single-chain antibody is 26553.5 Da. Figure 12 is the result diagram of the molecular weight of the antibody after binding to vcMMAE. The molecular weights of the antibody after binding to vcMMAE are 27988.0 Da and 29186.3 Da. The differences were calculated as the molecular weights of 1 molecule and 2 molecules of vcMMAE respectively, which proved by calculation that each double-mutant single-chain antibody successfully bound to 1.7 or more molecules of vcMMAE.Typical mass spectra of the double mutant bispecific antibodies before and after binding are shown in Figures 13 and 14. Figure 13 shows the molecular weight results of the double mutant bispecific antibodies before binding, and the molecular weight of the single chain antibody is 52588.5 Da. Figure 14 shows the molecular weight results of the antibodies after binding to vcMMAE, and the molecular weights of the antibodies after binding to vcMMAE are 53903.7 Da and 55220.6 Da. The difference was calculated as the molecular weight of one molecule and two molecules of vcMMAE, respectively. The calculation proved that each double mutant bispecific antibody successfully bound to more than 1.5 vcMMAE molecules.

[0086] Example 12. Binding and detection of other antibodies single chain antibodies to mPEG2000-MAL In this example, experiments were performed using the mutants of trastuzumab single-chain antibody, rituximab single-chain antibody, muromonab single-chain antibody, and ramucirumab single-chain antibody obtained in Example 8 as examples, and mPEG2000-MAL (purchased from Shanghai Zhenquan Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) was used as the conjugate moiety. Here, cysteine ​​mutations were performed on the amino acids at positions 46 and 87 of the light chain of Pertuzumab, cysteine ​​mutations were performed on the amino acid at position 46 of the light chain of Rituximab, cysteine ​​mutations were performed on the amino acid at position 46 of the light chain of Muromonab, and cysteine ​​mutations were performed on the ammonia amino acid at position 87 of the light chain of Ramucirumab. The binding reaction conditions for the obtained antibody mutant and the conjugate portion were antibody: conjugate portion (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature 37 ° C, reaction time 3 h, and reaction buffer PBS (pH 7.2). SDS-PAGE electrophoresis diagrams of the mutant single-chain antibody bound to mPEG2000-MAL were shown in Figures 15 to 17. SDS-PAGE experiments were employed to detect the binding of the mutant single-chain antibodies to mPEG2000-MAL, and the protein bands after binding shifted upward, indicating an increase in molecular weight, and the results indicated that the mutant single-chain antibodies of the four antibodies could be successfully bound to mPEG2000-MAL.

[0087] Example 13. Detection of cytostatics The present invention selected two cell lines, breast cancer cells SK-BR-3 and MCF-7 (purchased from Procell), and adopted a CCK8 kit (purchased from APExBIO) as a proliferation detection reagent. The inhibitory effects of drug molecule MMAE, anti-HER2 trastuzumab single-chain antibody cysteine mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, and HC61C-LC87C, and anti-HER2 bispecific antibody mutant BiTE-87-87, and the antibody-drug conjugates obtained after the above antibody mutants were conjugated to vcMMAE on cell proliferation were determined, and the half-maximal inhibitory concentration (IC 50 ) was calculated. SK-BR-3 is a HER2-high-expressing cell line, and MCF-7 is a HER2-low-expressing cell line. The IC 50 calculated when MMAE acted on SK-BR-3 was 0.20 nM, and the IC 50 calculated when MMAE acted on MCF-7 was 0.35 nM. The IC 50 calculated when the single-chain antibody cysteine mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C, and anti-HER2 bispecific antibody BiTE-87-87 acted on SK-BR-3 was >1000 nM, and the IC 50 calculated when the single-chain antibody cysteine mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C, and anti-HER2 bispecific antibody BiTE-87-87 acted on MCF-7 was >1000 nM. The IC 50were 23.95 nM, 15.55 nM, 19.76 nM, 17.28 nM, 13.18 nM, and 1.703 nM, respectively, and were calculated IC for antibody-drug complexes obtained after binding of anti-HER2 trastuzumab single-chain antibody cysteine ​​mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, and HC61C-LC87C to vcMMAE and the anti-HER2 bispecific antibody BiTE-87-87 on MCF-7. 50 were greater than 50 nM. Taken together, the above results show that vcMMAE has no targeting ability and produces very strong nonspecific killing ability in both cells. In the SK-BR-3 cell line, which has high expression of HER2, the antibody can exert stronger cytotoxicity after binding to vcMMAE than the single-chain antibody alone. On the other hand, in the MCF-7 cell line, the expression level of HER2 is too low, and antigen-mediated antibody binding and internalization are insufficient, so only weak cytotoxicity occurs after the antibody is bound to vcMMAE. Compared with MMAE, the magnitude of cytotoxicity by the antibody-drug conjugate at the same molar concentration shows HER2 receptor expression level dependence, proving that MMAE has a certain targeting ability after binding to the single-chain antibody cysteine ​​mutant, which can reduce toxicity and expand the drug therapeutic window.

[0088] As described above, by performing cysteine ​​mutations at positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79 and / or 114 in the chain variable region of the single-chain antibody, and / or positions 19, 21, 44, 46, 47, 48, 62, 71, 75, 78 and / or 87 in the light chain variable region, it is possible to obtain free sulfhydryl groups for binding, which can be successfully bound to the conjugate moiety and drug molecule, thereby obtaining an antibody-drug conjugate and thereby providing the drug with targeting ability.

[0089] Example 14. Screening of trastuzumab antibody cysteine ​​mutant cell lines Examples 1 to 7 verified each mutation site using the anti-HER2 trastuzumab single-chain antibody as an example. Furthermore, the present invention mutated the corresponding sites of its full antibody (i.e., IgG antibody) trastuzumab and verified that the above mutation sites have generality in different forms of the antibody. Specifically, the present invention performed cysteine mutations on the amino acids at positions 46 and 87 of the light chain of the trastuzumab antibody, and named the mutants trastuzumab-46C and trastuzumab-87C, respectively. The sequence listing is as follows:

[0090]

Table 9

[0091] Example 15. Binding and Detection of Trastuzumab Cysteine-Mutated Antibody to mPEG2000-MAL This example used the cysteine mutants at positions 46 and 87 of the light chain of the trastuzumab antibody prepared in Example 14 above, and conducted experiments with mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) as the conjugate part. The binding reaction conditions between the antibody mutant and the conjugate part were antibody:conjugate part (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature 37 °C, reaction time 3 h, and reaction buffer PBS (pH 7.2). For the reduced SDS-PAGE electrophoresis diagram of the binding of the mutant antibody to mPEG2000-MAL, refer to Figure 18. The reduced SDS-PAGE experiment was adopted to detect the binding of the mutant to mPEG2000-MAL. The protein band of the light chain after binding migrated upward, indicating an increase in molecular weight. As a result, it was shown that the mutants of the trastuzumab antibody (i.e., in IgG antibody form) could successfully bind to mPEG2000-MAL. The mutation sites had technical effects similar to those of single-chain antibodies and bispecific antibodies even in the IgG antibody form.

[0092] Example 16. Screening of Cysteine-Mutated Cell Lines of BiTE Form Bispecific Antibody Against EGFR and c-Met In Examples 9 and 11, after verifying the binding performance of the BiTE-form bispecific antibody constructed with the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody, furthermore, the present invention constructed a BiTE-form bispecific antibody named BiTE-E / M-WT based on the anti-EGFR cetuximab single-chain antibody and the anti-c-Met single-chain antibody. Moreover, cysteine mutations were respectively introduced at position 46 or 87 of the light chain on the anti-EGFR single-chain antibody and the anti-c-Met single-chain antibody to construct a cysteine-mutated bispecific antibody in BiTE form. For the cysteine-mutated bispecific antibody in BiTE form, with the VL mutation site of the anti-EGFR single-chain antibody in front and the VL mutation site of the anti-c-Met single-chain antibody behind, they were respectively named BiTE-E / M-46-46, BiTE-E / M-46-87, BiTE-E / M-87-46 and BiTE-E / M-87-87, and the sequences are shown in the following table.

[0093]

Table 10

[0094] Example 17. Binding and Detection of Cysteine-Mutated Anti-EGFR and c-Met BiTE-Form Bispecific Antibody to mPEG2000-MAL In this example, taking the cysteine-mutated antibody of the anti-EGFR and c-Met BiTE-form bispecific antibody constructed in Example 16 above as an example, experiments were carried out using mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) as the conjugate part. The binding reaction conditions between the antibody mutant and the conjugate part were antibody: conjugate part (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature 37 °C, reaction time 3 h, and reaction buffer PBS (pH 7.2). Taking BiTE-E / M-87-87 as an example, the binding of the mutant to mPEG2000-MAL was detected by SDS-PAGE experiment, and the protein band after binding migrated upward, indicating an increase in molecular weight. As a result, Figure 19 showed that the mutant of the anti-EGFR and c-Met BiTE-form bispecific antibody could successfully bind to mPEG2000-MAL.

[0095] Example 18. Screening of anti-HER2 single-chain bispecific antibody single-site cysteine mutants with different array forms Furthermore, based on the anti-HER2 trastuzumab single-chain antibody and anti-HER2 pertuzumab single-chain antibody, the present invention constructs bispecific antibodies with different compositions. The linking forms and names are shown in Table 5. This table includes representative examples linked with linkers of different lengths, with the heavy and light chains linked in different orders, and the antibodies linked in different orders (here, "Bi" indicates the same sequence order of the heavy and light chains as in Example 9, "Tan" indicates that the sequence orders of the heavy and light chains of the two antibodies are the same, and "LH" / "HL" indicates, in the "Tan" form, the number of repetitions of the G4S linker that links antibodies with different terminal numbers with the light chain / heavy chain in the front).

[0096] [Table 11]

[0097] The coding sequences and amino acid sequences of different G4S linkers are shown in the following table:

[0098] [Table 12]

[0099] Among the bispecific antibodies in the above table, cysteine mutations were made at position 46 or 87 of the light chain of the trastuzumab single-chain antibody or pertuzumab single-chain antibody to construct bispecific antibody cysteine single-site mutants with different compositions. Different names were given according to the selection of mutant antibodies. For example: Bi-TP-1-WT-87, that is, similar to the sequence order, the trastuzumab corresponding to T maintains the wild type, and the 87th position of the light chain of the trastuzumab corresponding to P is mutated into the form of cysteine.

[0100] Example 19. Binding of anti-HER2 single-chain bispecific antibody single-site cysteine mutants with different array forms to the small molecule drug vcMMAE and its detection This example selected the bispecific antibody with a single-site mutation at position 87 of trastuzumab obtained in Example 18 above as an example, and used the drug molecule vcMMAE (purchased from Nanjing Adooq Bioscience Co., Ltd., product identification number A14362) as the conjugate part to perform binding experiments with antibody mutants Bi-TP-1-WT-87, Bi-TP-2-WT-87, Bi-TP-3-WT-87, Bi-TP-4-WT-87, Tan-HL-TP-4-WT-87, Tan-LH-TP-4-WT-87, Tan-LH-PT-4-87-WT. Here, the binding reaction conditions between the antibody and the conjugate part were antibody: conjugate part (vcMMAE) = 1:5 (molar ratio), reaction temperature 37 °C, reaction time 3 h, and reaction buffer PBS (pH 7.2).

[0101] The binding situations of bispecific antibody single-site mutants with different sequence forms are shown in Figures 20 to 35. By comparing Figures 20 and 21, Figures 22 and 23, Figures 24 and 25, Figures 26 and 27, Figures 28 and 29, Figures 30 and 31, Figures 32 and 33, Figures 34 and 35 respectively, it was found that the difference in molecular weight before and after binding was about 1316 Da in all cases, that is, the above-mentioned specific antibody single-site mutants with different sequences were all able to achieve binding.

[0102] Example 20. Binding and Detection of Antibody to Small Molecule Drug MC-2MMAE This example took the anti-HER2 single-chain bispecific antibody single-site cysteine mutant Bi-TP-4-WT-87 obtained in Example 18 as an example, and used the branched drug MC-2MMAE (purchased from Yantai MabPlex international Biopharmaceutical Co., Ltd.) as the conjugate part for experiments. Here, the binding reaction conditions between the antibody and the conjugate part were antibody: conjugate part (vcMMAE) = 1:2.5 (molar ratio), reaction temperature 4°C, reaction time 12 h, and reaction buffer PBS (pH 7.2). The typical spectra before and after the binding are shown in Figures 34 - 35. Referring to Figure 34, the molecular weight before binding was 53534.3 Da, and referring to Figure 35, the molecular weight after drug binding was 56587.6 Da. The difference in molecular weight before and after binding was 3053.3 Da, that is, the previous MC-2MMAE molecule was bound, achieving the same technical effect as in Example 11. That is, the antibody mutant of the present invention can further bind to the multi-branched drug, and through the multi-branched drug linker, more load connection can be realized.

[0103] Example 21. Screening of a single-site cysteine mutant cell line of an anti-c-MET single-chain antibody Examples 1 - 8 verified the mutation sites such as positions 46 and 87 of the light chain using the trastuzumab single-chain antibody, trastuzumab single-chain antibody, rituximab single-chain antibody, muromonab single-chain antibody, and ramucirumab single-chain antibody as examples. Based on Examples 1 - 8, the present invention further mutated the anti-c-MET antibody to verify that the above mutation sites have generality for various antibodies. Specifically, the present invention performed a cysteine mutation at position 46 of the light chain of the anti-c-MET single-chain antibody, and named the mutant c-MET-46C. Its sequence listing is shown below.

[0104]

Table 13

[0105] Example 22. Binding and detection of a single-site cysteine mutant of an anti-c-MET single-chain antibody to vcMMAE This example selected the anti-c-MET single-chain antibody 46-site single-site antibody produced in Example 21 as an example, and performed a conjugation experiment with the drug molecule vcMMAE (purchased from Nanjing Adooq Bioscience Co, Ltd., product identification number A14362) as the conjugate part. Here, the binding reaction conditions between the antibody and the conjugate part were antibody: conjugate part (vcMMAE) = 1:5 (molar ratio), reaction temperature 37 °C, reaction time 3 h, and reaction buffer PBS (pH 7.2).

[0106] The spectra before and after binding are shown in Figures 36 and 37. Figure 36 is the result diagram of the molecular weight of the single-chain antibody before binding, and the molecular weight is 26001.1 Da. Figure 37 is the result diagram of the molecular weight of the antibody after binding to vcMMAE. The molecular weight of the antibody after binding to vcMMAE is 27318.1 Da, and the difference is calculated as the molecular weight of one molecule of vcMMAE. That is, each antibody variant successfully bound to one vcMMAE.

[0107] Example 22. Screening of anti-EGFR single-chain antibody single-site cysteine mutant cell lines Examples 1 to 8 and 21 verified the mutation sites such as positions 46 and 87 of different single-chain antibodies. On this basis, the present invention further mutated the 46th position of the light chain on the anti-EGFR zalutumumab single-chain antibody to verify that the above mutation site has universality for various antibodies. Specifically, the present invention performed a cysteine mutation at the 46th position of the light chain of the zalutumumab single-chain antibody, and named the mutants Zalu-46C respectively. The sequence listing is shown below.

[0108]

Table 14

[0109] Example 23. Binding and detection of anti-EGFR single-chain antibody single-site cysteine mutants to vcMMAE This example selected the single-site antibody at position 46 of the zalutumumab single-chain antibody obtained in Example 22 as an example, and performed a conjugation experiment with the drug molecule vcMMAE (purchased from Nanjing Adooq Bioscience Co., Ltd., product identification number A14362) as the conjugate part. Here, the binding reaction conditions between the antibody and the conjugate part were antibody: conjugate part (vcMMAE) = 1:5 (molar ratio), reaction temperature 37 °C, reaction time 3 h, and reaction buffer PBS (pH 7.2).

[0110] For the spectra before and after binding, refer to Figures 38 and 39. Figure 38 is the result diagram of the molecular weight of the single-chain antibody before binding, and the molecular weight is 27179.4 Da. Figure 39 is the result diagram of the molecular weight of the antibody after binding to vcMMAE. The molecular weight of the antibody after binding to vcMMAE is 28496.4 Da, and the difference is calculated as the molecular weight of one molecule of vcMMAE. That is, each antibody variant successfully bound to one vcMMAE.

[0111] The specific examples described above further detailed the object, technical solution, and beneficial effects of the present invention. However, what is described above is only the specific examples of the present invention and is not intended to limit the present invention. Modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should all be included within the protection scope of the present invention.

[0112] References 1. A Potent Anti-CD70 Antibody-Drug Conjugate Combining a DimericPyrrolobenzodiazepine Drug with Site-Specific Conjugation Technology[J]. Bioconjugate Chemistry, 2013, 24(7):1256-1263. 2. Crivianu-Gaita V, Thompson M. Aptamers, antibody scFv, and antibody Fab0 fragments: an overview and comparison of three of the most versatile biosensor biorecognition elements. BiosensBioelectron 2016;85:32-45. 3. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M. ScFv antibody: principles and clinical application. Clin Dev Immunol 2012;2012. Article ID 980250. 4.Safdari Y, Ahmadzadeh V. Use of single-chain antibody derivatives for targeted drug delivery. Mol Med 2016;22:258-70. 5. Deonarain MP. Fragment antibody fragment drug conjugates(FDCs): a unique drug class or just smaller ADCs?In: Proceedings of the protein engineering summit(PEGS) conference; 2017. 6.Deonarain M, Yahioglu G, Stamati I, Pomowski A, Clarke J, Edwards B, et al. Small-format drug conjugates: a viable alternative to ADCs for solid tumours? Antibodies 2018;7:16. 7. Deonarain MP. Fragment antibody fragment drug conjugates(FDCs): a unique drug class or just smaller ADCs?In: Proceedings of the Protein Engineering Summit(PEGS) Conference; 2017. Sequence Listing

[0113]

Table 15

Claims

1. A cysteine ​​engineered antibody, characterized in that it has an engineered cysteine ​​residue at any one or more positions selected from the following, based on the Kabat numbering system: Position 87 of the light chain variable region, position 46 of the light chain variable region, position 12 of the heavy chain variable region, position 62 of the light chain variable region, position 51 of the heavy chain variable region, position 35 of the heavy chain variable region, position 114 of the heavy chain variable region, position 60 of the heavy chain variable region, position 61 of the heavy chain variable region, positions 19, 21, 44, 47, 48, 71, 75, 78 of the light chain variable region, positions 34, 38, 44, 47, 67, 69, 78, 79 of the heavy chain variable region, or any combination thereof.

2. 2. The antibody of claim 1, having an engineered cysteine ​​residue at any one or more positions selected from the following, based on the Kabat numbering system: Positions 87 and 46 in the light chain variable region, positions 12, 51, 35, 61, 69, and 78 in the heavy chain variable region, or any combination thereof.

3. 2. The antibody of claim 1, having an engineered cysteine ​​residue at any one or more positions selected from the following, based on the Kabat numbering system: Positions 87 and 46 in the light chain variable region, positions 12, 51, 35 and 61 in the heavy chain variable region, or any combination thereof.

4. The antibody according to claim 1, which is obtained based on the following wild-type antibody: An antibody comprising a heavy chain variable region shown in SEQ ID NO: 4 and a light chain variable region shown in SEQ ID NO: 5; an antibody comprising a heavy chain variable region set forth in SEQ ID NO: 75 and a light chain variable region set forth in SEQ ID NO: 92; an antibody comprising a heavy chain variable region shown in SEQ ID NO: 81 and a light chain variable region shown in SEQ ID NO: 95; an antibody comprising a heavy chain variable region shown in SEQ ID NO: 85 and a light chain variable region shown in SEQ ID NO: 96; an antibody comprising a heavy chain variable region set forth in SEQ ID NO: 89 and a light chain variable region set forth in SEQ ID NO: 97; an antibody comprising a heavy chain as set forth in SEQ ID NO: 100 and a light chain as set forth in SEQ ID NO: 101; an antibody comprising a heavy chain variable region shown in SEQ ID NO: 108 and a light chain variable region shown in SEQ ID NO: 109; an antibody comprising a heavy chain variable region set forth in SEQ ID NO: 113 and a light chain variable region set forth in SEQ ID NO: 114, and / or An antibody comprising a heavy chain variable region shown in SEQ ID NO:130 and a light chain variable region shown in SEQ ID NO:

133.

5. the light chain is of the λ or κ type; optionally an anti-HER2 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-VEGFR-2 antibody, an anti-EGFR antibody and / or an anti-c-Met antibody; 2. The antibody of claim 1, which is optionally a single chain antibody, an IgG antibody, or a bispecific antibody, such as a bispecific antibody in the form of a BiTE / DART / Diabody.

6. 6. The antibody of claim 5, wherein the antibody, when a single-chain antibody, further comprises a linking chain, preferably a linking chain as shown in SEQ ID NO:

6.

7. A conjugate comprising the antibody of any one of claims 1 to 6 and a conjugate moiety.

8. the conjugate moiety is selected from polyethylene glycol, a cytotoxic agent, an active peptide, a nanobody, a single domain antibody, a Fab fragment, a Fab′ fragment, a scFv, a small molecule drug (such as a topoisomerase inhibitor, tubulysin A, DM1, PBD, MMAE, or MMAF), a chemotherapeutic agent, or a radiotherapeutic agent; the conjugate moiety is conjugated to the cysteine ​​engineered antibody via a linker; The conjugate of claim 7, wherein the linker preferably comprises an electrophilic group (preferably a maleimide group or a halogenated acetamide group), and optionally the linker is mc-VC-PAB.

9. The conjugate according to claim 8, wherein the polyethylene glycol is maleimide monomethoxypolyethylene glycol, preferably mPEG2000-Mal, mPEG5000-Mal, or mPEG10000-Mal.

10. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 6, and optionally a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising the conjugate of claim 7, and optionally a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 10 for treating cancer (e.g., HER2-positive cancer, preferably breast cancer).

13. A kit comprising the antibody of any one of claims 1 to 6.

14. A kit comprising the conjugate described in claim 7.

15. A kit comprising the pharmaceutical composition described in claim 10.

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