WO2024002258A1 - 抗体可变区的突变体及其应用 - Google Patents

抗体可变区的突变体及其应用 Download PDF

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WO2024002258A1
WO2024002258A1 PCT/CN2023/103976 CN2023103976W WO2024002258A1 WO 2024002258 A1 WO2024002258 A1 WO 2024002258A1 CN 2023103976 W CN2023103976 W CN 2023103976W WO 2024002258 A1 WO2024002258 A1 WO 2024002258A1
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antibody
cysteine
variable region
chain variable
seq
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PCT/CN2023/103976
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English (en)
French (fr)
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马宁宁
李明莹
余悦
柳思旭
张楠
李佳云
徐伟伟
郑波波
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沈阳药科大学
北京斯普瑞格生物技术有限公司
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Publication of WO2024002258A1 publication Critical patent/WO2024002258A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to the field of antibodies.
  • it relates to cysteine mutants of antibodies, compositions and/or conjugates containing the same, and uses thereof.
  • Antibody-drug conjugates are antibody products with drug molecules connected to them, obtained by connecting antibodies and drugs through biological or chemical means.
  • Antibody-conjugated drugs use the specificity of monoclonal antibodies to reduce the killing effect of drugs on normal cells and are mainly used in the treatment of tumors.
  • the preparation of antibody-conjugated drugs mainly involves coupling reactions with amino residues of lysine or sulfhydryl residues of cysteine. Whether coupling through lysine residues or coupling through cysteine residues, traditional non-site-specific coupling methods have some drawbacks. Site-specific coupling through site-specific coupling technology can improve antibody coupling. Drug homogeneity [1] .
  • Antibody molecules contain four polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (HC), while the two chains with smaller molecular weight are called light chains (LC).
  • the two H chains and the two L chains in the same antibody molecule have exactly the same amino acid composition.
  • variable region accounting for 1/4 and 1/2 of the heavy chain and light chain respectively
  • constant region C
  • Single chain antibody fragment is an antibody composed of an antibody heavy chain variable region and a light chain variable region connected by an amino acid short peptide (linker).
  • Single-chain antibodies are antibody variable regions in which the heavy chain and light chain are separated by a short amino acid and connected by a disulfide bond [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 conjugated drugs have great potential. The half-life of scFv is short, but the tumor penetration ability seems to be greater than that of Fab fragments. Therefore, single-chain antibody conjugated drugs have better efficacy and fewer side effects [4] . Data show that single-chain antibody-drug conjugates are better tolerated than IgG antibody-drug conjugates [5,6] .
  • cysteine The most common reactive amino acid in site-directed coupling technology is cysteine.
  • site-directed mutagenesis technology antibody-conjugated drugs with controllable drug-to-antibody coupling ratios can be developed.
  • the sulfhydryl group on the mutated cysteine can always remain free during the expression and purification process and can be directly used in downstream coupling reactions.
  • maleimide is the most popular linker group [7] , and then the antibody is connected to another functional molecule (antibody fragment, peptide or small molecule drugs) for conjugation.
  • sulfhydryl groups on cysteine introduced into antibodies through cysteine engineering will be oxidized during cell culture, such as reacting with sulfhydryl groups on free cysteine in the cell culture medium to form disulfide bonds.
  • the oxidized sulfhydryl group needs to be reduced before it becomes active and can be used for subsequent coupling reactions.
  • the reduction reaction will also open the disulfide bonds in the antibody.
  • These opened disulfide bonds in the antibody need to be re-oxidized to restore the disulfide bonds in the antibody.
  • Disulfide bond structure A series of reprocessing steps after the expression of these antibodies will complicate the production process of antibody-conjugated drugs and pose quality control challenges.
  • the present invention mainly provides a cysteine-engineered antibody, which according to the Kabat numbering system has engineered cysteine residues at any one or more positions selected from the following: heavy chain variable region Positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114 or any combination thereof, the corresponding IMGT numbers are 13, 39, 40, 43, 49 respectively , 52, 56, 67, 68, 75, 77, 86, 87, 122; or position 19, 21, 44, 46, 47, 48, 62, 71, 75, 78, 87 of the light chain variable region or its In any combination, the corresponding IMGT numbers are 19, 21, 50, 52, 53, 54, 76, 87, 91, 94, and 103.
  • the sulfhydryl group on the engineered cysteine is changed during the expression of the antibody. Can remain partially active.
  • the thiol group that remains active can be directly used to react with other active groups
  • the present invention provides the following aspects:
  • 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 following:
  • cysteine-engineered antibody according to item 1 wherein the cysteine-engineered antibody is 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 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 the heavy chain variable region shown in SEQ ID NO: 85 and the light chain variable region shown in SEQ ID NO: 96;
  • An antibody comprising the heavy chain variable region shown in SEQ ID NO: 89 and the 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 the heavy chain variable region shown in SEQ ID NO: 108 and the light chain variable region shown 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
  • Antibodies comprising the heavy chain variable region shown in SEQ ID NO: 130 and the light chain variable region shown in SEQ ID NO: 133.
  • cysteine-engineered antibody according to item 1, wherein the light chain is of lambda or kappa type;
  • the anti-HER2 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-VEGFR-2 antibody, anti-EGFR antibody and/or anti-c-Met antibody are optionally, the anti-HER2 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-VEGFR-2 antibody, anti-EGFR antibody and/or anti-c-Met antibody;
  • the antibody is a single chain antibody, an IgG antibody, or a bispecific antibody in the form of a BiTE/DART/Diabody.
  • the cysteine-engineered antibody according to item 5 characterized in that when the antibody is a single-chain antibody, the antibody further includes a connecting chain.
  • the sequence of the connecting chain is SEQ. ID NO.6.
  • a conjugate comprising the cysteine-engineered antibody described in any one of items 1 to 6, and a conjugate.
  • conjugate of item 7 wherein the conjugate is selected from the group consisting of polyethylene glycol, cytotoxic agents, active peptides, Nanobodies, single domain antibodies, Fab fragments, Fab' fragments, scFv, small molecules Drugs (for example, topase inhibitors, tubulysin A, DM1, PBD, MMAE or MMAF, etc.), chemotherapeutic agents or radiotherapy agents;
  • the conjugate is selected from the group consisting of polyethylene glycol, cytotoxic agents, active peptides, Nanobodies, single domain antibodies, Fab fragments, Fab' fragments, scFv, small molecules Drugs (for example, topase inhibitors, tubulysin A, DM1, PBD, MMAE or MMAF, etc.), chemotherapeutic agents or radiotherapy agents;
  • the conjugate is conjugated to the cysteine-engineered antibody through a linker
  • the linker contains an electrophilic group (preferably a maleimide group or a haloacetamide group); optionally, the linker is mc-VC-PAB.
  • an electrophilic group preferably a maleimide group or a haloacetamide group
  • the linker is mc-VC-PAB.
  • polyethylene glycol is maleimide monomethoxy polyethylene glycol, preferably mPEG2000-Mal, mPEG5000-Mal, mPEG10000-Mal.
  • composition characterized by comprising the cysteine-engineered antibody described in any one of items 1-6 or the conjugate described in any one of items 7-9, and optionally, pharmaceutically acceptable carrier.
  • cysteine-engineered antibody described in any one of Items 1-6, the conjugate described in any one of Items 7-9, or the pharmaceutical combination described in Item 10 is prepared for use in the treatment of cancer (for example, application in medicines or kits for HER2-positive cancers, preferably breast cancer.
  • a kit comprising the cysteine-engineered antibody described in any one of Items 1-6, the conjugate described in any one of Items 7-9, or the pharmaceutical composition described in Item 10.
  • site-directed mutation can improve the uniformity of antibody conjugated drugs.
  • the selected cysteine mutation site can retain the activity of part of the sulfhydryl groups after the antibody or antibody fragment is expressed, so it can be used for coupling reactions without treatment.
  • the site-specific cysteine mutation in this article does not change the activity of the antibody. After conjugation with the drug, the drug efficacy can be improved and the toxic and side effects can be reduced.
  • the mutation sites in the present invention are suitable for most antibodies and have certain versatility.
  • the present invention can utilize site mutations of different antibodies to construct bispecific antibody-conjugated drugs.
  • Figure 1 shows 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 rice 114C; LC: 21C, 47C, 48C, 71C, 75C, 19C, 46C, 62C, 78C and 87C) SDS-PAGE electrophoresis chart. It can be seen from the electrophoresis chart that the antibody band is single and the purity is in line with the follow-up Experimental requirements.
  • WT trastuzumab single chain antibody
  • mutant single chain antibodies HC: 12C, 35C, 61C, 34C, 38C, 44C, 47C, 51C, 60C, 67C, 68C , 78C, 79C rice 114C
  • LC 21C, 47C, 48C, 71C, 75C,
  • Figure 2 shows the SDS-PAGE electrophoresis pattern of anti-HER2 trastuzumab single-chain antibody light chain variable region mutant LC46C and heavy chain variable region mutant HC12C coupled with mPEG2000-MAL. It can be seen from this figure that the mutant LC46C of the light chain variable region and the mutant HC12C of the heavy chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 3 shows the SDS-PAGE electrophoresis pattern of the anti-HER2 trastuzumab single chain antibody light chain variable region mutant LC62C coupled with mPEG2000-MAL. It can be seen from this figure that the mutant LC62C of the light chain variable region can be successfully coupled with the conjugate mPEG2000-MAL.
  • Figure 4 shows the SDS-PAGE electrophoresis pattern of anti-HER2 trastuzumab single-chain antibody light chain variable region mutant LC87C and heavy chain variable region mutants HC38C, HC67C, and HC35C coupled with mPEG2000-MAL. It can be seen from this figure that the mutant LC87C of the light chain variable region and the mutants HC38C, HC67C, and HC35C of the heavy chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 5 shows the SDS-PAGE electrophoresis pattern of the anti-HER2 trastuzumab single-chain antibody heavy chain variable region mutant HC78C coupled with mPEG2000-MAL. It can be seen from this figure that the mutant HC78C of the heavy chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 6 shows the SDS-PAGE electrophoresis pattern of the anti-HER2 trastuzumab single-chain antibody heavy chain variable region mutant HC61C coupled with mPEG2000-MAL. It can be seen from this figure that the mutant HC61C of the heavy chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 7 shows the SDS-PAGE electrophoresis pattern of anti-HER2 trastuzumab single-chain antibody heavy chain variable region mutants HC69C and HC51C and light chain variable region mutant LC71C coupled with mPEG2000-MAL. It can be seen from this figure that the mutants HC69C and HC51C of the heavy chain variable region and the mutant LC71C of the light chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 8 shows the SDS-PAGE electrophoresis pattern of the anti-HER2 trastuzumab single chain antibody light chain variable region mutant LC48C coupled with mPEG2000-MAL. It can be seen from this figure that the mutant LC48C of the light chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 9 shows the deconvolution results of the molecular weight of the mutant LC87C detected by mass spectrometry in the light chain variable region of the anti-HER2 trastuzumab single-chain antibody. It can be seen from the figure that the molecular weight of the mutant 87C is 26521.3 Da.
  • Figure 10 shows the deconvolution results of the molecular weight of the mutant LC87C coupled vcMMAE detected by mass spectrometry in the light chain variable region of the anti-HER2 trastuzumab single chain antibody. It can be seen from the figure that the molecular weight of the mutant after coupling is 27838.2 Da.
  • Figure 11 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.
  • Figure 12 shows the mass spectrometry detection results of the molecular weight deconvolution of the anti-HER2 trastuzumab single-chain antibody double-site cysteine mutant coupled to vcMMAE. It can be seen from the figure that the molecular weight of the mutant after coupling is mainly 27988.0Da and 29186.3Da.
  • Figure 13 shows the deconvolution results of the molecular weight of the dual-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.
  • Figure 14 shows the molecular weight deconvolution results of dual-site mutation bispecific antibody coupling vcMMAE detected by mass spectrometry. It can be seen from the figure that the molecular weights of the mutants after coupling are mainly 53903.7Da and 55220.6Da.
  • Figure 15 shows the SDS-PAGE electrophoresis pattern of the anti-HER2 Pertuzumab single chain antibody light chain variable region mutants LC46C and LC87C coupled with mPEG2000-MAL. It can be seen from this figure that the mutants LC46C and LC87C of the light chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 16 shows the SDS- PAGE electropherogram. It can be seen from this figure that the mutant LC46C of the light chain variable region of both the rituximab single chain antibody and the moromona single chain antibody can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 17 shows the SDS-PAGE electrophoresis pattern of the light chain variable region mutant LC87C of the anti-VEGFR-2 ramucirut single-chain antibody coupled with mPEG2000-MAL. It can be seen from this figure that the mutant LC87C of the light chain variable region of the ramuclide single-chain antibody can be successfully coupled to the conjugate mPEG2000.
  • Figure 18 shows the SDS-PAGE electrophoresis pattern of mutants 46C and 87C of the trastuzumab light chain variable region coupled with mPEG2000-MAL. It can be seen from this figure that the mutants 46C and 87C of the trastuzumab light chain variable region can be successfully coupled to the conjugate mPEG2000-MAL.
  • Figure 19 shows the SDS-PAGE electrophoresis pattern of the anti-EGFR&c-MET bispecific antibody light chain 87 double-site mutant BiTE-E/M-87-87 coupled with mPEG2000-MAL. It can be seen from this figure that BiTE-E/M-87-87 and the conjugate mPEG2000-MAL were successfully coupled.
  • Figure 20 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Bi-TP-1-WT-87 cysteine mutant detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 52588.4 Da.
  • Figure 21 shows the mass spectrometry detection results of the 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.
  • Figure 22 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Bi-TP-2-WT-87 cysteine mutant detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 52002.6 Da.
  • Figure 23 shows the mass spectrometry detection results of the 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.
  • Figure 24 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Bi-TP-3-WT-87 cysteine mutant detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53316.1 Da.
  • Figure 25 shows the mass spectrometry detection results of the 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.
  • Figure 26 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Bi-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 53633.6 Da.
  • 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.
  • 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.
  • 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. From the figure, it can be seen that the molecular weight of the mutant after coupling is 54851.2Da.
  • Figure 30 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Tan-HL-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.
  • 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. From the figure, it can be seen that the molecular weight of the mutant after coupling is 54851.4Da.
  • Figure 32 shows the deconvolution results of the molecular weight of the anti-HER2 bispecific antibody Tan-LH-PT-4-87-WT cysteine mutant detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 53532.4 Da.
  • 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. From the figure, it can be seen that the molecular weight of the mutant after coupling is 54849.8Da.
  • Figure 34 shows the deconvolution results of mass spectrometry detection of the molecular weight of the 87-position mutated anti-HER2 bispecific antibody Bi-TP-4-WT-87 cysteine mutant of Pertuzumab. It can be seen from the figure that the molecular weight of the mutant is 53534.3 Da.
  • Figure 35 shows the mass spectrometry detection results of the molecular weight deconvolution of Pertuzumab 87 mutation anti-HER2 bispecific antibody Bi-TP-4-WT-87 cysteine mutant coupled MC-2MMAE. It can be seen from the figure The molecular weight of the mutant after coupling was 56587.6 Da.
  • Figure 36 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.
  • Figure 37 shows the deconvolution results of the molecular weight of the anti-c-MET single-chain antibody cysteine mutant c-MET-46C coupled to VcMMAE detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant after coupling is 27318.1 Da.
  • Figure 38 shows the deconvolution results of the molecular weight of Zalumu single-chain antibody cysteine mutant Zalu-46C detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant is 27179.4Da.
  • Figure 39 shows the deconvolution results of the molecular weight of the anti-zalumu single-chain antibody cysteine mutant Zalu-46C coupled to VcMMAE detected by mass spectrometry. It can be seen from the figure that the molecular weight of the mutant after coupling is 28496.4 Da.
  • restriction endonuclease used in the examples was purchased from Thermo Fisher Scientific (China) Co., Ltd.
  • the reagents or materials used in the following examples, unless the source is explicitly mentioned, are all commonly purchased in this field. .
  • Single chain Fv also abbreviated as “sFv” or “scFv”
  • sFv is an antibody fragment containing VH and VL antibody domains linked into a single polypeptide chain.
  • the sFv polypeptide also contains a polypeptide linker between the VH and VL domains that enables the sFv to form an ideal structure for antigen binding.
  • the polypeptide connecting chain has various forms, and is divided into flexible connecting chain, rigid connecting chain and shearable connecting chain.
  • the most commonly used flexible linking chain is (GGGGS)n.
  • Other common flexible linking chains include KESGSSVSSEQLAQFRSLD and EGKSSGSGSESKST, (Gly)8 composed of pure glycine, or the slightly shorter (Gly)6.
  • Rigid linking chains include (EAAAK)n and (XP)n, where X can specify any amino acid. It is recommended to choose alanine (Ala), lysine (Lys) or glutamic acid (Glu).
  • the sequence of the cleavable connecting chain is LEAGCKNFFPR ⁇ SFTSCGSLE, etc. In this article, the connecting chain used is the most commonly used flexible connecting chain, and its sequence is GGGGSGGGSGGGGS.
  • the selected mutation sites are located in the framework region of the antibody variable region, and therefore can be applied to cysteine mutations at the sites of any antibody, including But it is not limited to anti-HER2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-VEGFR-2 antibodies, anti-EGFR antibodies or anti-c-Met antibodies, etc.
  • HER2 human epidermal growth factor receptor-2, ErbB2/P185
  • EGFR human epidermal growth factor receptor
  • the encoding gene is located on human chromosome 17q21.
  • the molecular weight is 185kDa and it is a tyrosine kinase receptor membrane glycoprotein.
  • HER2 forms heterodimers with HER1, HER3 or HER4, leading to the activation of downstream signaling pathways such as MAPK and PI3K, over-conducting growth signals and leading to malignant cell proliferation.
  • HER2 has been found in various 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.
  • the phenomenon of proto-oncogene amplification or protein overexpression is clinically called HER2-positive tumors.
  • the main manifestations are that the tumors are highly malignant, prone to progression and metastasis, insensitive to radiotherapy and chemotherapy, prone to recurrence, and short patient survival.
  • trastuzumab (Herceptin, English name: Trastuzumab/Herceptin) is an anti-HER2 humanized monoclonal antibody drug developed by Genetech. The U.S. FDA approved the market in 1998.
  • trastuzumab is combined with the chemotherapy drug (paclitaxel). etc.) has become the first-line treatment option for HER2-overexpressing advanced metastatic breast cancer and advanced gastric cancer. In the treatment of HER2-positive metastatic breast cancer, the clinical effectiveness rate can reach 38%.
  • European EMEA has approved trastuzumab combined with chemotherapy drugs as a treatment for HER2 First-line treatment options for positive advanced gastric cancer.
  • linker means a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an antibody to a conjugate (eg, a drug moiety).
  • the linker is designated L.
  • a “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).
  • ADCs antibody-drug conjugates
  • ADCs can be conveniently prepared using linkers with reactive functional groups for binding to drugs and antibodies.
  • the cysteine thiols of cysteine-engineered antibodies can form bonds with electrophilic functional groups of linker reagents, drug moieties, or drug-linker intermediates.
  • the linker has a reactive site with 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 and forms a covalent bond with the linker.
  • Useful electrophilic groups include, but are not limited to, maleimide and haloacetamide groups.
  • Common joints are mainly divided into two types: non-cleavable type and cleavable type.
  • Not cleavable Type linkers are mainly thioether linkers; cleavable linkers can be divided into acid-cleavable type, reducible type and enzyme-cleavable type, among which the most commonly used cleavable linkers include Val-Cit dipeptide, etc.
  • mc-VC-PAB is selected as the connector, which is also a commonly used connector in ADC and can be purchased from MedChemExpress.
  • the drug part D used in the ADC can be topozyme inhibitors (such as etoposide, teniposide, doxorubicin, etc.), tubulysin A, DM1, MMAE, etc.
  • topozyme inhibitors such as etoposide, teniposide, doxorubicin, etc.
  • tubulysin A DM1, MMAE, etc.
  • tubulysin A is a product of mucus bacteria, which has anti-angiogenesis, anti-mitosis, and anti-proliferation effects in vitro.
  • TibA tubulysin A
  • DM1 is a derivative of the anti-microtubule drug maytansine (also translated as: maytansine, maytansine, maytansine, maytansine). It is a tubulin inhibitor that acts on microtubules. The ends of the tubes, which bind and inhibit the dynamics of microtubules, are antibody-conjugable maytansinoid alkaloids that can be used to overcome the systemic toxicity associated with maytansinoids and enhance tumor-specific delivery. The structure is shown below:
  • MMAE is a synthetic derivative of Aplysia toxin 10, which plays an effective mitotic inhibitory effect by inhibiting tubulin polymerization.
  • the structure is shown below:
  • PBD pyrrolobenzodiazepine
  • MMAF (Monomethylauristatin F) is an effective tubulin polymerization inhibitor used as an anti-tumor drug and can be purchased commercially.
  • active peptide is also called bioactive peptide, which refers to peptide compounds that are beneficial to the life activities of biological organisms or have physiological effects.
  • Subject refers to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs and other mammalian species. Terms do not necessarily indicate that the subject has been diagnosed with a specific disease.
  • patient refers to a subject under medical supervision.
  • a patient may be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, etc.
  • a "cancer patient” or “AML patient” may refer to an individual who has been diagnosed with cancer, is currently undergoing a therapeutic regimen, or is at risk of recurrence, such as after surgery to remove a tumor.
  • the cancer patient has been diagnosed with cancer and is a candidate for therapy. Cancer patients may include individuals who are not receiving treatment, those who are currently receiving treatment, those who have undergone surgery, and those who have discontinued treatment.
  • a subject in need of treatment may 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 treatments such as radiation or chemotherapy, etc. .
  • Cancer includes precancerous, neoplastic, and cancerous cells, and may refer to solid tumors or non-solid cancers. Cancer includes benign and malignant growths (abnormal growths).
  • cancer may refer to leukemias, carcinomas, sarcomas, adenocarcinomas, lymphomas, solid tumors, lymphomas, and the like.
  • types of cancer include, but are not limited to, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), B-cell lymphoma, non-Hodgkin's lymphoma, Burkholderia Burkitt's lymphoma, small cell lymphoma, large cell lymphoma, monocytic leukemia, myelogenous 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 (hepatocarcinoma), kidney cancer (renal cell carcinoma), bladder cancer, breast cancer, thyroid cancer, thoracic cancer, pancreatic cancer, uterine cancer Cancer, cervical cancer, testicular cancer, an
  • this article uses the discovery studio software to predict the four parameters of the antibody's residue solvent accessibility, sidechain solvent accessibility, percent solvent accessibility, and percent sidechain solvent accessibility, taking into account the half of nutrients and mutations during the cell culture process.
  • the cystine reaction affects subsequent coupling, and sites that are too exposed cannot be selected.
  • the inventor finally selected sites in the non-CDR region and the above four parameters are ⁇ 20 for mutation screening, that is, the heavy chain variable Region positions 12, 34, 35, 38, 44, 47, 51, 60, 61, 67, 69, 78, 79, 114; or positions 19, 21, 44, 46, 47, 48 of the light chain variable region ,62,71,75,78,87.
  • the specific heavy chain variable region sequence, light chain variable region sequence, and connecting chain nucleotide sequence are SEQ ID NO: 1-3
  • the amino acid sequence is SEQ ID NO: 4-6; on this basis, according to kabat numbering, the 12th amino acid of the heavy chain is replaced with cysteine to obtain the variant of the invention HC12C
  • the variant HC35C of the present invention is obtained by replacing the 35th amino acid of the heavy chain with cysteine
  • the variant HC61C of the present invention is obtained by replacing the 61st amino acid of the heavy chain with cysteine
  • the variant HC61C of the present invention is obtained by replacing the 21st amino acid of the light chain with cysteine.
  • the variant LC21C of the present invention is obtained by replacing the amino acid with cysteine
  • the variant LC47C of the present invention is obtained by replacing the 47th amino acid of the light chain with cysteine
  • the variant LC47C of the present invention is obtained by replacing the 48th amino acid of the light chain with cysteine.
  • the 71st amino acid of the light chain is replaced with cysteine to obtain the variant LC71C of the present invention
  • the 75th amino acid of the light chain is replaced with cysteine to obtain the variant LC75C of the present invention.
  • the variant HC34C of the present invention is obtained by replacing the 34th amino acid of the heavy chain with cysteine
  • the variant HC38C of the present invention is obtained by replacing the 38th amino acid of the heavy chain with cysteine
  • the 44th amino acid of the heavy chain is replaced with half
  • the variant HC44C of the present invention is obtained by replacing cystine.
  • the variant HC47C of the present invention is obtained by replacing the 47th amino acid of the heavy chain with cysteine.
  • the variant HC47C of the present invention is obtained by replacing the 51st amino acid of the heavy chain with cysteine.
  • Variant HC51C the 60th amino acid of the heavy chain is replaced with cysteine to obtain the variant HC60C of the present invention
  • the 61st amino acid of the heavy chain is replaced with cysteine to obtain the variant HC61C of the present invention
  • the heavy chain No The variant HC67C of the present invention is obtained by replacing the amino acid at position 67 with cysteine.
  • the variant HC69C of the present invention is obtained by replacing the amino acid at position 69 of the heavy chain with cysteine.
  • the variant HC69C of the present invention is obtained by replacing the amino acid at position 78 of the heavy chain with cysteine.
  • the variant HC78C of the present invention is obtained by substitution, and the 79th amino acid of the heavy chain is replaced with cysteine to obtain the variant of the present invention.
  • HC79C the variant HC114C of the present invention is obtained by replacing the 114th amino acid of the heavy chain with cysteine
  • the variant LC19C of the present invention is obtained by replacing the 19th amino acid of the light chain with cysteine
  • the 44th amino acid of the light chain is obtained
  • the variant LC44C of the present invention is obtained by replacing the amino acid at position 46 with cysteine.
  • the variant LC46C is obtained by replacing the amino acid at position 46 of the light chain with cysteine.
  • the amino acid at position 62 of the light chain is replaced with cysteine.
  • the variant LC62C of the present invention is obtained.
  • the 78th amino acid of the light chain is replaced with cysteine to obtain the variant LC78C of the present invention.
  • the 87th amino acid of the light chain is replaced with cysteine to obtain the variant LC87C of the present invention.
  • the above-mentioned antibody DNA was chemically synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the single-chain antibody gene was double-digested with HindIII and XhoI, and the single-chain antibody gene was connected to the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated with HindIII and XhoI using T4 ligase. After the connection was successful, , that is, the expression vector of anti-HER2 single-chain antibody and its mutants was successfully constructed.
  • the successfully constructed expression vector was transformed into DH5 ⁇ competent E. coli strain (purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.).
  • the transformed DH5 ⁇ strain was verified by double digestion with HindIII and XhoI enzymes and sequenced, and positive clones were selected.
  • Use a plasmid extraction kit (purchased from Kangwei Century) to lyse the obtained E. coli and extract the plasmid to obtain a purified expression vector.
  • the purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, spread into 96-well plates, grown for 15 days, single clones were picked, and the cell antibody production was determined by Western blotting. The first 20% was transferred to a 24-well plate. After 7 days of growth, the cell antibody production was detected. The first 5 to 6 strains were selected and transferred to shake flasks for culture. After the serum-free culture was stable, the materials were collected after continuous culture for 7 days to achieve anti-HER2 single chain antibody and Expression of its mutant in CHO-K1 cells, purification of the antibody and subsequent experiments.
  • the present invention uses Ellman's method (Thermo Fisher Scientific (China) Co., Ltd.) to detect the free sulfhydryl content of the antibody product, thereby selecting cell lines suitable for further coupling.
  • At the wavelength of 412nm detect the OD value. Establish a standard curve based on the OD value and calculate the thiol content of the sample.
  • the wild type basically contains no free sulfhydryl groups, and the mutants engineered with cysteine all have free sulfhydryl groups that can be used for coupling.
  • Biofilm layer interference (BLI) technology was used to determine the affinity of wild-type antibodies, cysteine-engineered antibody mutants and the antigen HER-FC with Pro A probe (ForteBio).
  • HER-FC (Beijing Yiqiao Shenzhou Technology Co., Ltd.) in PBS, with a working concentration of 10 ⁇ g/mL; dissolve the antibody in PBS2, and gradually dilute it to concentrations of 18.75nM, 37.5nM, 75nM, 150nM, and 300nM.
  • the working volume is 200 ⁇ L.
  • the OCTET system and data processing software are used to fit the data graph, and the software calculates the intermolecular force between the antibody and HER2, expressed as a KD value.
  • the results are shown in Table 2.
  • Enzyme-linked immunosorbent assay was used to determine the binding capacity of wild-type antibodies, cysteine-engineered antibody mutants, and the antigen HER-Fc (Beijing Yiqiao Shenzhou Technology Co., Ltd.). See Table 3 for results.
  • the present invention also carried out double-site cysteine mutations for the anti-HER2 trastuzumab single-chain antibody as shown in the table below.
  • the specific heavy chain variable region sequence, light chain variable region sequence, and connecting chain nucleotide sequence are SEQ ID NO: 1-3
  • the amino acid sequence is SEQ ID NO: 4-6; on this basis, according to the kabat number, the 12th amino acid of the heavy chain and the 35th amino acid of the heavy chain are replaced with cysteine
  • the variant 12C-35C of the present invention is obtained by replacing the amino acid at position 12 of the heavy chain and the amino acid at position 61 of the heavy chain 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 are obtained.
  • the variant 35C-61C of the present invention is obtained by replacing the amino acid at position 51 with cysteine.
  • the variant 51C-61C of the present invention is obtained by replacing the amino acid at position 51 of the heavy chain and the amino acid at position 61 of the heavy chain with cysteine.
  • the 51st amino acid and the 46th amino acid of the light chain are replaced with cysteine to obtain the variant 51C-46C of the present invention.
  • the 61st amino acid of the heavy chain and the 46th amino acid of the light chain are replaced with cysteine to obtain the variant of the present invention.
  • Variant 61C-46C the amino acid at position 61 of the heavy chain and the amino acid at position 87 of the light chain are replaced with cysteine to obtain the variant 61C-87C of the invention, and the amino acid at position 21 of the light chain and the amino acid at position 47 of the light chain are replaced with cysteine
  • the variant 21C-47C of the present invention is obtained by amino acid substitution.
  • the amino acid at position 21 of the light chain and the amino acid at position 48 of the light chain are replaced with cysteine to obtain the variant 21C-48C of the present invention.
  • the amino acid at position 21 of the light chain and the amino acid at position 48 of the light chain are The 75th amino acid of the light chain is replaced with cysteine to obtain the variant 21C-75C of the present invention, and the 46th amino acid of the light chain and the 87th amino acid of the light chain are replaced with cysteine to obtain the variant 46C-87C of the present invention.
  • the DNA of the above antibody was chemically synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the sequence list is as follows.
  • the single-chain antibody gene was double-digested with HindIII and XhoI, and the single-chain antibody gene was connected to the eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated with HindIII and XhoI using T4 ligase. After the connection was successful, , that is, the expression vector of anti-HER2 single-chain antibody and its double-site mutant was successfully constructed.
  • the successfully constructed expression vector was transformed into DH5 ⁇ competent E. coli strain.
  • the transformed DH5 ⁇ strain was verified by double digestion with HindIII and XhoI enzymes and sequenced, and positive clones were selected.
  • Use a plasmid extraction kit (purchased from Kangwei Century) to lyse the obtained E. coli and extract the plasmid to obtain a purified expression vector.
  • the purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation, spread into 96-well plates, grown for 15 days, single clones were picked, and the cell antibody production was determined by Western blotting. The first 20% was transferred to a 24-well plate. After 7 days of growth, the cell antibody production was detected. The first 5 to 6 strains were selected and transferred to shake flasks for culture. After the serum-free culture was stable, the materials were collected after continuous culture for 7 days to achieve the production of anti-HER2 single-chain antibodies. Expression in CHO-K1 cells, purification of the antibody and subsequent experiments.
  • the double-site mutant of the anti-HER2 trastuzumab single-chain antibody also has similar technical effects to the single-site mutant described in Examples 1-6.
  • For its coupling effect please refer to Example 11 "Antibody Coupling The experimental results of "vcMMAE and Detection” and Example 13 "Detection of Cell Proliferation Inhibition”.
  • Examples 1-7 used anti-HER2 trastuzumab single-chain antibody as an example to verify each mutation site.
  • the present invention also performs mutations on the corresponding sites of other antibodies, verifying that the above-mentioned mutation sites are universal in various antibodies.
  • the present invention performs cysteine mutations on amino acids at position 46 and position 87 of the light chain of the Pertuzumab single-chain antibody, and the mutants are named Per-46C and Per-87C respectively;
  • the 46th amino acid of the light chain of Rituximab single chain antibody was subjected to cysteine mutation, and the mutant was named Rit-46C; the 46th amino acid of the light chain of Muromonab single chain antibody was cysteine mutated.
  • Mutants of the corresponding sites of pertuzumab single chain antibody, rituximab single chain antibody, moromona single chain antibody, and ramucirud single chain antibody also have similar effects to the anti-HER2 trastuzumab single chain antibody.
  • Regarding its coupling effect please refer to the experimental results of "Example 12 Other Antibodies Single Chain Antibody Coupling and Detection of mPEG2000-MAL". Therefore, the above-mentioned mutation sites screened in the present invention are universal in various antibodies.
  • the present invention constructs a bispecific antibody in the form of BiTE based on the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody, which is named BiTE-WT.
  • cysteine mutations were carried out at position 46 or 87 of the light chain of the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody to construct BiTE cysteine mutants.
  • Bispecific antibodies For the BiTE form of cysteine-mutated bispecific antibodies, the trastuzumab VL mutation site is in the front and the pertuzumab VL mutation site is in the back, and they are named BiTE-46-46 and BiTE-46 respectively. -87, BiTE-87-46 and BiTE-87-87.
  • connection form of BiTE is Herceptin VL-GGGGSGGGGSGGGGGS-Herceptin VH-GGGGS-Perjeta VH-GGGGSGGGGSGGGGS-Perjeta VL.
  • the sequence is as follows.
  • Anti-HER2 single-chain bispecific antibody double-site mutants also have similar technical effects to the monospecific antibody single-site mutants described in Examples 1-6.
  • For its coupling effect please refer to Example 11 "Antibody Experimental results of "Coupling vcMMAE and Detection” and Example 13 "Detection of Cell Proliferation Inhibition”.
  • This example takes the purified single-site mutant of the anti-HER2 trastuzumab single chain antibody obtained in Example 3 as an example, and performs experiments using mPEG2000-MAL as the conjugate.
  • the SDS-PAGE electrophoresis diagrams of wild-type or mutant single-chain antibodies coupled with mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification number ZZP-MPEG-MAL-2K-01) are shown in Figure 2-Fig. 8.
  • This article uses SDS-PAGE experiments to detect the coupling of mutant single-chain antibodies with mPEG2000-MAL.
  • the protein bands after coupling move up, indicating an increase in molecular weight.
  • the results show that each mutant can be successfully coupled with mPEG2000-MAL.
  • the mutated single-chain antibody in this article has a reactive thiol group, which can react with a drug linker with a functional group that reacts with the thiol group (for example, a maleimide functional group). Therefore, it is reasonable to expect that it can also realize the connection of antibodies and drug molecules.
  • the single-site mutant (LC87) of the purified anti-HER2 trastuzumab single-chain antibody obtained in Example 3 and the double-site mutant of the purified anti-HER2 trastuzumab single-chain antibody obtained in Example 7 were selected.
  • One sample each of the point mutant (HC61C-LC87C) and the purified double-site mutation bispecific antibody (BiTE-87-87) obtained in Example 9 was taken as an example, and the drug molecule vcMMAE (purchased from Nanjing Baixin Deno Biotechnology Co., Ltd., product identification number A14362) was used as a conjugate for experiments.
  • Typical mass spectra of single-point mutation single-chain antibodies before and after coupling with vcMMAE are shown in Figures 9 and 10.
  • Figure 9 shows the molecular weight results of the single-chain antibody before coupling.
  • the molecular weight of the single-chain antibody is 26521.3 Da.
  • Figure 10 shows the coupling Antibody analysis after vcMMAE
  • the sub-weight result chart shows that the molecular weight of the antibody after coupling 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 to one molecule of vcMMAE.
  • Typical mass spectra of the double-site mutant before and after coupling are shown in Figure 11 and Figure 12.
  • Figure 11 is the molecular weight result of the single-chain antibody before coupling.
  • the molecular weight of the single-chain antibody is 26553.5Da.
  • Figure 12 is the result of vcMMAE after coupling.
  • the antibody molecular weight result chart shows that the molecular weight of the antibody after conjugation with vcMMAE is 27988.0Da and 29186.3Da.
  • the calculated difference is the molecular weight of 1 molecule and 2 molecules of vcMMAE respectively.
  • This example takes the pertuzumab single chain antibody, rituximab single chain antibody, moromona single chain antibody, and ramucirub single chain antibody mutants 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) was used as a conjugate for experiments. Among them, the 46th and 87th amino acids of the light chain of Pertuzumab were mutated with cysteine, and the 46th amino acid of the light chain of Rituximab was mutated with cysteine.
  • a cysteine mutation was performed on the 46th amino acid of the light chain of Muromonab, and a cysteine mutation was performed on the 87th amino acid of the light chain of Ramucirumab; the obtained antibody mutant was identical to
  • the SDS-PAGE electrophoresis pictures of the mutant single-chain antibody coupled with mPEG2000-MAL are shown in Figures 15 to 17.
  • An SDS-PAGE experiment was used to detect the coupling of the mutant single-chain antibodies with mPEG2000-MAL.
  • the coupled protein band moved upward, indicating an increase in molecular weight.
  • the results showed that the mutant single-chain antibodies of the four antibodies could successfully bind to mPEG2000-MAL. coupling.
  • the present invention selects two cell lines of breast cancer cells, SK-BR-3 and MCF-7 (purchased from Pronoce), and uses CCK8 kit (purchased from APExBIO) as a proliferation detection reagent to determine the drug molecule MMAE, anti-HER2 trastuzumab Bead single chain antibody cysteine mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C and HC61C-LC87C and anti-HER2 bispecific antibody mutant BiTE-87-87, as well as the above antibody mutants after conjugation with vcMMAE
  • the obtained antibody-conjugated drug inhibited cell proliferation, and the half inhibitory concentration (IC 50 ) was calculated.
  • SK-BR-3 is a cell line with high expression of HER2
  • MCF-7 is a cell line with low expression of HER2.
  • the calculated IC 50 of MMAE acting on SK-BR-3 is 0.20nM; the calculated IC 50 of MMAE acting on MCF-7 is 0.35nM.
  • the calculated IC 50 of single-chain antibody cysteine mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C and anti-HER2 bispecific antibody BiTE-87-87 on MCF-7 is >1000nm.
  • Antibody conjugation obtained by conjugating vcMMAE with anti-HER2 trastuzumab single chain antibody cysteine mutants LC46C, LC87C, HC61C-LC46, LC46C-LC87C, HC61C-LC87C and anti-HER2 bispecific antibody BiTE-87-87 The calculated IC 50 of the drug acting on SK-BR-3 are 23.95nM, 15.55nM, 19.76nM, 17.28nM, 13.18nM and 1.703nM respectively; anti-HER2 trastuzumab single chain antibody cysteine mutants LC46C and LC87C , HC61C-LC46, LC46C-LC87C, HC61C-LC87C and the anti-HER2 bispecific antibody BiTE-87-87, which were obtained after coupling vcMMAE, the calculated IC 50 of the antibody conjugated drugs acting on MCF-7 was greater than 50nM.
  • vcMMAE is not targeted and has a very strong non-specific killing ability on both types of cells.
  • the antibody coupled with vcMMAE can exert more powerful cytotoxicity; while for the MCF-7 cell line, the expression of HER2 is too low, and the antigen-mediated The induced antibody binding and internalization were insufficient, and the antibody conjugated to vcMMAE only produced a weak cytotoxic effect.
  • MMAE Compared with MMAE, the cytotoxicity produced by antibody-conjugated drugs at the same molar concentration showed a dependence on the expression of HER2 receptor, proving that MMAE and Single-chain antibody cysteine mutants have certain targeting properties after conjugation, which can reduce toxicity and expand the drug treatment window.
  • Examples 1-7 used anti-HER2 trastuzumab single-chain antibody as an example to verify each mutation site.
  • the present invention also carried out mutations on 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.
  • the present invention carries out cysteine mutations at amino acid position 46 and 87 of the light chain of the trastuzumab antibody, and the mutants are named trastuzumab-46C and trastuzumab-87C respectively.
  • the sequence list is as follows:
  • This example takes the cysteine mutants at position 46 and position 87 of the light chain of the trastuzumab antibody prepared in the above Example 14 as an example, and uses mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., Product identification number ZZP-MPEG-MAL-2K-01) was used as the conjugate for experiments.
  • the reduced SDS-PAGE electrophoresis diagram of the mutant antibody coupled to mPEG2000-MAL is shown in Figure 18.
  • the reducing SDS-PAGE experiment was used to detect the coupling of the mutation with mPEG2000-MAL.
  • the protein band of the coupled light chain moved up, indicating an increase in molecular weight.
  • the results showed the mutation of the trastuzumab antibody (i.e., IgG antibody form).
  • the body can be made Functionally coupled to mPEG2000-MAL. Mutation sites also have similar technical effects to single-chain antibodies and bispecific antibodies in the form of IgG antibodies.
  • the present invention constructs a bispecific antibody in the form of BiTE based on anti-EGFR cetuximab (cetuximab) single-chain antibody and anti-c-Met single-chain antibody, named BiTE-E/M-WT. Furthermore, cysteine mutations were carried out at position 46 or 87 of the light chain on the anti-EGFR single-chain antibody and the anti-c-Met single-chain antibody respectively to construct BiTE-form cysteine-mutated bispecific antibodies.
  • the VL mutation site of the anti-EGFR single-chain antibody is in the first place, and the VL mutation site of the anti-c-Met single-chain antibody is in the back, 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, the sequences are as follows.
  • Example 17 BiTE format bispecific antibody against EGFR and c-Met cysteine mutation coupled to mPEG2000-MAL and detection
  • This example takes the BiTE form bispecific antibody cysteine mutation antibody constructed in Example 16 above as an example, and uses mPEG2000-MAL (purchased from Shanghai Zhenzhun Biotechnology Co., Ltd., product identification No. ZZP-MPEG-MAL-2K-01) was used as the conjugate for experiments.
  • conjugate (mPEG2000-MAL) 1:30 (molar ratio)
  • the reaction temperature is 37°C
  • the reaction time is 3 hours
  • the reaction buffer is PBS (pH 7. 2).
  • SDS-PAGE experiment was used to detect the coupling of the mutant with mPEG2000-MAL.
  • the protein band after coupling moved up, indicating an increase in molecular weight.
  • the results are shown in Figure 19 of anti
  • the present invention also constructs bispecific antibodies with different configurations based on the anti-HER2 trastuzumab single-chain antibody and the anti-HER2 pertuzumab single-chain antibody.
  • the connection forms and configuration names are shown in Table 5.
  • this table covers representative cases in which linkers of different lengths are used to connect, light and heavy chains are connected in different orders, and antibodies are connected in different orders (where “Bi” represents the same sequence of light and heavy chains as in Example 9, and “Tan” represents two antibodies The light and heavy chains are arranged 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 G 4 S linker repeats connecting different antibodies).
  • cysteine at position 46 or 87 of the light chain of the trastuzumab single-chain antibody or pertuzumab single-chain antibody in the bispecific antibodies in the table above to construct bispecifics with different configurations.
  • Antibody cysteine single point mutants are named differently according to the selection of the mutated antibody, for example: Bi-TP-1-WT-87, that is, in the same order, the trastuzumab corresponding to T remains wild type, and P The corresponding position 87 of the Pertuzumab light chain was mutated to cysteine.
  • This example uses the bispecific antibody with a single point mutation at position 87 of Pertuzumab obtained in the above Example 18 as an example, and takes the drug molecule vcMMAE (purchased from Nanjing Baixin Denuo Biotechnology Co., Ltd., product identification number A14362) As conjugates, 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, Tan-LH-PT-4-87-WT were used for coupling experiments.
  • vcMMAE purchased from Nanjing Baixin Denuo Biotechnology Co., Ltd., product identification number A14362
  • Example 20 Coupling and detection of antibodies and small molecule drug MC-2MMAE
  • This example uses the single-site cysteine mutant Bi-TP-4-WT-87 of the anti-HER2 single-chain bispecific antibody obtained in Example 18 as an example, and uses the dual-branch drug MC-2MMAE (purchased from Yantai Mubari International Biopharmaceutical Co., Ltd.) was used as the conjugate for experiments.
  • the reaction temperature is 4°C
  • the reaction time is 12h
  • the reaction buffer is PBS (pH7.2) .
  • the typical spectra before and after coupling are shown in Figure 34-35.
  • the molecular weight before coupling is shown in Figure 34, which is 53534.3Da.
  • the molecular weight after coupling the drug is shown in Figure 35, which is 56587.6Da.
  • the molecular weight difference before and after coupling is 3053.3Da, that is, the previous one
  • the MC-2MMAE molecule achieves the same technical effect as Example 11, that is, the antibody mutant of the present invention can also be coupled with multi-branched drugs, thereby realizing the connection of more loads through multi-branched drug linkers.
  • Examples 1-8 use trastuzumab single chain antibody, pertuzumab single chain antibody, rituximab single chain antibody, moromona single chain antibody, and ramucirud single chain antibody as examples to detect light chain positions 46 and 87. The mutation sites were verified. On the basis of Examples 1-8, the present invention also conducted mutations on the anti-c-MET antibody, verifying that the above mutation sites are universal in various antibodies. Specifically, the present invention carries out cysteine mutation at position 46 of the light chain of the anti-c-MET single-chain antibody, and the mutant is named c-MET-46C. Its sequence listing is shown below.
  • This example uses the anti-c-MET single-chain antibody 46-position single-site antibody prepared in Example 21 as an example, and uses the drug molecule vcMMAE (purchased from Nanjing Baixin Denuo Biotechnology Co., Ltd., product identification number A14362) as the coupling Conduct coupling experiments.
  • Figures 36 and 37 The spectra before and after coupling are shown in Figures 36 and 37.
  • Figure 36 is the molecular weight result of the single-chain antibody before coupling, and the molecular weight is 26001.1Da.
  • Figure 37 is the molecular weight result of the antibody after vcMMAE after coupling. After coupling, The molecular weight of the antibody after vcMMAE is 27318.1Da. The calculated difference is the molecular weight of 1 molecule of vcMMAE. That is, each antibody mutant can successfully couple to 1 vcMMAE.
  • Examples 1-8 and 21 verified the mutation sites such as positions 46 and 87 of different single-chain antibodies.
  • the present invention also mutated position 46 of the light chain of the anti-EGFR Zalutumumab single-chain antibody, verifying that the above mutation site is universal in various antibodies.
  • the present invention carries out cysteine mutation at position 46 of the light chain of Zalumu single-chain antibody, and the mutants are respectively named Zalu-46C. Its sequence listing is shown below.
  • Figure 38 is the molecular weight result of the single-chain antibody before coupling.
  • the molecular weight is 27179.4Da.
  • Figure 39 is the molecular weight result of the antibody after vcMMAE after coupling. After coupling, The molecular weight of the antibody after vcMMAE is 28496.4Da.
  • the calculated difference is the molecular weight of 1 molecule of vcMMAE, that is, each antibody mutant can successfully couple to 1 vcMMAE.

Abstract

本发明提供了一种抗体的突变体及其应用，具体地，根据Kabat编号系统，所述抗体的突变体在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：重链可变区的位置12、34、35、38、44、47、51、60、61、67、69、78、79、114或其任意组合；或轻链可变区的位置19、21、44、46、47、48、62、71、75、78、87或其任意组合。所述工程化的半胱氨酸上的巯基在抗体表达过程中后可以保持部分活性。保持活性的巯基可以不经过还原处理，直接用于与其它活性基团反应。本发明的半胱氨酸工程改造的抗体可获得进行偶联药物的活性基团，有利于简化抗体偶联药物生产工艺，提高抗体偶联药物的均一性，提高药效，降低毒副作用。

Description

抗体可变区的突变体及其应用 技术领域
本发明涉及抗体领域。具体地，涉及抗体的半胱氨酸突变体，包含其的组合物和/或缀合物，及其用途。
背景技术
抗体偶联药物(Antibody-drug conjugates，ADC)是将抗体与药物通过生物或化学手段进行连接获得的连接有药物分子的抗体产物。抗体偶联药物通过单克隆抗体的特异性降低药物对于正常细胞的杀伤作用，主要应用于肿瘤的治疗。抗体偶联药物的制备主要是通过赖氨酸的氨基残基或半胱氨酸的巯基残基进行偶联反应。无论是通过赖氨酸残基偶联还是通过半胱氨酸残基偶联，传统的非定点偶联方法都存在一些弊端，通过定点偶联技术实现位点特异性偶联可以提高抗体偶联药物均一性[1]。
抗体分子含有四条多肽链，其中，分子量较大的两条链称为重链(heavy chain，HC)，而分子量较小的两条链称为轻链(Light chain，LC)。同一抗体分子中的两条H链和两条L链的氨基酸组成完全相同。通过分析不同抗体重链和轻链的氨基酸序列发现，重链和轻链靠近N端的氨基酸序列变化很大，其他部分氨基酸序列相对恒定。因此，将抗体轻链和重链中靠近N端氨基酸序列变化较大的区域称为可变区(variable region，V)，分别占重链和轻链的1/4和1/2；将靠近C端的氨基酸序列相对稳定的区域，称为恒定区(constant region，C)，分别占重链和轻链的3/4和1/2。
单链抗体(single chain antibody fragment，scFv)是由抗体重链可变区和轻链可变区经氨基酸短肽(linker)连接而成的抗体。单链抗体(scFv)是抗体可变区重链和轻链由一个短的氨基酸间隔，通过二硫键连接[2]。ScFv分子量较小，适合在酵母或者细菌中表达，有助于快速大批量生产，是理想的高通量选择技术的候选者，如噬菌体展示，细胞展示，酵母展示，核糖体展示[3]。单链抗体偶联药物有很大的潜力，scFv半衰期较短，但肿瘤穿透能力似乎大于Fab片段，因此单链抗体偶联药物药效较好，且副作用低[4]。数据表明，单链抗体偶联药物比IgG抗体偶联药物耐受性更好[5，6]。
定点偶联技术除应用于完整IgG抗体分子，同样适用于scFv及以scFv形式为基础的双特异性抗体偶联药物等。定点偶联技术中的反应性氨基酸，最常见的是半胱氨酸，通过定点诱变技术，可以开发药物抗体偶联比可控的抗体偶联药物。选择适当的位点进行半胱氨酸突变，可以使其突变得到的半胱氨酸上的巯基在表达纯化过程中始终保持游离的状态，直接应用于下游的偶联反应。由于半胱氨酸的普遍应用，马来酰亚胺为最受欢迎的连接子基团[7]，然后通过含有马来酰亚胺的接头将抗体与另外一个功能分子(抗体片段，多肽或小分子药物)进行偶联。
大多数通过半胱氨酸工程改造引入抗体的半胱氨酸上的巯基在细胞培养过程中会被氧化，如与细胞培养基中的游离半胱氨酸上的巯基反应，形成二硫键。被氧化的巯基需要被还原后，才具有活性，用于后续偶联反应，但还原反应会同时打开抗体内的二硫键，这些抗体内打开的二硫键需要被重新氧化，恢复抗体内的二硫键结构。这些抗体表达后的一系列再处理步骤，会造成抗体偶联药物的生产工艺复杂，也为质量控制造成挑战。
发明内容
本发明主要提供了一种半胱氨酸工程改造的抗体，按照Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：重链可变区的位置12、34、35、38、44、47、51、60、61、67、69、78、79、114或其任意组合，其对应的IMGT编号分别为13、39、40、43、49、52、56、67、68、75、77、86、87、122；或轻链可变区的位置19、21、44、46、47、48、62、71、75、78、87或其任意组合，其对应的IMGT编号分别为19、21、50、52、53、54、76、87、91、94、103，所述工程化的半胱氨酸上的巯基在抗体表达过程中后可以保持部分活性。保持活性的巯基可以不经过还原处理，直接用于与其它活性基团反应。
具体地，本发明提供了以下方面：
1.一种半胱氨酸工程改造的抗体，其特征在于，根据Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：
重链可变区的位置12、34、35、38、44、47、51、60、61、67、69、78、79、114或其任意组合；或
轻链可变区的位置19、21、44、46、47、48、62、71、75、78、87或其任意组合。
2.根据项目1所述的半胱氨酸工程改造的抗体，其中，根据Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：
重链可变区的位置12、35、61、51、69、78或其任意组合；或
轻链可变区的位置46、87或其任意组合。
3.根据项目1所述的半胱氨酸工程改造的抗体，其中，根据Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：
重链可变区的位置12、35、61、51或其任意组合；或
轻链可变区的位置46、87或其任意组合。
4.根据项目1所述的半胱氨酸工程改造的抗体，其中，所述半胱氨酸工程改造的抗体是基于以下野生型抗体获得的：
包含SEQ ID NO：4中所示的重链可变区和SEQ ID NO：5中所示的轻链可变区的抗体；
包含SEQ ID NO：75中所示的重链可变区和SEQ ID NO：92中所示的轻链可变区的抗体；
包含SEQ ID NO：81中所示的重链可变区和SEQ ID NO：95中所示的轻链可变区的抗体；
包含SEQ ID NO：85中所示的重链可变区和SEQ ID NO：96中所示的轻链可变区的抗体；
包含SEQ ID NO：89中所示的重链可变区和SEQ ID NO：97中所示的轻链可变区的抗体；
包含SEQ ID NO：100中所示的重链和SEQ ID NO：101中所示的轻链的抗体；
包含SEQ ID NO：108中所示的重链可变区和SEQ ID NO：109中所示的轻链可变区的抗体；
包含SEQ ID NO：113中所示的重链可变区和SEQ ID NO：114中所示的轻链可变区的抗体；和/或
包含SEQ ID NO：130中所示的重链可变区和SEQ ID NO：133中所示的轻链可变区的抗体。
5.根据项目1所述的半胱氨酸工程改造的抗体，其中，所述轻链为λ或κ类型；
任选地，所述为抗HER2抗体、抗CD3抗体、抗CD20抗体、抗VEGFR-2抗体、抗EGFR抗体和/或抗c-Met抗体；
任选地，所述抗体为单链抗体，IgG抗体，或双特异性抗体例如BiTE/DART/Diabody形式的双特异性抗体。
6.根据项目5所述的半胱氨酸工程改造的抗体，其特征在于，当所述抗体为单链抗体时，所述抗体还包括连接链，优选地，所述连接链的序列为SEQ ID NO.6。
7.缀合物，其包含项目1-6任一项所述的半胱氨酸工程改造的抗体，和偶联物。
8.项目7所述的缀合物，其中所述偶联物选自聚乙二醇，细胞毒性剂，活性肽，纳米抗体，单结构域抗体，Fab片段，Fab’片段，scFv，小分子药物(例如，拓扑酶抑制剂、tubulysin A、DM1、PBD、MMAE或MMAF等)，化疗剂或放疗剂；
所述偶联物通过接头与所述半胱氨酸工程改造的抗体缀合；
优选地，所述接头含有亲电子基团(优选马来酰亚胺基团或卤代乙酰胺基团)；任选地，所述接头为mc-VC-PAB。
9.根据项目8所述的缀合物，其中，所述聚乙二醇为马来酰亚胺单甲氧基聚乙二醇，优选mPEG2000-Mal，mPEG5000-Mal，mPEG10000-Mal。
10.药物组合物，其特征在于包含项目1-6任一项所述的半胱氨酸工程改造的抗体或项目7-9任一项所述的缀合物，以及任选地，药用载体。
11.项目1-6任一项所述的半胱氨酸工程改造的抗体，项目7-9任一项所述的缀合物，或项目10所述的药物组合在制备用于治疗癌症(例如HER2阳性的癌症，优选乳腺癌)的药物或试剂盒中的应用。
12.试剂盒，其包含项目1-6任一项所述的半胱氨酸工程改造的抗体，项目7-9任一项所述的缀合物，或项目10所述的药物组合物。
本发明的技术效果
1.利用半胱氨酸突变，获得可以进行偶联药物的活性基团，应用广泛，便于偶联。
2.与非定点突变相比，定点突变可提高抗体偶联药物的均一性。
3.选定的半胱氨酸突变位点，在抗体或抗体片段表达后可以保持部分巯基的活性，从而不需要处理即可用于偶联反应。
4.本文中的定点半胱氨酸突变不改变抗体的活性，偶联药物后，可提高药效，降低毒副作用。
5.本发明中突变位点适用于大多数抗体，具有一定的通用性。
6.本发明可利用不同抗体的进行位点突变构建双特异性抗体偶联药物。
附图说明
图1显示了纯化后的野生型抗HER2曲妥珠单链抗体(WT)和突变型单链抗体(HC：12C、35C、61C、34C、38C、44C、47C、51C、60C、67C、68C、78C、79C稻114C；LC：21C、47C、48C、71C、75C、19C、46C、62C、78C和87C)SDS-PAGE电泳图，从电泳图中可看出抗体条带单一，纯度符合后续实验需求。
图2显示了抗HER2曲妥珠单链抗体轻链可变区的突变体LC46C、重链可变区的突变体HC12C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，轻链可变区的突变体LC46C和重链可变区的突变体HC12C可与缀合物mPEG2000-MAL偶联成功。
图3显示了抗HER2曲妥珠单链抗体轻链可变区的突变体LC62C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，轻链可变区的突变体LC62C可与缀合物mPEG2000-MAL偶联成功。
图4显示了抗HER2曲妥珠单链抗体轻链可变区的突变体LC87C、重链可变区的突变体HC38C、HC67C、HC35C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，轻链可变区的突变体LC87C、重链可变区的突变体HC38C、HC67C、HC35C可与缀合物mPEG2000-MAL偶联成功。
图5显示了抗HER2曲妥珠单链抗体重链可变区的突变体HC78C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，重链可变区的突变体HC78C可与缀合物mPEG2000-MAL偶联成功。
图6显示了抗HER2曲妥珠单链抗体重链可变区的突变体HC61C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，重链可变区的突变体HC61C可与缀合物mPEG2000-MAL偶联成功。
图7显示了抗HER2曲妥珠单链抗体重链可变区的突变体HC69C和HC51C以及轻链可变区的突变体LC71C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，重链可变区的突变体HC69C和HC51C以及轻链可变区的突变体LC71C可与缀合物mPEG2000-MAL偶联成功。
图8显示了抗HER2曲妥珠单链抗体轻链可变区的突变体LC48C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，轻链可变区的突变体LC48C可与缀合物mPEG2000-MAL偶联成功。
图9显示了质谱检测抗HER2曲妥珠单链抗体轻链可变区的突变体LC87C分子量去卷积结果，从图中可以看出突变体87C分子量为26521.3Da。
图10显示了质谱检测抗HER2曲妥珠单链抗体轻链可变区的突变体LC87C偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为27838.2Da。
图11显示了质谱检测抗HER2曲妥珠单链抗体双位点半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为26553.5Da。
图12显示了质谱检测抗HER2曲妥珠单链抗体双位点半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为主要为27988.0Da和29186.3Da。
图13显示了质谱检测双位点突变双特异性抗体分子量去卷积结果，从图中可以看出突变体分子量为52588.5Da。
图14显示了质谱检测双位点突变双特异性抗体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为主要为53903.7Da和55220.6Da。
图15显示了抗HER2帕妥珠单链抗体轻链可变区的突变体LC46C和LC87C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，轻链可变区的突变体LC46C、LC87C可与缀合物mPEG2000-MAL偶联成功。
图16显示了抗CD20利妥昔单链抗体轻链可变区的突变体LC46C和抗CD3莫罗莫那单链抗体轻链可变区的突变体LC46C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，利妥昔单链抗体和莫罗莫那单链抗体的轻链可变区的突变体LC46C均可与缀合物mPEG2000-MAL偶联成功。
图17显示了抗VEGFR-2雷莫芦单链抗体轻链可变区的突变体LC87C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，雷莫芦单链抗体轻链可变区的突变体LC87C可与缀合物mPEG2000偶联成功。
图18显示了曲妥珠单抗轻链可变区的突变体46C和87C与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，曲妥珠抗体轻链可变区的突变体46C和87C可与缀合物mPEG2000-MAL偶联成功。
图19显示了抗EGFR&c-MET双特异性抗体轻链87双位点突变体BiTE-E/M-87-87与mPEG2000-MAL偶联后的SDS-PAGE电泳图。从该图可看出，BiTE-E/M-87-87与缀合物mPEG2000-MAL偶联成功。
图20显示了质谱检测抗HER2双特异性抗体Bi-TP-1-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为52588.4Da。
图21显示了质谱检测抗HER2双特异性抗体Bi-TP-1-WT-87半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为53904.2Da。
图22显示了质谱检测抗HER2双特异性抗体Bi-TP-2-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为52002.6Da。
图23显示了质谱检测抗HER2双特异性抗体Bi-TP-2-WT-87半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为54319.4Da。
图24显示了质谱检测抗HER2双特异性抗体Bi-TP-3-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为53316.1Da。
图25显示了质谱检测抗HER2双特异性抗体Bi-TP-3-WT-87半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为54632.8Da。
图26显示了质谱检测抗HER2双特异性抗体Bi-TP-4-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为53633.6Da。
图27显示了质谱检测抗HER2双特异性抗体Bi-TP-4-WT-87半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为54949.5Da。
图28显示了质谱检测抗HER2双特异性抗体Tan-LH-TP-4-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为53533.3Da。
图29显示了质谱检测抗HER2双特异性抗体Tan-LH-TP-4-WT-87半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为54851.2Da。
图30显示了质谱检测抗HER2双特异性抗体Tan-HL-TP-4-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为53533.3Da。
图31显示了质谱检测抗HER2双特异性抗体Tan-LH-TP-4-WT-87半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为54851.4Da。
图32显示了质谱检测抗HER2双特异性抗体Tan-LH-PT-4-87-WT半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为53532.4Da。
图33显示了质谱检测抗HER2双特异性抗体Tan-LH-PT-4-87-WT半胱氨酸突变体偶联vcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为54849.8Da。
图34显示了质谱检测帕妥珠87位突变抗HER2双特异性抗体Bi-TP-4-WT-87半胱氨酸突变体分子量去卷积结果，从图中可以看出突变体分子量为53534.3Da。
图35显示了质谱检测帕妥珠87位突变抗HER2双特异性抗体Bi-TP-4-WT-87半胱氨酸突变体偶联MC-2MMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为56587.6Da。
图36显示了质谱检测抗c-MET单链抗体半胱氨酸突变体c-MET-46C分子量去卷积结果，从图中可以看出突变体分子量为26001.1Da。
图37显示了质谱检测抗c-MET单链抗体半胱氨酸突变体c-MET-46C偶联VcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为27318.1Da。
图38显示了质谱检测扎鲁木单链抗体半胱氨酸突变体Zalu-46C分子量去卷积结果，从图中可以看出突变体分子量为27179.4Da。
图39显示了质谱检测抗扎鲁木单链抗体半胱氨酸突变体Zalu-46C偶联VcMMAE分子量去卷积结果，从图中可以看出突变体偶联后分子量为28496.4Da。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
实施例中所用限制性内切核酸酶购自赛默飞世尔科技(中国)有限公司公司，以下实施例中所用的试剂或材料，如未明确提及来源，均是本领域可以常规购买的。
定义
“单链Fv”也缩写为“sFv”或“scFv”，是包含连接成单个多肽链的VH和VL抗体结构域的抗体片段。优选地，sFv多肽还在VH和VL结构域之间包含多肽连接链，所述多肽连接链使得sFv能够形成用于抗原结合的理想的结构。
所述多肽连接链有多种形式，分为柔性连接链、刚性连接链和可剪切连接链。最常用的柔性连接链是(GGGGS)n，其他常见的柔性连接链还有KESGSVSSEQLAQFRSLD和EGKSSGSGSESKST，纯甘氨酸组成的(Gly)8，或稍短的(Gly)6。刚性连接链有(EAAAK)n，(XP)n，其中X可指定任何氨基酸，推荐选择丙氨酸(Ala)，赖氨酸(Lys)或谷氨酸(Glu)。可剪切连接链序列为LEAGCKNFFPR↓SFTSCGSLE等。在本文中，所使用的连接链为最常用的柔性连接链，其序列是GGGGSGGGGSGGGGS。
在本发明的半胱氨酸工程改造的抗体中，所选的突变位点均位于抗体可变区的框架区，因此可适用于对任何抗体的所述位点进行半胱氨酸突变，包括但不限于抗HER2抗体、抗CD3抗体、抗CD20抗体、抗VEGFR-2抗体、抗EGFR抗体或抗c-Met抗体等。
其中，HER2(human epidermal growth factor receptor-2，ErbB2/P185)是人表皮生长因子受体(EGFR)家族的第二个成员，由原癌基因erbB2/Her2编码，编码基因定位于人染色体17q21，分子量为185kDa，是一种酪氨酸激酶受体膜糖蛋白。HER2通过与HER1、HER3或HER4形成异二聚体，导致下游MAPK和PI3K等信号通路活化，过度传导生长信号从而导致细胞恶性增殖。在人类多种肿瘤中，包括25-30％乳腺癌、35-45％胰腺癌及90％的结肠直肠癌、16-57％的非小细胞肺癌、9-38％的胃癌等都发现有HER2原癌基因扩增或者蛋白过表达现象，临床上称为HER2阳性肿瘤，主要表现为肿瘤恶性程度高，易进展及转移，对放化疗不敏感，易复发，患者生存期短。
1975年，Kohler与Milstein发明了用来制备单克隆抗体(Monoclonal antibody，mAb)的杂交瘤技术，之后单抗药物迅速发展并应用于临床。近年来，靶向HER2的抗体药物也已经成为HER2阳性肿瘤治疗新热点。曲妥珠单抗(赫塞汀，英文名Trastuzumab/Herceptin)是Genetech公司开发的抗HER2人源化单克隆抗体药物，美国FDA于1998年批准上市，目前曲妥珠单抗联合化疗药物(紫杉醇等)已经成为HER2过表达晚期转移性乳腺癌和晚期胃癌的一线治疗方案，在HER2阳性转移性乳腺癌的治疗中，临床有效率可达38％，欧洲EMEA已经批准Trastuzumab联合化疗药物作为治疗HER2阳性进展期胃癌的一线治疗方案。
本文中所使用的术语“接头”意指包含将抗体共价附接至偶联物(例如药物部分)的共价键或原子链的化学部分。在各种实施方案中，接头被指定为L。“接头”(L)是双功能部分或多功能部分，其可用于连接一个或多个药物部分(D)和抗体单元(Ab)以形成抗体-药物缀合物(ADC)。抗体-药物缀合物(ADC)可以使用具有用于与药物和抗体结合的反应性官能团的接头来方便地制备。经半胱氨酸工程化的抗体(CYSMAB)的半胱氨酸硫醇可以与接头试剂、药物部分或药物-接头中间体的亲电子官能团形成键。
在一方面，接头具有反应性位点，其具有与存在于抗体上的亲核半胱氨酸反应的亲电子基团。抗体的半胱氨酸硫醇与接头上的亲电子基团反应并与接头形成共价键。有用的亲电子基团包括但不限于马来酰亚胺和卤代乙酰胺基团。常见的接头主要分为不可裂解型和可裂解型两类。不可裂解 型接头主要为硫醚连接子；可裂解接头可分为酸可裂解型，可还原型和酶裂解型等，其中最常用的可裂解连接子包括Val-Cit二肽等。在本文中，选用mc-VC-PAB作为接头，其也是ADC中常用的接头，可购自MedChemExpress公司。
在本文中，ADC中使用的药物部分D可以是拓扑酶抑制剂(例如依托泊苷、替尼泊苷、多柔比星等)，tubulysin A，DM1，MMAE等。
其中，tubulysin A(TubA)是黏液性细菌的产物，其在体外具有抗血管生成、抗有丝分裂、抗增殖的作用。结构如下图所示：
其中，DM1是抗微管药物美登素(又译：美坦辛、美坦新、美登木素、美坦生，maytansine)的衍生物，是一种微管蛋白抑制剂，它在微管的末端结合并抑制微管的动态性，是抗体可缀合的美登木素生物碱，可以用于克服与美登素相关的全身毒性并增强肿瘤特异性递送。结构如下图所示：
其中，MMAE是海兔毒素10的合成衍生物，通过抑制微管蛋白聚合而起到有效的有丝分裂抑制作用。结构如下图所示：
其中，PBD也称吡咯并苯并二氮杂卓(pyrrolobenzodiazepine，PBD)，其能够通过与DNA小沟相结合，形成有效的细胞毒性DNA链间交联，从而能够阻断细胞分裂并杀死癌细胞。
其中，MMAF(Monomethylauristatin F)是一种有效的微管蛋白聚合(tubulin polymerization)抑制剂，用作抗肿瘤药物，其可通过市售购买获得。
在本文中，术语“活性肽”也称为生物活性肽，指对生物机体的生命活动有益或具有生理作用的肽类化合物。
“受试者”、“患者”、“个体”及类似术语可互换使用，并且除非另有说明，否则是指哺乳动物诸如人和非人灵长类，以及兔、大鼠、小鼠、山羊、猪和其它哺乳动物物种。术语并不一定指示受试者已经被诊断出患有特定疾病。术语“患者”是指处于医疗监督下的受试者。患者可以是寻求治疗、监测、调整或修改现有治疗方案等的个体。“癌症患者”或“AML患者”可以是指被诊断为患有癌症、目前正在接受治疗性方案或者如在去除肿瘤手术后处于复发风险的个体。在一些实施方案中，癌症患者已经被诊断为患有癌症并且是疗法的候选人。癌症患者可以包括未接受治疗、目前正在接受治疗、已经进行手术的个体及已经停止治疗的那些个体。
在治疗癌症的上下文中，需要治疗的受试者可以是指患有癌症或癌前病况、已经患有癌症且处于复发风险、疑似患有癌症、正在经历标准癌症治疗诸如放疗或化疗等的个体。
“癌症”、“肿瘤”及类似术语包括癌前、赘生性和癌性细胞，并且可以是指实体瘤或非实体癌。癌症包括良性和恶性赘生物(异常生长)。
术语“癌症”可以是指白血病、癌、肉瘤、腺癌、淋巴瘤、实体瘤和淋巴癌等。不同类型的癌症的实例包括但不限于急性骨髓性白血病(AML)、慢性骨髓性白血病(CML)、B-细胞淋巴瘤、非霍奇金氏淋巴瘤(non-Hodgkin′s lymphoma)、伯基特氏淋巴瘤(Burkitt′slymphoma)、小细胞淋巴瘤、大细胞淋巴瘤、单核细胞性白血病、骨髓性白血病、急性淋巴细胞性白血病、多发性骨髓瘤、肺癌(如非小细胞肺癌或NSCLC)、卵巢癌、前列腺癌、结肠直肠癌、肝癌(1iver cancer)(即肝癌(hepatocarcinoma))、肾癌(即肾细胞癌)、膀胱癌、乳腺癌、甲状腺癌、胸腔癌、胰腺癌、子宫癌、宫颈癌、睾丸癌、肛门癌、胰腺癌、胆管癌、胃肠道类癌肿瘤、食管癌、胆囊癌、阑尾癌、小肠癌、胃(胃部)癌、中枢神经系统癌、皮肤癌、绒毛膜癌、尿路上皮癌；头颈癌、骨原性肉瘤、纤维肉瘤、神经母细胞瘤、神经胶质瘤和黑素瘤。
实施例1抗HER2曲妥珠单链抗体突变位点的筛选
本文根据蛋白数据库PDB中4X4X模型，利用discovery studio软件对该抗体的residue solvent accessibility、sidechain solvent accessibility、percent solvent accessibility、percent sidechain solvent accessibility四个参数进行预测，考虑细胞培养过程中营养物与突变的半胱氨酸反应影响后续偶联，不能选择过于暴露的位点，本发明人经过综合考量，最终挑选了非CDR区且上述四个参数均≤20的位点进行突变筛选，即重链可变区的位置12、34、35、38、44、47、51、60、61、67、69、78、79、114；或轻链可变区的位置19、21、44、46、47、48、62、71、75、78、87。
实施例2.抗HER2曲妥珠单链抗体半胱氨酸突变细胞株筛选和表达
(1)抗HER2曲妥珠单链抗体及突变体表达载体的构建
使用编码本文所述的抗体(其对应的野生型序列来自于USP Medicines Compendium)的DNA，其中，具体的重链可变区序列、轻链可变区序列、连接链的核苷酸序列为SEQ ID NO：1-3所示，氨基酸序列为SEQ ID NO：4-6所示；在此基础上，根据kabat编号，将重链第12位氨基酸用半胱氨酸置换获得本发明的变体HC12C，将重链第35位氨基酸用半胱氨酸置换获得本发明的变体HC35C，将重链第61位氨基酸用半胱氨酸置换获得本发明的变体HC61C，将轻链第21位氨基酸用半胱氨酸置换获得本发明的变体LC21C，将轻链第47位氨基酸用半胱氨酸置换获得本发明的变体LC47C，将轻链第48位氨基酸用半胱氨酸置换获得本发明的变体LC48C，将轻链第71位氨基酸用半胱氨酸置换获得本发明的变体LC71C，将轻链第75位氨基酸用半胱氨酸置换获得本发明的变体LC75C，将重链第34位氨基酸用半胱氨酸置换获得本发明的变体HC34C，将重链第38位氨基酸用半胱氨酸置换获得本发明的变体HC38C，将重链第44位氨基酸用半胱氨酸置换获得本发明的变体HC44C，将重链第47位氨基酸用半胱氨酸置换获得本发明的变体HC47C，将重链第51位氨基酸用半胱氨酸置换获得本发明的变体HC51C，将重链第60位氨基酸用半胱氨酸置换获得本发明的变体HC60C，将重链第61位氨基酸用半胱氨酸置换获得本发明的变体HC61C，将重链第67位氨基酸用半胱氨酸置换获得本发明的变体HC67C，将重链第69位氨基酸用半胱氨酸置换获得本发明的变体HC69C，将重链第78位氨基酸用半胱氨酸置换获得本发明的变体HC78C，将重链第79位氨基酸用半胱氨酸置换获得本发明的变 体HC79C，将重链第114位氨基酸用半胱氨酸置换获得本发明的变体HC114C，将轻链第19位氨基酸用半胱氨酸置换获得本发明的变体LC19C，将轻链第44位氨基酸用半胱氨酸置换获得本发明的变体LC44C，将轻链第46位氨基酸用半胱氨酸置换获得本发明的变体LC46C，将轻链第62位氨基酸用半胱氨酸置换获得本发明的变体LC62C，将轻链第78位氨基酸用半胱氨酸置换获得本发明的变体LC78C，将轻链第87位氨基酸用半胱氨酸置换获得本发明的变体LC87C。将上述提及的抗体DNA进行化学合成(苏州金唯智生物科技有限公司)。
然后将单链抗体的基因用HindIII和XhoI进行双酶切，用T4连接酶将单链抗体基因连接到经HindIII和XhoI处理过的真核表达载体pCGS3(Biovector NTCC Inc.)中，连接成功后，即成功构建抗HER2单链抗体及其突变体的表达载体。

(2)抗HER2曲妥珠单链抗体及突变体表达载体的筛选和纯化
将构建成功的表达载体转入DH5α感受态大肠杆菌菌株(购自北京庄盟国际生物基因科技有限公司)。转化后的DH5α菌株通过用HindIII和XhoI两种酶进行双酶切，并进行测序来验证，选取阳性克隆。使用质粒提取试剂盒(购自康为世纪)对所得的大肠杆菌裂解并提取质粒，获得纯化的表达载体。
(3)抗HER2曲妥珠单链抗体及突变体在CHO细胞中的表达
将纯化后的表达载体用电穿孔法转染到CHO-K1(购自ATCC)细胞中，铺至96孔板，生长15天，挑取单克隆，通过蛋白质印迹的方式测定细胞抗体产量，选取前20％转入24孔板，生长7天后，检测细胞抗体产量，选取前5到6株转至摇瓶培养，无血清培养稳定后，连续培养7天后收料，实现抗HER2单链抗体及其突变体在CHO-K1细胞中的表达，对抗体进行纯化与后续实验。
实施例3.Ni亲和层析柱纯化
将细胞及培养基转移进50mL离心管，置于高速冷冻离心机中，8000r条件离心15min，保留上清液，弃去细胞碎片；将培养基用3倍体积的平衡液稀释，之后用0.22μm微孔滤膜过滤一遍，收集滤液备用；用10倍柱体积超纯水、PBS清洗泵与进样器、纯化柱后等待上样；上样；用含有250mM咪唑的洗脱液洗脱并将纯化后的抗体蛋白溶液接取至EP管中，置于2-8℃保存。SDS-PAGE纯度检测结果见图1，从电泳图中可看出抗体条带单一，纯度符合后续实验需求。
实施例4.抗体自由巯基占比检测
本发明采用Ellman’s方法(赛默飞世尔科技(中国)有限公司)检测抗体产物游离巯基含量，从而选择适合进行进一步偶联的细胞株。配置0.1M Na2HPO4溶液、DTNB(10mM)溶液，以及半胱氨酸标准溶液，使其终浓度为0.0059M～0.375M。同时准备浓度为5mg/mL的待测抗体样品。分别在装有半胱氨酸标准溶液以及待测样品的1.5mL EP管中加入250μL 0.1M Na2HPO4、5μL DTNB溶液，涡旋震荡混匀后，室温下避光孵育5min。在波长412nm处，检测OD值。根据OD值建立标准曲线，并计算样品巯基含量。
表1.使用Ellman’s方法检测抗体产物中游离巯基的含量

由表1可知，野生型基本不含游离巯基，经半胱氨酸工程改造的突变体都存在游离的可用于偶联的巯基。
实施例5.抗体与抗原亲和力测定
采用生物膜层干涉技术(BLI)，用Pro A探针(ForteBio)测定野生型抗体、经半胱氨酸工程改造的抗体突变体与抗原HER-FC的亲和力。
将HER-FC(北京义翘神州科技有限公司)溶于PBS，工作浓度为10μg/mL；将抗体溶于PBS2中，梯度稀释至浓度分别为18.75nM、37.5nM、75nM、150nM、300nM。工作体积均为200μL。
通过OCTET系统与数据处理软件拟合数据图，软件计算得出抗体等与HER2的分子间作用力，以KD值表示，结果见表2。
表2.野生型抗体、突变体对于抗原的亲和力

由表中数据可以看出野生型抗体、突变体对于抗原的亲和力几乎没有差别，选取的突变位点并不改变抗体的作用效果。
实施例6抗体与抗原结合测定
采用酶联免疫吸附法(ELISA)测定野生型抗体、经半胱氨酸工程改造的抗体突变体与抗原HER-Fc(北京义翘神州科技有限公司)的结合力。结果参见表3。
表3.野生型抗体、突变体对于抗原的结合力
由表中数据可以看出野生型抗体、突变体对于抗原的结合力几乎没有差别，因此选取的突变位点并不改变抗体的作用效果。
实施例7抗HER2曲妥珠单链抗体双位点半胱氨酸突变细胞株的筛选
在实施例1-6的基础上，本发明还对抗HER2曲妥珠单链抗体进行了如下表所示双位点的半胱氨酸突变。
表4.抗体双位点突变及其命名

(1)抗HER2曲妥珠单链抗体及双位点突变体表达载体的构建
使用编码本文所述的抗体(其对应的野生型序列来自于USP Medicines Compendium)的DNA，其中，具体的重链可变区序列、轻链可变区序列、连接链的核苷酸序列为SEQ ID NO：1-3所示，氨基酸序列为SEQ ID NO：4-6所示；在此基础上，根据kabat编号，将重链第12位氨基酸和重链35位氨基酸用半胱氨酸置换获得本发明的变体12C-35C，将重链第12位氨基酸和重链61位氨基酸用半胱氨酸置换获得本发明的变体12C-61C，将重链第35位氨基酸和重链61位氨基酸用半胱氨酸置换获得本发明的变体35C-61C，将重链第51位氨基酸和重链61位氨基酸用半胱氨酸置换获得本发明的变体51C-61C，将重链第51位氨基酸和轻链46位氨基酸用半胱氨酸置换获得本发明的变体51C-46C，将重链第61位氨基酸和轻链46位氨基酸用半胱氨酸置换获得本发明的变体61C-46C，将重链第61位氨基酸和轻链87位氨基酸用半胱氨酸置换获得本发明的变体61C-87C，将轻链第21位氨基酸和轻链47位氨基酸用半胱氨酸置换获得本发明的变体21C-47C，将轻链第21位氨基酸和轻链48位氨基酸用半胱氨酸置换获得本发明的变体21C-48C，将轻链第21位氨基酸和轻链75位氨基酸用半胱氨酸置换获得本发明的变体21C-75C，将轻链第46位氨基酸和轻链87位氨基酸用半胱氨酸置换获得本发明的变体46C-87C，将上述抗体的DNA进行化学合成(苏州金唯智生物科技有限公司)，序列表如下所示。
然后将单链抗体的基因用HindIII和XhoI进行双酶切，用T4连接酶将单链抗体基因连接到经HindIII和XhoI处理过的真核表达载体pCGS3(Biovector NTCC Inc.)中，连接成功后，即成功构建抗HER2单链抗体及其双位点突变体的表达载体。
(2)抗HER2曲妥珠单链抗体及双位点突变体表达载体的筛选和纯化
将构建成功的表达载体转入DH5α感受态大肠杆菌菌株。转化后的DH5α菌株通过用HindIII和XhoI两种酶进行双酶切，并进行测序来验证，选取阳性克隆。使用质粒提取试剂盒(购自康为世纪)对所得的大肠杆菌裂解并提取质粒，获得纯化的表达载体。
(3)抗HER2曲妥珠单链抗体及双位点突变体在CHO细胞中的表达
将纯化后的表达载体用电穿孔法转染到CHO-K1(购自ATCC)细胞中，铺至96孔板，生长15天，挑取单克隆，通过蛋白质印迹的方式测定细胞抗体产量，选取前20％转入24孔板，生长7天后，检测细胞抗体产量，选取前5到6株转至摇瓶培养，无血清培养稳定后，连续培养7天后收料，实现抗HER2单链抗体在CHO-K1细胞中的表达，对抗体进行纯化与后续实验。
抗HER2曲妥珠单链抗体的双位点突变体也具有与实施例1-6中所述的单位点突变体相似的技术效果，关于其偶联效果可参见实施例11“抗体偶联联vcMMAE与检测”和实施例13“细胞增殖抑制作用检测”的实验结果。
实施例8其他单链抗体半胱氨酸突变细胞株的筛选
实施例1-7以抗HER2曲妥珠单链抗体为示例对各突变位点进行了验证。本发明还在其他抗体的相应位点上进行了突变，验证了上述突变位点在各种抗体上具有通用性。具体地，本发明在帕妥珠(Pertuzumab)单链抗体的轻链46位和轻链87位氨基酸进行半胱氨酸突变，分别对其突变体命名为Per-46C和Per-87C；在利妥昔(Rituximab)单链抗体的轻链46位氨基酸进行半胱氨酸突变，对其突变体命名为Rit-46C；莫罗莫那(Muromonab)单链抗体的轻链46位氨基酸进行半胱氨酸突变，对其突变体命名为Mur-46C；在雷莫芦(Ramucirumab)单链抗体的轻链87位氨基酸进行半胱氨酸突变，对其突变体命名为Ram-87C。其序列表如下所示。
帕妥珠单链抗体、利妥昔单链抗体、莫罗莫那单链抗体、雷莫芦单链抗体的相应位点的突变体也具有与抗HER2曲妥珠单链抗体相似的效果，关于其偶联效果可参见“实施例12其他抗体单链抗体偶联mPEG2000-MAL与检测”的实验结果。因此，本发明筛选的上述突变位点在各种抗体上具有通用性。
实施例9抗HER2单链双特异性抗体双位点半胱氨酸突变细胞株的筛选
本发明根据抗HER2曲妥珠单抗单链抗体和抗HER2帕妥珠单抗单链抗体构建BiTE形式的双特异性抗体，命名为BiTE-WT。并且，分别在抗HER2曲妥珠单抗单链抗体和抗HER2帕妥珠单抗单链抗体上的轻链46位或87位进行半胱氨酸突变构建BiTE形式的半胱氨酸突变型双特异性抗体。对于BiTE形式的半胱氨酸突变型双特异性抗体，以曲妥珠抗体VL突变位点在前，以帕妥珠VL突变位点在后，分别命名为BiTE-46-46、BiTE-46-87、BiTE-87-46和BiTE-87-87。
BiTE的连接形式为Herceptin VL-GGGGSGGGGSGGGGS-Herceptin VH-GGGGS-Perjeta VH-GGGGSGGGGSGGGGS-Perjeta VL，序列如下表。
抗HER2单链双特异性抗体双位点突变体也具有与实施例1-6中所述的单特异性抗体单位点突变体相似的技术效果，关于其偶联效果可参见实施例11“抗体偶联联vcMMAE与检测”和实施例13“细胞增殖抑制作用检测”的实验结果。
实施例10.抗HER2曲妥珠单链抗体偶联mPEG2000-MAL与检测
本实施例以实施例3所获得的纯化后的抗HER2曲妥珠单链抗体的单位点突变体为示例，并以mPEG2000-MAL作为偶联物进行实验。其中，抗体与偶联物的偶联反应条件为抗体：偶联物(mPEG2000-MAL)＝1∶30(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。其中野生型或突变单链抗体与mPEG2000-MAL(购自上海甄准生物科技有限公司，产品识别编号ZZP-MPEG-MAL-2K-01)偶联的SDS-PAGE电泳图分别参见图2-图8。
本文采用SDS-PAGE实验检测突变单链抗体与mPEG2000-MAL的偶联，偶联后的蛋白条带上移，表明分子量的增加，结果显示各突变体均可成功与mPEG2000-MAL偶联。根据抗体与mPEG2000-MAL偶联的结果，可知本文中突变的单链抗体具有反应性巯基，其可以与带有和巯基反应的官能团(例如，马来酰亚胺官能团)的药物接头进行反应，从而可以合理预期其也可以实现抗体与药物分子的连接。
实施例11.抗体偶联vcMMAE与检测
本实施例选用实施例3所获得的纯化后的抗HER2曲妥珠单链抗体的单位点突变体(LC87)、实施例7所获得的纯化后的抗HER2曲妥珠单链抗体的双位点突变体(HC61C-LC87C)和实施例9所获得的纯化后的双位点突变双特异性抗体(BiTE-87-87)各一个样品为示例，并以药物分子vcMMAE(购自南京百鑫德诺生物科技有限公司，产品识别编号A14362)作为偶联物进行实验。其中，抗体与偶联物的偶联反应条件为抗体：偶联物(vcMMAE)＝1∶5(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。单位点突变单链抗体与vcMMAE偶联前后的典型质谱图参见图9和图10，其中图9为偶联前的单链抗体分子量结果图，单链抗体分子量为26521.3Da，图10为偶联后的vcMMAE后的抗体分 子量结果图，偶联后的vcMMAE后的抗体分子量为27838.2Da，经计算差值为1316.9Da，为vcMMAE的分子量，证明每个突变单链抗体成功偶联一分子vcMMAE。双位点突变体偶联前后的典型质谱图参见图11和图12，其中图11为偶联前的单链抗体分子量结果图，单链抗体分子量为26553.5Da，图12偶联后的vcMMAE后的抗体分子量结果图，偶联后的vcMMAE后的抗体分子量为27988.0Da和29186.3Da，计算差值，分别为1分子和2分子vcMMAE的分子量，经计算证明每个双突变单链抗体成功偶联1.7个以上vcMMAE。双位点突变双特异性抗体偶联前后的典型质谱图参见图13和图14，其中图13为偶联前的双位点突变双特异性抗体分子量结果图，单链抗体分子量为52588.5Da，图14为偶联后的vcMMAE后的抗体分子量结果图，偶联后的vcMMAE后的抗体分子量为53903.7Da和55220.6Da，计算差值，分别为1分子和2分子vcMMAE的分子量，经计算证明每个双位点突变双特异性抗体成功偶联1.5个以上vcMMAE。
实施例12.其他抗体单链抗体偶联mPEG2000-MAL与检测
本实施例以实施例8所获得的帕妥珠单链抗体、利妥昔单链抗体、莫罗莫那单链抗体、雷莫芦单链抗体的突变体为示例，并以mPEG2000-MAL(购自上海甄准生物科技有限公司，产品识别编号ZZP-MPEG-MAL-2K-01)作为偶联物进行实验。其中，将帕妥珠单抗(Pertuzumab)的轻链46位和轻链87位氨基酸进行半胱氨酸突变，利妥昔单抗(Rituximab)的轻链46位氨基酸进行半胱氨酸突变，莫罗莫那(Muromonab)的轻链46位氨基酸进行半胱氨酸突变，在雷莫芦单抗(Ramucirumab)的轻链87位氨氨基酸进行半胱氨酸突变；所获得的抗体突变体与偶联物的偶联反应条件为抗体：偶联物(mPEG2000-MAL)＝1∶30(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。其中突变单链抗体与mPEG2000-MAL偶联的SDS-PAGE电泳图参见图15-图17。采用SDS-PAGE实验检测突变单链抗体与mPEG2000-MAL的偶联，偶联后的蛋白条带上移，表明分子量的增加，结果显示四种抗体的突变单链抗体可成功与mPEG2000-MAL 偶联。
实施例13细胞增殖抑制作用检测
本发明选用乳腺癌细胞SK-BR-3和MCF-7两个细胞系(购自普诺赛)，采用CCK8试剂盒(购自APExBIO)作为增殖检测试剂，判断药物分子MMAE，抗HER2曲妥珠单链抗体半胱氨酸突变体LC46C、LC87C、HC61C-LC46、LC46C-LC87C和HC61C-LC87C以及抗HER2双特异性抗体突变体BiTE-87-87，以及上述抗体突变体与vcMMAE偶联后获得的抗体偶联药物对细胞增殖抑制作用，并计算半数抑制浓度(IC50)。其中SK-BR-3是HER2高表达细胞株，MCF-7是HER2低表达细胞株。MMAE作用于SK-BR-3计算出的IC50为0.20nM；MMAE作用于MCF-7计算出的IC50为0.35nM。单链抗体半胱氨酸突变体LC46C、LC87C、HC61C-LC46、LC46C-LC87C、HC61C-LC87C和抗HER2双特异性抗体BiTE-87-87作用于SK-BR-3计算出的IC50＞1000nm；单链抗体半胱氨酸突变体LC46C、LC87C、HC61C-LC46、LC46C-LC87C、HC61C-LC87C和抗HER2双特异性抗体BiTE-87-87作用于MCF-7计算出的IC50＞1000nm。抗HER2曲妥珠单链抗体半胱氨酸突变体LC46C、LC87C、HC61C-LC46、LC46C-LC87C、HC61C-LC87C和抗HER2双特异性抗体BiTE-87-87偶联vcMMAE后获得的抗体偶联药物作用于SK-BR-3计算出的IC50分别为23.95nM、15.55nM、19.76nM、17.28nM、13.18nM和1.703nM；抗HER2曲妥珠单链抗体半胱氨酸突变体LC46C、LC87C、HC61C-LC46、LC46C-LC87C、HC61C-LC87C和抗HER2双特异性抗体BiTE-87-87偶联vcMMAE后获得的抗体偶联药物作用于MCF-7计算出的IC50均大于50nM。综合以上结果，vcMMAE其不具备靶向性，对两种细胞都产生了非常强的非特异性的杀伤能力。对于HER2高表达的SK-BR-3细胞系，与单独单链抗体相比，抗体偶联vcMMAE后能够发挥更强大的细胞毒性；而对于MCF-7细胞系，HER2表达量过少，抗原介导的抗体结合和内化不足，抗体偶联vcMMAE后只产生了微弱的细胞毒性作用。与MMAE相比，相同摩尔浓度的抗体偶联药物产生的细胞毒性大小呈现出HER2受体表达量依赖性，证明MMAE与 单链抗体半胱氨酸突变体偶联后具有一定的靶向性，可以降低毒性，扩大药物治疗窗口。
综上，对单链抗体的链可变区的位置12、34、35、38、44、47、51、60、61、67、69、78、79和/或114，和/或轻链可变区的位置19、21、44、46、47、48、62、71、75、78和/或87进行半胱氨酸突变，可获得用于偶联的游离巯基，从而可成功与偶联物以及药物分子进行偶联，获得抗体偶联药物，从而使得药物具有靶向性。
实施例14曲妥珠抗体半胱氨酸突变细胞株的筛选
实施例1-7以抗HER2曲妥珠单链抗体为示例对各突变位点进行了验证。在此基础上，进一步地，本发明还在其完整抗体(即，IgG抗体)曲妥珠单抗的相应位点上进行了突变，验证了上述突变位点在抗体的不同形式上具有通用性。具体地，本发明在曲妥珠抗体的轻链46位和轻链87位氨基酸进行半胱氨酸突变，分别对其突变体命名为trastuzumab-46C和trastuzumab-87C。序列表如下：
实施例15.曲妥珠半胱氨酸突变抗体偶联mPEG2000-MAL与检测
本实施例以上述实施例14所制备的曲妥珠抗体的轻链46位和轻链87位半胱氨酸突变体为示例，并以mPEG2000-MAL(购自上海甄准生物科技有限公司，产品识别编号ZZP-MPEG-MAL-2K-01)作为偶联物进行实验。抗体突变体与偶联物的偶联反应条件为抗体：偶联物(mPEG2000-MAL)＝1∶30(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。其中突变抗体与mPEG2000-MAL偶联的还原型SDS-PAGE电泳图参见图18。采用还原型SDS-PAGE实验检测突变与mPEG2000-MAL的偶联，偶联后的轻链的蛋白条带上移，表明分子量的增加，结果显示曲妥珠抗体(即，IgG抗体形式)的突变体可成 功与mPEG2000-MAL偶联。突变位点在IgG抗体形式上也具有与单链抗体、双特异性抗体相似的技术效果。
实施例16.抗EGFR和c-Met的BiTE形式双特异性抗体半胱氨酸突变细胞株的筛选
在实施例9和实施例11对抗HER2曲妥珠单抗单链抗体和抗HER2帕妥珠单抗单链抗体构建BiTE形式的双特异性抗体的偶联性能进行验证的基础上，进一步地，本发明根据抗EGFR西妥昔单抗(cetuximab)单链抗体和抗c-Met单链抗体构建了BiTE形式的双特异性抗体，命名为BiTE-E/M-WT。并且，分别在抗EGFR单链抗体和抗c-Met单链抗体上的轻链46位或87位进行半胱氨酸突变构建BiTE形式的半胱氨酸突变型双特异性抗体。对于BiTE形式的半胱氨酸突变型双特异性抗体，以抗EGFR单链抗体VL突变位点在前，以抗c-Met单链抗体VL突变位点在后，分别命名为BiTE-E/M-46-46、BiTE-E/M-46-87、BiTE-E/M-87-46和BiTE-E/M-87-87，序列如下表。
实施例17.抗EGFR和c-Met的BiTE形式双特异性抗体半胱氨酸突变偶联mPEG2000-MAL与检测
本实施例以上述实施例16构建的抗EGFR和c-Met的BiTE形式双特异性抗体半胱氨酸突变抗体为示例，并以mPEG2000-MAL(购自上海甄准生物科技有限公司，产品识别编号ZZP-MPEG-MAL-2K-01)作为偶联物进行实验。抗体突变体与偶联物的偶联反应条件为抗体：偶联物(mPEG2000-MAL)＝1∶30(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。以BiTE-E/M-87-87为例，采用SDS-PAGE实验检测突变体与mPEG2000-MAL的偶联，偶联后的蛋白条带上移，表明分子量的增加，结果图19显示抗EGFR和c-Met的BiTE形式双特异性抗体的突变体可成功与mPEG2000-MAL偶联。
实施例18不同排列形式抗HER2单链双特异性抗体单位点半胱氨酸突变细胞株的筛选
进一步地，本发明根据抗HER2曲妥珠单抗单链抗体和抗HER2帕妥珠单抗单链抗体还构建了不同构型的双特异性抗体，连接形式与构型名称如表5所示，该表格涵盖用不同长度连接子连接、轻重链按照不同顺序连接以及抗体按照不同顺序连接的代表案例(其中“Bi”表示和实施例9相同的轻重链排列顺序，“Tan”表示两个抗体的轻重链排列顺序相同，“LH”/“HL”表示在“Tan”形式下，轻链/重链在前，尾端的数字表示连接不同抗体的G4S连接子重复个数)。
表5.抗HER2双特异性抗体名称汇总

不同G4S连接子的编码序列与氨基酸序列如下表所示：
将上表双特异性抗体中的曲妥珠单抗单链抗体或帕妥珠单抗单链抗体上的轻链46位或87位进行半胱氨酸突变，构建不同构型的双特异性抗体半胱氨酸单位点突变体，并根据突变抗体的选择，予以不同命名，例如：Bi-TP-1-WT-87，即和排列顺序一样，T对应的曲妥珠保持野生型，P对应的帕妥珠轻链87位突变为半胱氨酸的形式。
实施例19.不同排列形式抗HER2单链双特异性抗体单位点半胱氨酸突变体偶联小分子药物vcMMAE及其检测
本实施例选用上述实施例18所获得的帕妥珠87位单位点突变的双特异性抗体为例，并以药物分子vcMMAE(购自南京百鑫德诺生物科技有限公司，产品识别编号A14362)作为偶联物，和抗体突变体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进行偶联实验。其中，抗体与偶联物的偶联反应条件为抗体：偶联物(vcMMAE)＝1∶5(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。
不同排列形式的双特异性抗体单位点突变体偶联情况为图20-图35。分别将图20与图21、图22与图23、图24与图25、图26与图27、图28与图29、图30与图31、图32与图33和图34与图35进行对比， 可以发现偶联前后分子量的差值均为1316Da左右，即不同排列的上述特异性抗体单位点突变体均可实现偶联。
实施例20.抗体与小分子药物MC-2MMAE的偶联与检测
本实施例以实施例18获得抗HER2单链双特异性抗体单位点半胱氨酸突变体Bi-TP-4-WT-87示例，并以双分支药物MC-2MMAE(购自烟台迈百瑞国际生物医药股份有限公司)作为偶联物进行实验。其中，抗体与偶联物的偶联反应条件为抗体：偶联物(vcMMAE)＝1∶2.5(摩尔比)，反应温度为4℃，反应时长12h，反应缓冲液为PBS(pH7.2)。其偶联前后典型图谱参见图34-35，偶联前分子量参见图34，为53534.3Da，偶联药物后分子量参见图35，为56587.6Da，偶联前后分子量相差3053.3Da，即偶联上一个MC-2MMAE分子，实现实例11相同的技术效果，即本发明的抗体突变体还可以与多分支药物进行偶联，从而可通过多分枝药物连接子，实现更多载荷的连接。
实施例21.抗c-MET单链抗体单位点半胱氨酸突变细胞株的筛选
实施例1-8以曲妥珠单链抗体、帕妥珠单链抗体、利妥昔单链抗体、莫罗莫那单链抗、雷莫芦单链抗体为示例对轻链46、87位等突变位点进行了验证。在实施例1-8的基础上，本发明还在抗c-MET抗体上进行了突变，验证了上述突变位点在各种抗体上具有通用性。具体地，本发明在抗c-MET单链抗体的轻链46位进行半胱氨酸突变，对其突变体命名为c-MET-46C。其序列表如下所示。
实施例22抗c-MET单链抗体单位点半胱氨酸突变体偶联vcMMAE与检测
本实施例选用实施例21制备的抗c-MET单链抗体46位单位点抗体为例，并以药物分子vcMMAE(购自南京百鑫德诺生物科技有限公司，产品识别编号A14362)作为偶联物进行偶联实验。其中，抗体与偶联物的偶联反应条件为抗体：偶联物(vcMMAE)＝1∶5(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。
偶联前后的图谱参见图36和37，其中图36为偶联前的单链抗体分子量结果图，分子量为26001.1Da，图37为偶联后的vcMMAE后的抗体分子量结果图，偶联后的vcMMAE后的抗体分子量为27318.1Da计算差值，为1分子vcMMAE的分子量，即每个抗体突变体可成功偶联1个vcMMAE。
实施例22抗EGFR单链抗体单位点半胱氨酸突变细胞株的筛选
实施例1-8、21对不同单链抗体的46、87位等突变位点进行了验证。在此基础上，本发明还在抗EGFR扎鲁木单抗(Zalutumumab)单链抗体上轻链46位进行了突变，验证了上述突变位点在各种抗体上具有通用性。具体地，本发明在扎鲁木单链抗体的轻链46位进行半胱氨酸突变，分别对其突变体命名为Zalu-46C。其序列表如下所示。
实施例23抗EGFR单链抗体单位点半胱氨酸突变体偶联vcMMAE与检测
本实施例选用实施例22获得的扎鲁木单链抗体46位单位点抗体为例，并以药物分子vcMMAE(购自南京百鑫德诺生物科技有限公司，产品识别编号A14362)作为偶联物进行偶联实验。其中，抗体与偶联物的偶联反应条件为抗体：偶联物(vcMMAE)＝1∶5(摩尔比)，反应温度为37℃，反应时长3h，反应缓冲液为PBS(pH7.2)。
偶联前后的图谱参见图38和39，其中图38为偶联前的单链抗体分子量结果图，分子量为27179.4Da，图39为偶联后的vcMMAE后的抗体分子量结果图，偶联后的vcMMAE后的抗体分子量为28496.4Da，计算差值，为1分子vcMMAE的分子量，即每个抗体突变体可成功偶联1个vcMMAE。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施 例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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Claims (12)

  1. 一种半胱氨酸工程改造的抗体，其特征在于，根据Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：
    重链可变区的位置12、34、35、38、44、47、51、60、61、67、69、78、79、114或其任意组合；或
    轻链可变区的位置19、21、44、46、47、48、62、71、75、78、87或其任意组合。
  2. 根据权利要求1所述的半胱氨酸工程改造的抗体，其中，根据Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：
    重链可变区的位置12、35、61、51、69、78或其任意组合；或
    轻链可变区的位置46、87或其任意组合。
  3. 根据权利要求1所述的半胱氨酸工程改造的抗体，其中，根据Kabat编号系统，其在选自以下的任意一个或多个位置处具有工程化的半胱氨酸残基：
    重链可变区的位置12、35、61、51或其任意组合；或
    轻链可变区的位置46、87或其任意组合。
  4. 根据权利要求1所述的半胱氨酸工程改造的抗体，其中，所述半胱氨酸工程改造的抗体是基于以下野生型抗体获得的：
    包含SEQ ID NO：4中所示的重链可变区和SEQ ID NO：5中所示的轻链可变区的抗体；
    包含SEQ ID NO：75中所示的重链可变区和SEQ ID NO：92中所示的轻链可变区的抗体；
    包含SEQ ID NO：81中所示的重链可变区和SEQ ID NO：95中所示的轻链可变区的抗体；
    包含SEQ ID NO：85中所示的重链可变区和SEQ ID NO：96中所示的轻链可变区的抗体；
    包含SEQ ID NO：89中所示的重链可变区和SEQ ID NO：97中所示的轻链可变区的抗体；
    包含SEQ ID NO：100中所示的重链和SEQ ID NO：101中所示的轻链的抗体；
    包含SEQ ID NO：108中所示的重链可变区和SEQ ID NO：109中所示的轻链可变区的抗体；
    包含SEQ ID NO：113中所示的重链可变区和SEQ ID NO：114中所示的轻链可变区的抗体；和/或
    包含SEQ ID NO：130中所示的重链可变区和SEQ ID NO：133中所示的轻链可变区的抗体。
  5. 根据权利要求1所述的半胱氨酸工程改造的抗体，其中，所述轻链为λ或κ类型；
    任选地，所述抗体为抗HER2抗体、抗CD3抗体、抗CD20抗体、抗VEGFR-2抗体、抗EGFR抗体和/或抗c-Met抗体；
    任选地，所述抗体为单链抗体，IgG抗体，或双特异性抗体例如BiTE/DART/Diabody形式的双特异性抗体。
  6. 根据权利要求5所述的半胱氨酸工程改造的抗体，其特征在于，当所述抗体为单链抗体时，所述抗体还包括连接链，优选地，所述连接链的序列为SEQ ID NO.6。
  7. 缀合物，其包含权利要求1-6任一项所述的半胱氨酸工程改造的抗体，和偶联物。
  8. 权利要求7所述的缀合物，其中所述偶联物选自聚乙二醇，细胞毒性剂，活性肽，纳米抗体，单结构域抗体，Fab片段，Fab’片段，scFv，小分子药物(例如，拓扑酶抑制剂、tubulysin A、DM1、PBD、MMAE或MMAF等)，化疗剂或放疗剂；
    所述偶联物通过接头与所述半胱氨酸工程改造的抗体缀合；
    优选地，所述接头含有亲电子基团(优选马来酰亚胺基团或卤代乙酰胺基团)；任选地，所述接头为mc-VC-PAB。
  9. 根据权利要求8所述的缀合物，其中，所述聚乙二醇为马来酰亚胺单甲氧基聚乙二醇，优选mPEG2000-Mal，mPEG5000-Mal，mPEG10000-Mal。
  10. 药物组合物，其特征在于包含权利要求1-6任一项所述的半胱氨酸工程改造的抗体或权利要求7-9任一项所述的缀合物，以及任选地，药用载体。
  11. 权利要求1-6任一项所述的半胱氨酸工程改造的抗体，权利要求7-9任一项所述的缀合物，或权利要求10所述的药物组合在制备用于治疗癌症(例如HER2阳性的癌症，优选乳腺癌)的药物或试剂盒中的应用。
  12. 试剂盒，其包含权利要求1-6任一项所述的半胱氨酸工程改造的抗体，权利要求7-9任一项所述的缀合物，或权利要求10所述的药物组合物。
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KATHRYN E. TILLER, RATUL CHOWDHURY, TONG LI, SETH D. LUDWIG, SABYASACHI SEN, COSTAS D. MARANAS, PETER M. TESSIER: "Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis", FRONTIERS IN IMMUNOLOGY, vol. 8, 4 September 2017 (2017-09-04), XP055573977, DOI: 10.3389/fimmu.2017.00986 *
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