Mutant of antibody variable region and application thereof

Abstract

The invention provides a mutant of an antibody and application thereof, and particularly, according to a Kabat numbering system, the mutant of the antibody has engineered cysteine residues at any one or more positions selected from the group consisting of positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 of a heavy chain variable region or any combination thereof; or positions 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 or any combination thereof of the light chain variable region. The sulfydryl on the engineered cysteine can maintain partial activity after the expression process of the antibody. The sulfydryl keeping activity can be directly used for reacting with other active groups without being subjected to reduction treatment. The cysteine engineering modified antibody provided by the invention can obtain an active group for coupling a drug, and is beneficial to simplifying the production process of the antibody coupling drug, improving the uniformity of the antibody coupling drug, improving the drug effect and reducing the toxic and side effects.

Description

Mutants of antibody variable regions and their applications Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to cysteine ​​mutants of antibodies, compositions and / or conjugates comprising the same, and uses thereof. Background Art

[0002] Antibody-drug conjugates (ADCs) are antibody products connected to drug molecules obtained by connecting antibodies and drugs through biological or chemical means. Antibody-drug conjugates reduce the killing effect of drugs on normal cells through the specificity of monoclonal antibodies and are mainly used in the treatment of tumors. The preparation of antibody-drug conjugates is mainly through coupling reactions with amino residues of lysine or sulfhydryl residues of cysteine. Whether it is coupled through lysine residues or cysteine ​​residues, traditional non-site-specific coupling methods have some disadvantages. Achieving site-specific coupling through site-specific coupling technology can improve the uniformity of antibody-drug conjugates. [1] .

[0003] Antibody molecules contain four polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (HC), and the two chains with smaller molecular weight are called light chains (LC). The two H chains and two L chains in the same antibody molecule have exactly the same amino acid composition. By 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, while the amino acid sequences of other parts are relatively constant. Therefore, the region of the antibody light and heavy chains with the most variable amino acid sequence near the N-terminus is called the variable region (V), which accounts for 1 / 4 and 1 / 2 of the heavy and light chains, respectively; the region with relatively stable amino acid sequence near the C-terminus is called the constant region (C), which accounts for 3 / 4 and 1 / 2 of the heavy and light chains, respectively.

[0004] Single chain antibody fragment (scFv) is an antibody composed of the variable region of the heavy chain and the variable region of the light chain of the antibody connected by a short amino acid peptide (linker). Single chain antibody (scFv) is an antibody in which the heavy chain and the light chain of the variable region of the antibody are separated by a short amino acid and connected by disulfide bonds. [2] ScFv has a small molecular weight and is suitable for expression in yeast or bacteria, which facilitates rapid mass production and is an ideal candidate for high-throughput selection technologies such as phage display, cell display, yeast display, and ribosome display. [3]Single-chain antibody-drug conjugates have great potential. scFv has a shorter half-life, but its tumor penetration ability seems to be greater than that of Fab fragments. Therefore, single-chain antibody-drug conjugates have better efficacy and fewer side effects. [4] Data suggest that single-chain antibody-drug conjugates are better tolerated than IgG antibody-drug conjugates [5，6] .

[0005] In addition to being applied to intact IgG antibody molecules, site-directed conjugation technology is also applicable to scFv and bispecific antibody-drug conjugates based on scFv. The most common reactive amino acid in site-directed conjugation technology is cysteine. Through site-directed mutagenesis technology, antibody-drug conjugates with controllable drug-antibody conjugation ratios can be developed. Selecting appropriate sites for cysteine ​​mutation can keep the sulfhydryl group on the mutated cysteine ​​free during the expression and purification process and can be directly used in downstream conjugation reactions. Due to the widespread use of cysteine, maleimide is the most popular linker group. [7] , and then the antibody is coupled to another functional molecule (antibody fragment, peptide or small molecule drug) through a maleimide-containing linker.

[0006] Most thiol groups on cysteines introduced into antibodies through cysteine ​​engineering are oxidized during cell culture, reacting with thiol groups on free cysteines in the cell culture medium to form disulfide bonds. Oxidized thiol groups need to be reduced to become active for subsequent conjugation reactions, but the reduction reaction also opens disulfide bonds within the antibody, which then need to be reoxidized to restore the disulfide bond structure. This series of post-expression processing steps complicates the production process of antibody-drug conjugates and poses challenges to quality control.

[0007] Summary of the Invention

[0008] The present invention mainly provides a cysteine ​​engineered antibody, which has an engineered cysteine ​​residue at any one or more positions selected from the following according to the Kabat numbering system: positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 of the heavy chain variable region or any combination thereof, corresponding to IMGT numbers 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 of the light chain variable region or any combination thereof, corresponding to IMGT numbers 19, 21, 50, 52, 53, 54, 76, 87, 91, 94, 103, respectively, wherein the thiol group on the engineered cysteine ​​can retain partial activity during antibody expression. The active thiol groups can be directly used to react with other active groups without reduction treatment.

[0009] Specifically, the present invention provides the following aspects:

[0010] 1. A cysteine ​​engineered antibody, characterized in that, according to the Kabat numbering system, it has an engineered cysteine ​​residue at any one or more positions selected from the group consisting of:

[0011] position 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 of the heavy chain variable region, or any combination thereof; or

[0012] Position 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 of the light chain variable region, or any combination thereof.

[0013] 2. The cysteine ​​engineered antibody according to item 1, wherein, according to the Kabat numbering system, it has an engineered cysteine ​​residue at any one or more positions selected from the group consisting of:

[0014] Position 12, 35, 61, 51, 69, 78 of the heavy chain variable region, or any combination thereof; or

[0015] Position 46, 87 or any combination thereof of the light chain variable region.

[0016] 3. The cysteine ​​engineered antibody according to item 1, wherein, according to the Kabat numbering system, it has an engineered cysteine ​​residue at any one or more positions selected from the group consisting of:

[0017] Position 12, 35, 61, 51, or any combination thereof, of the heavy chain variable region; or

[0018] Position 46, 87 or any combination thereof of the light chain variable region.

[0019] 4. The cysteine ​​engineered antibody according to item 1, wherein the cysteine ​​engineered antibody is obtained based on the following wild-type antibody:

[0020] 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;

[0021] an antibody comprising the heavy chain variable region set forth in SEQ ID NO: 75 and the light chain variable region set forth in SEQ ID NO: 92;

[0022] an antibody comprising the heavy chain variable region set forth in SEQ ID NO: 81 and the light chain variable region set forth in SEQ ID NO: 95;

[0023] an antibody comprising the heavy chain variable region set forth in SEQ ID NO: 85 and the light chain variable region set forth in SEQ ID NO: 96;

[0024] an antibody comprising the heavy chain variable region set forth in SEQ ID NO: 89 and the light chain variable region set forth in SEQ ID NO: 97;

[0025] an antibody comprising the heavy chain shown in SEQ ID NO: 100 and the light chain shown in SEQ ID NO: 101;

[0026] an antibody comprising the heavy chain variable region set forth in SEQ ID NO: 108 and the light chain variable region set forth in SEQ ID NO: 109;

[0027] An antibody comprising the heavy chain variable region shown in SEQ ID NO: 113 and the light chain variable region shown in SEQ ID NO: 114; and / or

[0028] An antibody comprising the heavy chain variable region set forth in SEQ ID NO: 130 and the light chain variable region set forth in SEQ ID NO: 133.

[0029] 5. The cysteine ​​engineered antibody according to item 1, wherein the light chain is of λ or κ type;

[0030] Optionally, the antibody is 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;

[0031] Optionally, the antibody is a single chain antibody, an IgG antibody, or a bispecific antibody such as a bispecific antibody in the form of BiTE / DART / Diabody.

[0032] 6. The cysteine ​​engineered antibody according to item 5, characterized in that, when the antibody is a single-chain antibody, the antibody further comprises a connecting chain, and preferably, the sequence of the connecting chain is SEQ ID NO.6.

[0033] 7. A conjugate comprising the cysteine ​​engineered antibody according to any one of items 1 to 6, and a conjugate.

[0034] 8. The conjugate of item 7, wherein the conjugate 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 (e.g., a topoisomerase inhibitor, tubulysin A, DM1, PBD, MMAE or MMAF, etc.), a chemotherapeutic agent or a radiotherapeutic agent;

[0035] The conjugate is conjugated to the cysteine ​​engineered antibody via a linker;

[0036] Preferably, the linker contains an electrophilic group (preferably a maleimide group or a haloacetamide group); optionally, the linker is mc-VC-PAB.

[0037] 9. The conjugate according to item 8, wherein the polyethylene glycol is maleimide monomethoxy polyethylene glycol, preferably mPEG2000-Mal, mPEG5000-Mal, or mPEG10000-Mal.

[0038] 10. A pharmaceutical composition, characterized by 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.

[0039] 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 drug combination according to item 10 in the preparation of a drug or kit for treating cancer (e.g., HER2-positive cancer, preferably breast cancer).

[0040] 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.

[0041] Technical effects of the present invention

[0042] 1. By utilizing cysteine ​​mutation, an active group that can be conjugated with drugs is obtained, which has a wide range of applications and is easy to conjugate.

[0043] 2. Compared with non-site-directed mutagenesis, site-directed mutagenesis can improve the uniformity of antibody-drug conjugates.

[0044] 3. The selected cysteine ​​mutation sites can maintain the activity of some sulfhydryl groups after the expression of the antibody or antibody fragment, so that it can be used for coupling reactions without treatment.

[0045] 4. The site-directed cysteine ​​mutations in this article do not change the activity of the antibody. After drug conjugation, they can improve drug efficacy and reduce toxic side effects.

[0046] 5. The mutation sites in the present invention are applicable to most antibodies and have a certain degree of versatility.

[0047] 6. The present invention can utilize site mutations of different antibodies to construct bispecific antibody-drug conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 shows the SDS-PAGE electrophoresis of the purified wild-type anti-HER2 trastuzumab single-chain antibody (WT) and mutant single-chain antibodies (HC: 12C, 35C, 61C, 34C, 38C, 44C, 47C, 51C, 60C, 67C, 68C, 78C, 79C and 114C; LC: 21C, 47C, 48C, 71C, 75C, 19C, 46C, 62C, 78C and 87C). It can be seen from the electrophoresis that the antibody bands are single and the purity meets the requirements of subsequent experiments.

[0049] Figure 2 shows an SDS-PAGE electrophoresis of the anti-HER2 trastuzumab single-chain antibody light chain variable region mutant LC46C and heavy chain variable region mutant HC12C after coupling with mPEG2000-MAL. This figure demonstrates that the light chain variable region mutant LC46C and the heavy chain variable region mutant HC12C can be successfully coupled to the conjugate mPEG2000-MAL.

[0050] Figure 3 shows an SDS-PAGE electrophoresis of the anti-HER2 trastuzumab single-chain antibody light chain variable region mutant LC62C after coupling with mPEG2000-MAL. As can be seen from the figure, the light chain variable region mutant LC62C can be successfully coupled with the conjugate mPEG2000-MAL.

[0051] Figure 4 shows an SDS-PAGE electrophoresis of the anti-HER2 trastuzumab single-chain antibody light chain variable region mutant LC87C and heavy chain variable region mutants HC38C, HC67C, and HC35C after coupling with mPEG2000-MAL. This figure demonstrates that the light chain variable region mutant LC87C and the heavy chain variable region mutants HC38C, HC67C, and HC35C can be successfully coupled to the conjugate mPEG2000-MAL.

[0052] Figure 5 shows an SDS-PAGE electrophoresis of the heavy chain variable region mutant HC78C of the anti-HER2 trastuzumab single-chain antibody after coupling with mPEG2000-MAL. As can be seen from the figure, the heavy chain variable region mutant HC78C can be successfully coupled with the conjugate mPEG2000-MAL.

[0053] Figure 6 shows an SDS-PAGE electrophoresis of the heavy chain variable region mutant HC61C of the anti-HER2 trastuzumab single-chain antibody after coupling with mPEG2000-MAL. As can be seen from the figure, the heavy chain variable region mutant HC61C can be successfully coupled with the conjugate mPEG2000-MAL.

[0054] Figure 7 shows an SDS-PAGE electrophoresis of the anti-HER2 trastuzumab single-chain antibody heavy chain variable region mutants HC69C and HC51C, and the light chain variable region mutant LC71C, after coupling with mPEG2000-MAL. This figure demonstrates that the heavy chain variable region mutants HC69C and HC51C, as well as the light chain variable region mutant LC71C, can be successfully coupled to the conjugate mPEG2000-MAL.

[0055] Figure 8 shows an SDS-PAGE electrophoresis of the anti-HER2 trastuzumab single-chain antibody light chain variable region mutant LC48C after coupling with mPEG2000-MAL. As can be seen from the figure, the light chain variable region mutant LC48C can be successfully coupled with the conjugate mPEG2000-MAL.

[0056] FIG9 shows the deconvolution results of the molecular weight of the mutant LC87C in the light chain variable region of the anti-HER2 trastuzumab single-chain antibody detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant 87C is 26521.3 Da.

[0057] Figure 10 shows the mass spectrometry detection of the molecular weight deconvolution results of the mutant LC87C of the light chain variable region of the anti-HER2 trastuzumab single-chain antibody coupled with vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 27838.2 Da.

[0058] FIG11 shows the deconvolution results of the molecular weight of the double-site cysteine ​​mutant of the anti-HER2 trastuzumab single-chain antibody detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 26553.5 Da.

[0059] Figure 12 shows the mass spectrometry detection results of molecular weight deconvolution of the dual-site cysteine ​​mutants of the anti-HER2 trastuzumab single-chain antibody coupled with vcMMAE. It can be seen from the figure that the molecular weights of the mutants after coupling are mainly 27988.0Da and 29186.3Da.

[0060] Figure 13 shows the molecular weight deconvolution results of the double-site mutation bispecific antibody detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 52588.5 Da.

[0061] Figure 14 shows the mass spectrometry detection of the molecular weight deconvolution results of the double-site mutant bispecific antibody coupled to vcMMAE. It can be seen from the figure that the molecular weights of the mutants after coupling are mainly 53903.7Da and 55220.6Da.

[0062] Figure 15 shows an SDS-PAGE electrophoresis of the anti-HER2 Pertuzumab single-chain antibody light chain variable region mutants LC46C and LC87C after coupling with mPEG2000-MAL. As can be seen from the figure, the light chain variable region mutants LC46C and LC87C can be successfully coupled to the conjugate mPEG2000-MAL.

[0063] Figure 16 shows an SDS-PAGE electrophoresis of the light chain variable region mutant LC46C of the anti-CD20 rituximab single-chain antibody and the light chain variable region mutant LC46C of the anti-CD3 muromona single-chain antibody after coupling with mPEG2000-MAL. As can be seen from this figure, both the light chain variable region mutant LC46C of the rituximab single-chain antibody and the muromona single-chain antibody were successfully coupled to the conjugate mPEG2000-MAL.

[0064] Figure 17 shows an SDS-PAGE electrophoresis of the light chain variable region mutant LC87C of the anti-VEGFR-2 ramucirumab single-chain antibody after coupling with mPEG2000-MAL. As can be seen from the figure, the light chain variable region mutant LC87C of the ramucirumab single-chain antibody can be successfully coupled with the conjugate mPEG2000.

[0065] Figure 18 shows an SDS-PAGE electrophoresis of trastuzumab light chain variable region mutants 46C and 87C after coupling with mPEG2000-MAL. As can be seen from the figure, trastuzumab light chain variable region mutants 46C and 87C can be successfully coupled with the conjugate mPEG2000-MAL.

[0066] Figure 19 shows an SDS-PAGE electrophoresis of the anti-EGFR & c-MET bispecific antibody light chain 87 double-site mutant BiTE-E / M-87-87 after coupling with mPEG2000-MAL. As can be seen from the figure, BiTE-E / M-87-87 was successfully coupled to the conjugate mPEG2000-MAL.

[0067] Figure 20 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Bi-TP-1-WT-87 detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 52588.4 Da.

[0068] Figure 21 shows the mass spectrometry detection results of molecular weight deconvolution of the anti-HER2 bispecific antibody Bi-TP-1-WT-87 cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 53904.2 Da.

[0069] Figure 22 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Bi-TP-2-WT-87 detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 52002.6 Da.

[0070] Figure 23 shows the mass spectrometry detection results of molecular weight deconvolution of the anti-HER2 bispecific antibody Bi-TP-2-WT-87 cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 54319.4 Da.

[0071] Figure 24 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Bi-TP-3-WT-87 detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53316.1 Da.

[0072] Figure 25 shows the mass spectrometry detection results of molecular weight deconvolution of the anti-HER2 bispecific antibody Bi-TP-3-WT-87 cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 54632.8 Da.

[0073] Figure 26 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Bi-TP-4-WT-87 detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53633.6 Da.

[0074] Figure 27 shows the mass spectrometry detection results of the molecular weight deconvolution of the anti-HER2 bispecific antibody Bi-TP-4-WT-87 cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 54949.5 Da.

[0075] Figure 28 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Tan-LH-TP-4-WT-87 cysteine ​​mutant detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53533.3 Da.

[0076] Figure 29 shows the mass spectrometry detection results of the molecular weight deconvolution of the anti-HER2 bispecific antibody Tan-LH-TP-4-WT-87 cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 54851.2 Da.

[0077] Figure 30 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Tan-HL-TP-4-WT-87 detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53533.3 Da.

[0078] Figure 31 shows the mass spectrometry detection results of the molecular weight deconvolution of the anti-HER2 bispecific antibody Tan-LH-TP-4-WT-87 cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 54851.4 Da.

[0079] Figure 32 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Tan-LH-PT-4-87-WT detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53532.4 Da.

[0080] Figure 33 shows the mass spectrometry detection results of the molecular weight deconvolution of the anti-HER2 bispecific antibody Tan-LH-PT-4-87-WT cysteine ​​mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 54849.8 Da.

[0081] Figure 34 shows the deconvolution results of the molecular weight of the cysteine ​​mutant of the anti-HER2 bispecific antibody Bi-TP-4-WT-87 with mutation at position 87 detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53534.3 Da.

[0082] Figure 35 shows the mass spectrometry detection results of molecular weight deconvolution of the cysteine ​​mutant of Pertuzumab 87-mutated anti-HER2 bispecific antibody Bi-TP-4-WT-87 coupled with MC-2MMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 56587.6 Da.

[0083] FIG36 shows the deconvolution results of the molecular weight of the anti-c-MET single-chain antibody cysteine ​​mutant c-MET-46C detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 26001.1 Da.

[0084] Figure 37 shows the mass spectrometry detection results of the molecular weight deconvolution of the anti-c-MET single-chain antibody cysteine ​​mutant c-MET-46C coupled to VcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 27318.1 Da.

[0085] Figure 38 shows the deconvolution results of the molecular weight of Zalu-46C, a cysteine ​​mutant of the Zalu single-chain antibody, detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 27179.4 Da.

[0086] Figure 39 shows the mass spectrometry detection of the molecular weight deconvolution results of the anti-Zalumu single-chain antibody cysteine ​​mutant Zalu-46C coupled to VcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is 28496.4 Da. DETAILED DESCRIPTION

[0087] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0088] The restriction endonucleases used in the examples were purchased from Thermo Fisher Scientific (China) Co., Ltd. The reagents or materials used in the following examples, unless their sources are explicitly mentioned, are all commercially available in the art.

[0089] definition

[0090] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a connecting polypeptide between the VH and VL domains that enables the sFv to form an ideal structure for antigen binding.

[0091] The polypeptide connecting chain has various forms, which are divided into flexible connecting chains, rigid connecting chains and cleavable connecting chains. The most commonly used flexible connecting chain is (GGGGS)n, and other common flexible connecting chains also include KESGSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8 composed of pure glycine, or slightly shorter (Gly)6. Rigid connecting chains include (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). The cleavable connecting chain sequence is LEAGCKNFFPR↓SFTSCGSLE, etc. In this article, the connecting chain used is the most commonly used flexible connecting chain, and its sequence is GGGGSGGGGSGGGGS.

[0092] In the cysteine ​​engineered antibodies of the present invention, the selected mutation sites are all located in the framework region of the antibody variable region, and therefore can be applied to cysteine ​​mutation of the aforementioned 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, or anti-c-Met antibodies.

[0093] 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 encoding gene is located on human chromosome 17q21. With a molecular weight of 185 kDa, it is a tyrosine kinase receptor membrane glycoprotein. HER2 forms heterodimers with HER1, HER3, or HER4, leading to activation of downstream signaling pathways such as MAPK and PI3K. This over-transmission of growth signals leads to malignant cell proliferation. HER2 proto-oncogene amplification or protein overexpression has been found in a variety of human tumors, including 25-30% of breast cancer, 35-45% of pancreatic cancer, 90% of colorectal cancer, 16-57% of non-small cell lung cancer, and 9-38% of gastric cancer. Clinically, these tumors are termed HER2-positive and are characterized by high malignancy, high propensity for progression and metastasis, insensitivity to chemotherapy and radiotherapy, high recurrence rates, and short patient survival.

[0094] In 1975, Kohler and Milstein invented hybridoma technology for producing monoclonal antibodies (mAbs), which led to the rapid development and clinical application of mAb drugs. In recent years, antibody drugs targeting HER2 have become a new hot topic in the treatment of HER2-positive tumors. Trastuzumab (Herceptin) is a humanized anti-HER2 monoclonal antibody developed by Genetech and approved for marketing by the US FDA in 1998. Currently, trastuzumab combined with chemotherapy drugs (such as paclitaxel) has become a first-line treatment for HER2-overexpressing advanced metastatic breast cancer and advanced gastric cancer. In the treatment of HER2-positive metastatic breast cancer, the clinical efficacy can reach 38%. Trastuzumab combined with chemotherapy drugs has been approved by the European Union (EMEA) as a first-line treatment for HER2-positive advanced gastric cancer.

[0095] As used herein, the term "linker" means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a conjugate (e.g., a drug moiety). In various embodiments, a linker is designated as L." Linker" (L) is a bifunctional moiety or a multifunctional moiety that can be used to connect one or more drug moieties (D) and an antibody unit (Ab) to form an antibody-drug conjugate (ADC). Antibody-drug conjugates (ADC) can be easily prepared using a linker with a reactive functional group for combining the drug and the antibody. The cysteine ​​thiol of cysteine-engineered antibodies (CYSMAB) can form a key with the electrophilic functional group of a linker reagent, a drug moiety, or a drug-linker intermediate.

[0096] On the one hand, the linker has a reactive site having an electrophilic group that reacts with the nucleophilic cysteine ​​present on the antibody. The cysteine ​​thiol of the antibody reacts with the electrophilic group on the linker and forms 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 categories: non-cleavable and cleavable. Non-cleavable linkers are mainly thioether linkers; cleavable linkers can be divided into acid-cleavable, reducible and enzyme-cleavable types, among which the most commonly used cleavable linkers include Val-Cit dipeptide and the like. In this article, mc-VC-PAB was selected as the linker, which is also a commonly used linker in ADC and can be purchased from MedChemExpress.

[0097] Herein, the drug moiety D used in ADC can be a topoisomerase inhibitor (eg, etoposide, teniposide, doxorubicin, etc.), tubulysin A, DM1, MMAE, etc.

[0098] Tubulysin A (TubA) is a product of mucus-producing bacteria that has anti-angiogenic, anti-mitotic, and anti-proliferative effects in vitro. Its structure is shown in the figure below:

[0099] DM1 is a derivative of the anti-microtubule drug maytansine (also translated as maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, maytansine, DM1 is a tubulin inhibitor that binds to the ends of microtubules and inhibits their dynamics. It is an antibody-conjugated maytansine alkaloid that can be used to overcome the systemic toxicity associated with maytansine and enhance tumor-specific delivery. The structure is shown in the figure below:

[0100] Among them, MMAE is a synthetic derivative of Dolastatin 10, which effectively inhibits mitosis by inhibiting tubulin polymerization. The structure is shown in the figure below:

[0101] Among them, PBD, also known as pyrrolobenzodiazepine (PBD), can bind to the minor groove of DNA to form effective cytotoxic DNA interchain cross-links, thereby blocking cell division and killing cancer cells.

[0102] Among them, MMAF (Monomethylauristatin F) is a potent tubulin polymerization inhibitor used as an anti-tumor drug and is commercially available.

[0103] In this article, the term "active peptide" is also called bioactive peptide, which refers to a peptide compound that is beneficial to the life activities of biological organisms or has physiological effects.

[0104] "Subject," "patient," "individual" and similar terms are used interchangeably and, unless otherwise indicated, refer to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species. The terms do not necessarily indicate that a subject has been diagnosed with a specific disease. The term "patient" refers to a subject under medical supervision. A patient can be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, etc. A "cancer patient" or "AML patient" can refer to an individual who has been diagnosed with cancer, is currently receiving a therapeutic regimen, or is at risk of recurrence, such as after surgery to remove a tumor. In some embodiments, a cancer patient has been diagnosed with cancer and is a candidate for therapy. Cancer patients can include individuals who have not received treatment, are currently receiving treatment, have undergone surgery, and those who have stopped treatment.

[0105] In the context of treating cancer, a subject in need of treatment can refer to an individual who has cancer or a precancerous condition, has had cancer and is at risk of recurrence, is suspected of having cancer, is undergoing standard cancer treatment such as radiation therapy or chemotherapy, etc.

[0106] "Cancer," "tumor," and similar terms include precancerous, neoplastic, and cancerous cells and can refer to solid tumors or non-solid cancers. Cancer includes both benign and malignant neoplasms (abnormal growths).

[0107] The term "cancer" may refer to leukemia, carcinoma, sarcoma, adenocarcinoma, lymphoma, solid tumors, lymphoma, etc. Examples of different types of cancer include, but are not limited to, 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 (such as non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer, colorectal cancer, liver cancer (liver cancer), and ovarian cancer. 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, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, appendix 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, and melanoma.

[0108] Example 1 Screening of mutation sites of anti-HER2 trastuzumab single-chain antibody

[0109] Based on the 4X4X model in the protein database (PDB), the four parameters of the antibody, namely, residue solvent accessibility, sidechain solvent accessibility, percent solvent accessibility, and percent sidechain solvent accessibility, were predicted using the Discovery Studio software. Considering that the reaction between nutrients and mutated cysteines during cell culture may affect subsequent conjugation, overly exposed sites should not be selected. After comprehensive consideration, the inventors ultimately selected non-CDR regions where all four parameters were ≤20 for mutation screening, namely, positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, and 114 in the heavy chain variable region; and positions 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, and 87 in the light chain variable region.

[0110] Example 2. Screening and expression of anti-HER2 trastuzumab single-chain antibody cysteine ​​mutant cell lines

[0111] (1) Construction of anti-HER2 trastuzumab single-chain antibody and mutant expression vectors

[0112] DNA encoding the antibodies described herein (the corresponding wild-type sequences are from the USP Medicines Compendium) was used, wherein the specific heavy chain variable region sequences, light chain variable region sequences, and connecting chain nucleotide sequences are shown in SEQ ID NOs: 1-3, and the amino acid sequences are shown in SEQ ID NOs: 4-6; on this basis, according to Kabat numbering, the 12th amino acid of the heavy chain was substituted with cysteine ​​to obtain the variant HC12C of the present invention, the 35th amino acid of the heavy chain was substituted with cysteine ​​to obtain the variant HC35C of the present invention, the 61st amino acid of the heavy chain was substituted with cysteine ​​to obtain the variant HC61C of the present invention, the 21st amino acid of the light chain was substituted with cysteine ​​to obtain the variant LC21C of the present invention, the 47th amino acid of the light chain was substituted with cysteine ​​to obtain the variant LC47C of the present invention, and the 48th amino acid of the light chain was substituted with cysteine ​​to obtain the variant LC47C of the present invention. 8C, the 71st amino acid of the light chain was replaced by cysteine ​​to obtain the variant LC71C of the present invention, the 75th amino acid of the light chain was replaced by cysteine ​​to obtain the variant LC75C of the present invention, the 34th amino acid of the heavy chain was replaced by cysteine ​​to obtain the variant HC34C of the present invention, the 38th amino acid of the heavy chain was replaced by cysteine ​​to obtain the variant HC38C of the present invention, the 44th amino acid of the heavy chain was replaced by cysteine ​​to obtain the variant HC44C of the present invention, the 47th amino acid of the heavy chain was replaced by cysteine ​​to obtain the variant HC47C of the present invention, the 51st amino acid of the heavy chain was replaced by cysteine ​​to obtain The variant HC51C of the present invention is obtained by replacing the amino acid at position 60 of the heavy chain with cysteine ​​to obtain the variant HC60C of the present invention, replacing the amino acid at position 61 of the heavy chain with cysteine ​​to obtain the variant HC61C of the present invention, replacing the amino acid at position 67 of the heavy chain with cysteine ​​to obtain the variant HC67C of the present invention, replacing the amino acid at position 69 of the heavy chain with cysteine ​​to obtain the variant HC69C of the present invention, replacing the amino acid at position 78 of the heavy chain with cysteine ​​to obtain the variant HC78C of the present invention, replacing the amino acid at position 79 of the heavy chain with cysteine ​​to obtain the variant HC79C of the present invention, replacing the amino acid at position 114 ...9C of the present invention, The amino acid at position 1 of the light chain was replaced with cysteine ​​to obtain the variant HC114C of the present invention, the amino acid at position 19 of the light chain was replaced with cysteine ​​to obtain the variant LC19C of the present invention, the amino acid at position 44 of the light chain was replaced with cysteine ​​to obtain the variant LC44C of the present invention, the amino acid at position 46 of the light chain was replaced 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 DNAs were chemically synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.).

[0113] The single-chain antibody gene was then double-digested with HindIII and XhoI, and ligated with T4 ligase into the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated with HindIII and XhoI. After successful ligation, the expression vector for the anti-HER2 single-chain antibody and its mutants was successfully constructed.

[0114] (2) Screening and purification of anti-HER2 trastuzumab single-chain antibody and mutant expression vectors

[0115] The successfully constructed expression vector was transformed into a DH5α competent E. coli strain (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.). The transformed DH5α strain was double-digested with HindIII and XhoI, and sequenced for verification, and positive clones were selected. The resulting E. coli was lysed and the plasmid was extracted using a plasmid extraction kit (purchased from Kangwei Century) to obtain a purified expression vector.

[0116] (3) Expression of anti-HER2 trastuzumab single-chain antibody and mutants in CHO cells

[0117] The purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, plated into 96-well plates, grown for 15 days, and single clones were picked. The cell antibody production was determined by Western blotting. The top 20% were selected and transferred to 24-well plates. After 7 days of growth, the cell antibody production was detected, and the top 5 to 6 strains were selected and transferred to shake flask culture. After the serum-free culture stabilized, the culture was harvested after 7 days of continuous culture to achieve the expression of anti-HER2 single-chain antibodies and their mutants in CHO-K1 cells. The antibodies were purified and subsequently subjected to subsequent experiments.

[0118] Example 3. Ni affinity chromatography column purification

[0119] Transfer the cells and culture medium into a 50mL centrifuge tube and centrifuge at 8000r / min in a high-speed refrigerated centrifuge for 15 minutes. Retain the supernatant and discard the cell debris. Dilute the culture medium with 3 volumes of equilibration buffer and filter through a 0.22μm microporous filter. Collect the filtrate for later use. Rinse the pump, injector, and purification column with 10 column volumes of ultrapure water and PBS before loading the sample. Elute with elution buffer containing 250mM imidazole and transfer the purified antibody protein solution to an EP tube and store at 2-8°C. The SDS-PAGE purity test results are shown in Figure 1. The electrophoresis shows a single antibody band, indicating that the purity meets the requirements of subsequent experiments.

[0120] Example 4. Detection of free thiol ratio of antibodies

[0121] The present invention uses Ellman's method (Thermo Fisher Scientific (China) Co., Ltd.) to detect the free thiol content of the antibody product, thereby selecting a cell line suitable for further coupling. 0.1M Na2HPO4 solution, DTNB (10mM) solution, and cysteine ​​standard solution are configured to a final concentration of 0.0059M to 0.375M. At the same time, a 5mg / mL concentration of the antibody sample to be tested is prepared. 250μL 0.1M Na2HPO4 and 5μL DTNB solution are added to 1.5mL EP tubes containing cysteine ​​standard solution and the sample to be tested, respectively. After vortexing and mixing, the tubes are incubated at room temperature in the dark for 5min. The OD value is detected at a wavelength of 412nm. A standard curve is established based on the OD value, and the thiol content of the sample is calculated.

[0122] Table 1. Detection of free sulfhydryl groups in antibody products using Ellman's method

[0123] As shown in Table 1, the wild type contains almost no free thiol groups, while the mutants modified by cysteine ​​engineering all have free thiol groups that can be used for coupling.

[0124] Example 5. Antibody and antigen affinity determination

[0125] The affinity of wild-type antibody and cysteine-engineered antibody mutants to the antigen HER-FC was determined by biofilm interferometry (BLI) using a Pro A probe (ForteBio).

[0126] HER-FC (Beijing Sino Biological Technology Co., Ltd.) was dissolved in PBS at a working concentration of 10 μg / mL. 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 for each dilution was 200 μL.

[0127] The OCTET system and data processing software were used to fit the data graph. The software calculated the intermolecular interaction force between the antibody and HER2, which was expressed as the KD value. The results are shown in Table 2.

[0128] Table 2. Affinity of wild-type antibodies and mutants for antigens

[0129] From the data in the table, it can be seen that there is almost no difference in the affinity of wild-type antibodies and mutants for antigens, and the selected mutation sites do not change the effect of the antibody.

[0130] Example 6 Antibody-antigen binding assay

[0131] The binding affinity of wild-type antibodies and cysteine ​​engineered antibody mutants to the antigen HER-Fc (Beijing Sino Biological Technology Co., Ltd.) was determined by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 3.

[0132] Table 3. Binding ability of wild-type antibodies and mutants to antigens

[0133] From the data in the table, it can be seen that there is almost no difference in the binding ability of wild-type antibodies and mutants to antigens, so the selected mutation sites do not change the effect of the antibody.

[0134] Example 7 Screening of Anti-HER2 Trastuzumab Single-chain Antibody Dual-site Cysteine ​​Mutation Cell Lines

[0135] Based on Examples 1-6, the present invention also performed double-site cysteine ​​mutations on the anti-HER2 trastuzumab single-chain antibody as shown in the following table.

[0136] Table 4. Antibody double-site mutations and their nomenclature

[0137] (1) Construction of anti-HER2 trastuzumab single-chain antibody and double-site mutant expression vector

[0138] DNA encoding the antibody described herein (the corresponding wild-type sequence is from USP Medicines Compendium) is used, wherein the specific heavy chain variable region sequence, light chain variable region sequence, and connecting chain nucleotide sequence are shown in SEQ ID NO: 1-3, and the amino acid sequence is shown in SEQ ID NO: NO: 4-6; on this basis, according to Kabat numbering, the amino acid at position 12 of the heavy chain and the amino acid at position 35 of the heavy chain were replaced with cysteine ​​to obtain the variant 12C-35C of the present invention, the amino acid at position 12 of the heavy chain and the amino acid at position 61 of the heavy chain were replaced with cysteine ​​to obtain the variant 12C-61C of the present invention, the amino acid at position 35 of the heavy chain and the amino acid at position 61 of the heavy chain were replaced with cysteine ​​to obtain the variant 35C-61C of the present invention, the amino acid at position 51 of the heavy chain and the amino acid at position 61 of the heavy chain were replaced with cysteine ​​to obtain the 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 were replaced with cysteine ​​to obtain the 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 were replaced with cysteine ​​to obtain The variant 61C-46C of the present invention is obtained by replacing the 61st amino acid of the heavy chain and the 87th amino acid of the light chain with cysteine ​​to obtain the variant 61C-87C of the present invention, replacing the 21st amino acid of the light chain and the 47th amino acid of the light chain with cysteine ​​to obtain the variant 21C-47C of the present invention, replacing the 21st amino acid of the light chain and the 48th amino acid of the light chain with cysteine ​​to obtain the variant 21C-48C of the present invention, replacing the 21st amino acid of the light chain and the 75th amino acid of the light chain with cysteine ​​to obtain the variant 21C-75C of the present invention, and replacing the 46th amino acid of the light chain and the 87th amino acid of the light chain with cysteine ​​to obtain the variant 46C-87C of the present invention. The DNA of the above-mentioned antibody was chemically synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the sequence table is shown below.

[0139] The single-chain antibody gene was then double-digested with HindIII and XhoI, and ligated with T4 ligase into the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated with HindIII and XhoI. After successful ligation, the expression vector for the anti-HER2 single-chain antibody and its double-site mutant was successfully constructed.

[0140] (2) Screening and purification of anti-HER2 trastuzumab single-chain antibody and double-site mutant expression vectors

[0141] The successfully constructed expression vector was transformed into a DH5α competent E. coli strain. The transformed DH5α strain was double-digested with HindIII and XhoI, and sequenced for verification, with positive clones selected. The resulting E. coli was lysed and the plasmid extracted using a plasmid extraction kit (purchased from Kangwei Century) to obtain a purified expression vector.

[0142] (3) Expression of anti-HER2 trastuzumab single-chain antibody and double-site mutant in CHO cells

[0143] The purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, plated into 96-well plates, grown for 15 days, and single clones were picked. The cell antibody production was determined by Western blotting. The top 20% were selected and transferred to 24-well plates. After 7 days of growth, the cell antibody production was detected, and the top 5 to 6 strains were selected and transferred to shake flask culture. After the serum-free culture stabilized, the culture was harvested after 7 days of continuous culture to achieve the expression of the anti-HER2 single-chain antibody in CHO-K1 cells. The antibody was purified and subsequently subjected to subsequent experiments.

[0144] The double-site mutant of the anti-HER2 trastuzumab single-chain antibody also has similar technical effects as the single-site mutant described in Examples 1-6. For its coupling effect, please refer to the experimental results of Example 11 "Antibody Coupling with vcMMAE and Detection" and Example 13 "Cell Proliferation Inhibition Effect Detection".

[0145] Example 8 Screening of other single-chain antibody cysteine ​​mutant cell lines

[0146] Examples 1-7 use the anti-HER2 trastuzumab single-chain antibody as an example to verify the various mutation sites. The present invention also performs mutations on the corresponding sites of other antibodies, verifying that the above mutation sites are universal on various antibodies. Specifically, the present invention performs cysteine ​​mutations on amino acids at positions 46 and 87 of the light chain of the Pertuzumab single-chain antibody, and names the mutants Per-46C and Per-87C, respectively; performs cysteine ​​mutations on amino acids at positions 46 of the light chain of the Rituximab single-chain antibody, and names the mutants Rit-46C; performs cysteine ​​mutations on amino acids at positions 46 of the light chain of the Muromonab single-chain antibody, and names the mutants Mur-46C; performs cysteine ​​mutations on amino acids at positions 87 of the light chain of the Ramucirumab single-chain antibody, and names the mutants Ram-87C. The sequence table is shown below.

[0147] Mutants at the corresponding sites in pertuzumab single-chain antibody, rituximab single-chain antibody, muromona single-chain antibody, and ramucirug single-chain antibody also exhibited similar effects to the anti-HER2 trastuzumab single-chain antibody. For details on their coupling effects, see the experimental results in "Example 12: Conjugation of Other Antibody Single-chain Antibodies with mPEG2000-MAL and Detection." Therefore, the mutation sites screened by the present invention are universally applicable to various antibodies.

[0148] Example 9 Screening of Anti-HER2 Single-Chain Bispecific Antibody Dual-Site Cysteine ​​Mutation Cell Lines

[0149] The present invention constructs a bispecific antibody in the form of a BiTE based on an anti-HER2 trastuzumab single-chain antibody and an anti-HER2 pertuzumab single-chain antibody, named BiTE-WT. Furthermore, cysteine ​​mutations are performed at positions 46 or 87 of the light chains of the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody, respectively, to construct BiTE-type cysteine-mutated bispecific antibodies. The BiTE-type cysteine-mutated bispecific antibodies are named BiTE-46-46, BiTE-46-87, BiTE-87-46, and BiTE-87-87, respectively, with the trastuzumab VL mutation site first and the pertuzumab VL mutation site second.

[0150] The connection form of BiTE is Herceptin VL-GGGGSGGGGSGGGGS-Herceptin VH-GGGGS-Perjeta VH-GGGGSGGGGSGGGGS-Perjeta VL, and the sequence is shown in the following table.

[0151] The anti-HER2 single-chain bispecific antibody double-site mutant also has similar technical effects as the monospecific antibody single-site mutant described in Examples 1-6. For its coupling effect, please refer to the experimental results of Example 11 "Antibody Coupling with vcMMAE and Detection" and Example 13 "Cell Proliferation Inhibition Effect Detection".

[0152] Example 10. Anti-HER2 trastuzumab single-chain antibody coupled with mPEG2000-MAL and detection

[0153] This example uses the purified single-site mutant of the anti-HER2 trastuzumab single-chain antibody obtained in Example 3 as an example, and uses mPEG2000-MAL as a conjugate for experiments. The conjugation reaction conditions for the antibody and the conjugate are antibody: conjugate (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature of 37°C, reaction time of 3 hours, and reaction buffer of PBS (pH 7.2). The SDS-PAGE electrophoresis patterns of the wild-type or mutant single-chain antibody conjugated to mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) are shown in Figures 2 to 8, respectively.

[0154] This study used SDS-PAGE to examine the conjugation of mutant single-chain antibodies to mPEG2000-MAL. The upward shift of the conjugated protein bands indicates an increase in molecular weight, demonstrating that all mutants were successfully conjugated to mPEG2000-MAL. The conjugation results indicate that the mutant single-chain antibodies described herein possess reactive sulfhydryl groups, which can react with drug linkers containing sulfhydryl-reactive functional groups (e.g., maleimide functional groups). Consequently, it is reasonable to anticipate that these mutants can also be used to link antibodies to drug molecules.

[0155] Example 11. Antibody Conjugation to vcMMAE and Detection

[0156] This example uses one sample each of the purified single-site mutant (LC87) of the anti-HER2 trastuzumab single-chain antibody obtained in Example 3, the purified double-site mutant (HC61C-LC87C) of the anti-HER2 trastuzumab single-chain antibody obtained in Example 7, and the purified double-site mutant bispecific antibody (BiTE-87-87) obtained in Example 9 as examples, and uses the drug molecule vcMMAE (purchased from Nanjing Baixindenuo Biotechnology Co., Ltd., product identification number A14362) as a conjugate for experiments. The conjugation reaction conditions between the antibody and the conjugate are antibody:conjugate (vcMMAE) = 1:5 (molar ratio), reaction temperature is 37°C, reaction time is 3 hours, and the reaction buffer is PBS (pH 7.2). Typical mass spectra of the single-chain antibody before and after coupling with vcMMAE are shown in Figures 9 and 10, where Figure 9 shows the molecular weight result of the single-chain antibody before coupling, and the molecular weight of the single-chain antibody is 26521.3 Da, and Figure 10 shows the molecular weight result of the antibody after coupling with vcMMAE, and the molecular weight of the antibody after coupling with vcMMAE is 27838.2 Da. The calculated difference is 1316.9 Da, which is the molecular weight of vcMMAE, proving that each mutant single-chain antibody is successfully coupled with one molecule of vcMMAE. Typical mass spectra of the double-site mutants before and after coupling are shown in Figures 11 and 12, where Figure 11 shows the molecular weight of the single-chain antibody before coupling, with a molecular weight of 26553.5 Da, and Figure 12 shows the molecular weight of the antibody after coupling with vcMMAE, with molecular weights of 27988.0 Da and 29186.3 Da after coupling with vcMMAE. The calculated differences are the molecular weights of 1 and 2 vcMMAE molecules, respectively. Calculations show that each double-mutant single-chain antibody was successfully coupled with more than 1.7 vcMMAE molecules. Typical mass spectra of the double-site mutation bispecific antibody before and after conjugation are shown in Figures 13 and 14 . Figure 13 shows the molecular weight result of the double-site mutation bispecific antibody before conjugation, with the single-chain antibody molecular weight being 52588.5 Da. Figure 14 shows the molecular weight result of the antibody after conjugation with vcMMAE, with the molecular weights of the antibody after conjugation with vcMMAE being 53903.7 Da and 55220.6 Da. The calculated differences are the molecular weights of 1 and 2 vcMMAE molecules, respectively. Calculations indicate that each double-site mutation bispecific antibody was successfully conjugated with more than 1.5 vcMMAE molecules.

[0157] Example 12. Other Antibody Single Chain Antibodies Coupling mPEG2000-MAL and Detection

[0158] This example uses the mutants of the pertuzumab single-chain antibody, rituximab single-chain antibody, muromona single-chain antibody, and ramucirumab single-chain antibody obtained in Example 8 as examples, and uses mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) as a conjugate for experiments. The light chain amino acids 46 and 87 of pertuzumab were mutated to cysteine, the light chain amino acid 46 of rituximab was mutated to cysteine, the light chain amino acid 46 of muromonab was mutated to cysteine, and the light chain amino acid 87 of ramucirumab was mutated to cysteine. The conjugation reaction conditions for the obtained antibody mutants and the conjugate were: antibody:conjugate (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature 37°C, reaction time 3 hours, and reaction buffer PBS (pH 7.2). SDS-PAGE electrophoresis patterns of the mutant single-chain antibodies conjugated with mPEG2000-MAL are shown in Figures 15-17. SDS-PAGE experiments were used to detect the coupling of mutant single-chain antibodies with mPEG2000-MAL. The protein bands after coupling shifted upward, indicating an increase in molecular weight. The results showed that the mutant single-chain antibodies of the four antibodies could be successfully coupled with mPEG2000-MAL.

[0159] Example 13 Cell proliferation inhibition assay

[0160] The present invention selects two breast cancer cell lines, SK-BR-3 and MCF-7 (purchased from Pronocell), and uses a CCK8 kit (purchased from APExBIO) as a proliferation detection reagent to determine the inhibitory effects of the drug molecule MMAE, the anti-HER2 trastuzumab single-chain antibody cysteine ​​mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C and HC61C-LC87C, the anti-HER2 bispecific antibody mutant BiTE-87-87, and the antibody-drug conjugate obtained by conjugating the above antibody mutants with vcMMAE on cell proliferation, and calculates the half inhibitory concentration (IC50). 50 SK-BR-3 is a HER2-overexpressing cell line, and MCF-7 is a HER2-low expressing cell line. The IC 50 The IC value of MMAE on MCF-7 cells was 0.20 nM. 50The IC values ​​calculated for the single-chain antibody cysteine ​​mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C, and the anti-HER2 bispecific antibody BiTE-87-87 on SK-BR-3 were 0.35 nM. 50 >1000 nm; IC calculated for the single-chain antibody cysteine ​​mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C, and the anti-HER2 bispecific antibody BiTE-87-87 on MCF-7 cells 50 IC calculated for SK-BR-3 after the antibody-drug conjugates obtained by conjugating the anti-HER2 trastuzumab single-chain antibody cysteine ​​mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, and HC61C-LC87C and the anti-HER2 bispecific antibody BiTE-87-87 to vcMMAE 50 The IC values ​​of the antibody-drug conjugates obtained by coupling the anti-HER2 trastuzumab single-chain antibody cysteine ​​mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C and the anti-HER2 bispecific antibody BiTE-87-87 to vcMMAE on MCF-7 were calculated to be 23.95nM, 15.55nM, 19.76nM, 17.28nM, 13.18nM and 1.703nM, respectively. 50 Both are greater than 50nM. Based on the above results, vcMMAE has no targeting ability and has a very strong non-specific killing ability on both cell types. For the SK-BR-3 cell line with high HER2 expression, the antibody coupled to vcMMAE can exert more powerful cytotoxicity compared with the single-chain antibody alone; while for the MCF-7 cell line, the HER2 expression level is too low, the antigen-mediated antibody binding and internalization are insufficient, and the antibody coupled to vcMMAE only produces a weak cytotoxic effect. Compared with MMAE, the cytotoxicity generated by the antibody-drug conjugate at the same molar concentration shows a HER2 receptor expression level dependency, proving that MMAE has a certain targeting ability after coupling with the single-chain antibody cysteine ​​mutant, which can reduce toxicity and expand the drug treatment window.

[0161] In summary, by performing cysteine ​​mutations at positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79 and / or 114 of 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 of the light chain variable region, free thiol groups for conjugation can be obtained, thereby successfully conjugating with the conjugate and the drug molecule to obtain an antibody-drug conjugate, thereby making the drug targeted.

[0162] Example 14 Screening of Trastuzumab Antibody Cysteine ​​Mutant Cell Lines

[0163] Examples 1-7 used the anti-HER2 trastuzumab single-chain antibody as an example to verify the various mutation sites. On this basis, the present invention further mutated the corresponding sites of its complete antibody (i.e., IgG antibody) trastuzumab, verifying that the above mutation sites are universal in different forms of the antibody. Specifically, the present invention mutated cysteine ​​at amino acids 46 and 87 of the light chain of trastuzumab, and named the mutants trastuzumab-46C and trastuzumab-87C, respectively. The sequence table is as follows:

[0164] Example 15. Trastuzumab cysteine ​​mutant antibody coupled with mPEG2000-MAL and detection

[0165] This example uses the cysteine ​​mutants at positions 46 and 87 of the light chain of the trastuzumab antibody prepared in Example 14 above as examples, and uses mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) as a conjugate for experiments. The conjugation reaction conditions for the antibody mutant and the conjugate are antibody:conjugate (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature of 37°C, reaction time of 3 hours, and reaction buffer of PBS (pH 7.2). The reduced SDS-PAGE electrophoresis of the conjugated mutant antibody and mPEG2000-MAL is shown in Figure 18. The conjugation of the mutant and mPEG2000-MAL was tested using a reduced SDS-PAGE experiment. The protein band of the light chain after conjugation shifted upward, indicating an increase in molecular weight. The results show that the mutant of the trastuzumab antibody (i.e., IgG antibody form) can be successfully conjugated to mPEG2000-MAL. The mutation site also has similar technical effects in the IgG antibody format as single-chain antibodies and bispecific antibodies.

[0166] Example 16. Screening of Cysteine-mutated Cell Lines of BiTE-Format Bispecific Antibodies Against EGFR and c-Met

[0167] Based on the verification of the coupling performance of the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody to construct a BiTE-type bispecific antibody in Example 9 and Example 11, the present invention further constructs a BiTE-type bispecific antibody based on the anti-EGFR cetuximab single-chain antibody and the anti-c-Met single-chain antibody, and is named BiTE-E / M-WT. In addition, cysteine ​​mutations are performed at positions 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 BiTE-type cysteine-mutated bispecific antibody. For the BiTE-type cysteine-mutated bispecific antibody, the anti-EGFR single-chain antibody VL mutation site is placed first and the anti-c-Met single-chain antibody VL mutation site is placed last, and they are named BiTE-E / M-46-46, BiTE-E / M-46-87, BiTE-E / M-87-46, and BiTE-E / M-87-87, respectively, and the sequences are shown in the following table.

[0168] Example 17. Cysteine ​​mutation of BiTE-type bispecific antibody against EGFR and c-Met coupled with mPEG2000-MAL and detection

[0169] This example uses the cysteine-mutated bispecific antibody against EGFR and c-Met constructed in BiTE format as an example, and uses mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) as the conjugate. The conjugation reaction conditions for the antibody mutant and the conjugate are: antibody:conjugate (mPEG2000-MAL) = 1:30 (molar ratio), reaction temperature 37°C, reaction time 3 hours, and reaction buffer PBS (pH 7.2). Using BiTE-E / M-87-87 as an example, SDS-PAGE was used to detect the conjugation of the mutant with mPEG2000-MAL. The protein band after conjugation shifted upward, indicating an increase in molecular weight. Figure 19 shows that the mutant of the bispecific antibody against EGFR and c-Met in BiTE format was successfully conjugated to mPEG2000-MAL.

[0170] Example 18 Screening of cell lines containing single-site cysteine ​​mutations of anti-HER2 single-chain bispecific antibodies with different arrangements

[0171] Furthermore, the present invention also constructs bispecific antibodies of different configurations based on the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody. The connection form and configuration name are shown in Table 5, which covers representative cases of connection with linkers of different lengths, connection of light and heavy chains in different orders, and connection of antibodies in different orders (wherein "Bi" indicates the same light and heavy chain arrangement order as in Example 9, "Tan" indicates that the light and heavy chains of the two antibodies are in the same order, "LH" / "HL" indicates that in the "Tan" format, the light chain / heavy chain comes first, and the number at the end indicates the number of G4S linker repeats connecting different antibodies).

[0172] Table 5. Summary of anti-HER2 bispecific antibody names

[0173] The coding sequences and amino acid sequences of different G4S linkers are shown in the following table:

[0174] The cysteine ​​at position 46 or 87 of the light chain of the trastuzumab single-chain antibody or the pertuzumab single-chain antibody in the bispecific antibodies in the above table was mutated to construct bispecific antibody cysteine ​​single-site mutants of different configurations. These mutants were given different names according to the selection of mutant antibodies, for example: Bi-TP-1-WT-87, which means that the arrangement order is the same, the trastuzumab corresponding to T remains wild-type, and the pertuzumab light chain position 87 corresponding to P is mutated to cysteine.

[0175] Example 19. Different Arrangements of Anti-HER2 Single-chain Bispecific Antibodies with Single-site Cysteine ​​Mutants Conjugated to Small Molecule Drug vcMMAE and Detection

[0176] This example uses the bispecific antibody with a single-site mutation at position 87 of Pertuzumab obtained in Example 18 as an example, and uses the drug molecule vcMMAE (purchased from Nanjing Baixindenuo Biotechnology Co., Ltd., product identification number A14362) as a conjugate, and 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, and Tan-LH-PT-4-87-WT for conjugation experiments. The conjugation reaction conditions for the antibody and conjugate are: antibody:conjugate (vcMMAE) = 1:5 (molar ratio), reaction temperature: 37°C, reaction time: 3 h, and reaction buffer: PBS (pH 7.2).

[0177] Figures 20-35 illustrate the conjugation of bispecific antibody single-site mutants with different arrangements. Comparing Figures 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 30 and 31, 32 and 33, and 34 and 35, reveals that the molecular weight difference before and after conjugation is approximately 1316 Da, indicating that conjugation is possible for all of the aforementioned specific antibody single-site mutants with different arrangements.

[0178] Example 20. Coupling and detection of antibodies and small molecule drugs MC-2MMAE

[0179] This example uses the anti-HER2 single-chain bispecific antibody single-site cysteine ​​mutant Bi-TP-4-WT-87 obtained in Example 18 as an example, and uses the bi-branched drug MC-2MMAE (purchased from Yantai MyBioPharma International Biopharmaceutical Co., Ltd.) as a conjugate for experiments. Among them, the coupling reaction conditions of the antibody and the conjugate are antibody: conjugate (vcMMAE) = 1:2.5 (molar ratio), reaction temperature is 4°C, reaction time is 12h, and reaction buffer is PBS (pH 7.2). Its typical spectra before and after coupling are shown in Figures 34-35. The molecular weight before coupling is 53534.3Da, as shown in Figure 34, and the molecular weight after drug coupling is 56587.6Da, as shown in Figure 35. The molecular weight difference before and after coupling is 3053.3Da, that is, by coupling one MC-2MMAE molecule, the same technical effect as in Example 11 is achieved, that is, the antibody mutant of the present invention can also be coupled with multi-branched drugs, thereby enabling the connection of more payloads through multi-branched drug linkers.

[0180] Example 21. Screening of anti-c-MET single-chain antibody single-site cysteine ​​mutation cell lines

[0181] Examples 1-8 used trastuzumab single-chain antibody, pertuzumab single-chain antibody, rituximab single-chain antibody, moromonad single-chain antibody, and ramucirumab single-chain antibody as examples to verify the mutation sites at positions 46 and 87 of the light chain. Based on Examples 1-8, the present invention also performed mutations on anti-c-MET antibodies, verifying that the above mutation sites are universal across 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 the mutant was named c-MET-46C. The sequence listing is shown below.

[0182] Example 22: Anti-c-MET single-chain antibody single-site cysteine ​​mutant coupled to vcMMAE and detection

[0183] This example uses the anti-c-MET single-chain antibody prepared in Example 21, targeting position 46, as an example, and uses the drug molecule vcMMAE (purchased from Nanjing Baixindenuo Biotechnology Co., Ltd., product identification number A14362) as a conjugate for conjugation experiments. The conjugation reaction conditions for the antibody and conjugate are: antibody:conjugate (vcMMAE) = 1:5 (molar ratio), reaction temperature 37°C, reaction time 3 hours, and reaction buffer PBS (pH 7.2).

[0184] The spectra before and after coupling are shown in Figures 36 and 37, where Figure 36 is the molecular weight result of the single-chain antibody before coupling, with a molecular weight of 26001.1 Da, and Figure 37 is the molecular weight result of the antibody after coupling with vcMMAE, with a molecular weight of 27318.1 Da. The calculated difference is the molecular weight of 1 molecule of vcMMAE, that is, each antibody mutant can be successfully coupled with 1 vcMMAE.

[0185] Example 22 Screening of Anti-EGFR Single-chain Antibody Single-site Cysteine ​​Mutation Cell Lines

[0186] Examples 1-8 and 21 verify the mutation sites at positions 46 and 87 of different single-chain antibodies. On this basis, the present invention also mutates the light chain at position 46 of the anti-EGFR Zalutumumab single-chain antibody, verifying that the above mutation site has versatility on various antibodies. Specifically, the present invention performs cysteine ​​mutation at position 46 of the light chain of the Zalutumumab single-chain antibody, and names the mutant Zalu-46C as such. The sequence table is as follows.

[0187] Example 23: Coupling of anti-EGFR single-chain antibody single-site cysteine ​​mutants with vcMMAE and detection

[0188] This example uses the single-site antibody at position 46 of the Zarumu single-chain antibody obtained in Example 22 as an example, and uses the drug molecule vcMMAE (purchased from Nanjing Baixindenuo Biotechnology Co., Ltd., product identification number A14362) as a conjugate for a conjugation experiment. The conjugation reaction conditions for the antibody and conjugate are antibody:conjugate (vcMMAE) = 1:5 (molar ratio), reaction temperature of 37°C, reaction time of 3 hours, and reaction buffer of PBS (pH 7.2).

[0189] The spectra before and after coupling are shown in Figures 38 and 39, where Figure 38 is the molecular weight result of the single-chain antibody before coupling, with a molecular weight of 27179.4 Da, and Figure 39 is the molecular weight result of the antibody after coupling with vcMMAE, with a molecular weight of 28496.4 Da. The calculated difference is the molecular weight of 1 molecule of vcMMAE, that is, each antibody mutant can be successfully coupled with 1 vcMMAE.

[0190] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0191] References

[0192] 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.

[0193] 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.

[0194] 3.Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alilithen NBM, Hamid M. ScFv antibody: principles and clinical application. Clin Dev Immunol 2012; 2012. Article ID 980250.

[0195] 4.Safdari Y,Ahmadzadeh V.Use of single-chain antibody derivatives for targeted drug delivery.Mol Med 2016;22:258-70.

[0196] 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.

[0197] 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.

[0198] 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.

Claims

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

2. The cysteine ​​engineered antibody according to claim 1, wherein According to the Kabat numbering system, it has an engineered cysteine ​​residue at any one or more positions selected from the group consisting of: Position 12, 35, 61, 51, 69, 78 of the heavy chain variable region, or any combination thereof; or Position 46, 87 or any combination thereof of the light chain variable region.

3. The cysteine ​​engineered antibody according to claim 1, wherein According to the Kabat numbering system, it has an engineered cysteine ​​residue at any one or more positions selected from the group consisting of: Position 12, 35, 61, 51, or any combination thereof, of the heavy chain variable region; or Position 46, 87 or any combination thereof of the light chain variable region.

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

133.

5. The cysteine ​​engineered antibody according to claim 1, wherein The light chain is of λ or κ type; Optionally, the antibody is 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 antibody is a single chain antibody, an IgG antibody, or a bispecific antibody such as a bispecific antibody in the form of BiTE / DART / Diabody.

6. The cysteine ​​engineered antibody according to claim 5, characterized in that When the antibody is a single-chain antibody, the antibody further comprises a connecting chain. Preferably, the sequence of the connecting chain is SEQ ID NO.

6.

7. A conjugate comprising the cysteine ​​engineered antibody according to any one of claims 1 to 6, and a conjugate.

8. The conjugate of claim 7, wherein the conjugate 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 (e.g., a topoisomerase inhibitor, tubulysin A, DM1, PBD, MMAE or MMAF, etc.), a chemotherapeutic agent or a radiotherapeutic agent; The conjugate 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); optionally, the linker is mc-VC-PAB.

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

10. A pharmaceutical composition characterized in that The invention also comprises the cysteine ​​engineered antibody according to any one of claims 1 to 6 or the conjugate according to any one of claims 7 to 9, and optionally, a pharmaceutically acceptable carrier.

11. Use of the cysteine ​​engineered antibody according to any one of claims 1 to 6, the conjugate according to any one of claims 7 to 9, or the drug combination according to claim 10 in the preparation of a medicament or kit for treating cancer (e.g., HER2-positive cancer, preferably breast cancer).

12. A kit comprising the cysteine ​​engineered antibody according to any one of claims 1 to 6, the conjugate according to any one of claims 7 to 9, or the pharmaceutical composition according to claim 10.

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