WO2023231877A1 - 抗cd228抗体及其药物偶联物 - Google Patents

抗cd228抗体及其药物偶联物 Download PDF

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WO2023231877A1
WO2023231877A1 PCT/CN2023/096155 CN2023096155W WO2023231877A1 WO 2023231877 A1 WO2023231877 A1 WO 2023231877A1 CN 2023096155 W CN2023096155 W CN 2023096155W WO 2023231877 A1 WO2023231877 A1 WO 2023231877A1
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seq
antibody
antigen
binding fragment
light chain
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PCT/CN2023/096155
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English (en)
French (fr)
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宋德勇
韩静
刘红
沈楠
矫婕
王巧平
冯建霞
李敏
宗梦琦
李莹
窦昌林
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山东博安生物技术股份有限公司
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Publication of WO2023231877A1 publication Critical patent/WO2023231877A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to an anti-CD228 antibody and its drug conjugate.
  • CD228 (also known as melanotransferrin, MTF, melanoma-associated antigen p97, MFI2 or MAP97) is a 90-97kDa sialoglycoprotein member of the transferrin family. CD228 is typically found tethered to the cell membrane via a glycosylphosphatidylinositol anchor, and only a small amount of soluble protein is detected.
  • CD228 plays a certain role in cell proliferation, migration and tumorigenesis. Increased expression of CD228 can lead to accelerated melanoma tumor growth. In cell models, high expression of CD228 can increase cell proliferation, while downregulation of CD228 can lead to decreased cell proliferation.
  • CD228 is expressed in a variety of tumors, such as melanoma, mesothelioma, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer, etc., and has a wide range of indications.
  • CD228 is expressed in 72% of melanomas and 79% of pancreatic cancers. It is also expressed in 83% of mesothelioma and 100% of colon cancers, 57% of breast cancers, and 69% of squamous cell carcinomas. It has great clinical significance. need.
  • Antibody-drug conjugates couple antibodies with small molecule chemotherapy drugs through linkers. They have the high targeting properties of antibodies and give full play to the cytotoxicity of chemotherapy drugs to achieve the effect of killing tumor cells efficiently.
  • Antibody-drug conjugates have been successfully launched, products under development are gradually increasing, and the technology has matured.
  • CD228 is highly expressed in many tumor tissues and has low or no expression in normal tissues. Based on the difference in its expression, CD228 may be an ideal target for antibody-drug conjugates.
  • a new CD228 antibody-drug conjugate is provided as an effective anti-cancer drug and has wide application value in the medical field.
  • the invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, which can bind to CD228 protein; the invention also provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof; a cell containing the nucleic acid; and a cell containing the antibody or an antigen thereof.
  • the pharmaceutical composition of the binding fragment, the nucleic acid, and the cell containing the antibody or the antigen-binding fragment thereof, the nucleic acid, and the pharmaceutical composition; the antibody or the antigen-binding fragment thereof, the nucleic acid , the application of the pharmaceutical composition in preventing, treating, detecting or diagnosing diseases related to CD228, the application of CD228 antibodies or antigen-binding fragments thereof in the preparation of antibody drug conjugates (ADC), and anti-CD228 antibody conjugates .
  • ADC antibody drug conjugates
  • One aspect of the present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, said antibody or an antigen-binding fragment thereof comprising the following three light chain complementarity-determining regions and/or three heavy chain complementarity-determining regions,
  • the three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:14 and LCDR3 shown in SEQ ID NO:15, and/or
  • the three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:18;
  • the three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20 and LCDR3 shown in SEQ ID NO:21, and/or
  • the three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:22;
  • the three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:23, LCDR2 shown in SEQ ID NO:20 and LCDR3 shown in SEQ ID NO:21, and/or
  • the three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:22;
  • the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof include LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:26, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 27, and HCDR3 shown in SEQ ID NO: 28;
  • the three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:30, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:28; or
  • the three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:14 and LCDR3 shown in SEQ ID NO:21, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:32; or
  • the three light chain complementarity determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45, and/or
  • the three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:49; or,
  • the three light chain complementarity determining regions of the above-mentioned antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:46, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:48, and/or the
  • the three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in
  • the invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, which when combined with CD228 binds to the following residues shown in SEQ ID NO:41 At least one combination of: E312A, L313A, R282A, R275A.
  • the present invention provides an anti-CD228 antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof comprising:
  • the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO: 1, and/or the heavy chain variable region shown in SEQ ID NO: 2;
  • the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO: 3, and/or the heavy chain variable region shown in SEQ ID NO: 4;
  • the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO: 5, and/or the heavy chain variable region shown in SEQ ID NO: 6;
  • the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO:7, and/or the heavy chain variable region shown in SEQ ID NO:8;
  • the antibody or its antigen-binding fragment includes the light chain variable region shown in SEQ ID NO: 9, and/or the heavy chain variable region shown in SEQ ID NO: 10; the antibody or its antigen-binding fragment includes SEQ The light chain variable region shown in ID NO: 11, and/or the heavy chain variable region shown in SEQ ID NO: 12; the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO: 38 variable region, and/or the heavy chain variable region shown in SEQ ID NO:37; or
  • the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO: 40, and/or the heavy chain variable region shown in SEQ ID NO: 39.
  • sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 33.
  • sequence of the light chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 34.
  • the antibodies or antigen-binding fragments thereof of the present invention include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, scFv or dsFv fragments, etc.
  • a second aspect of the invention provides a nucleic acid encoding said antibody or antigen-binding fragment thereof.
  • a third aspect of the invention provides a vector comprising said nucleic acid encoding said antibody or antigen-binding fragment thereof.
  • the vector can be used to express the antibody or antigen-binding fragment thereof.
  • the vector can be a viral vector;
  • the viral vector includes but is not limited to lentiviral vector, adenoviral vector, adeno-associated virus vector or retroviral vector, etc.;
  • the vector can be non-viral Vector; preferably, the vector can be a mammalian cell expression vector; preferably, the expression vector can be a bacterial expression vector; preferably Optionally, the expression vector may be a fungal expression vector.
  • a fourth aspect of the invention provides a cell, said cell comprising said nucleic acid or said vector, said cell expressing said antibody or antigen-binding fragment thereof.
  • the cells are bacterial cells;
  • the bacterial cells are E. coli cells, etc.;
  • the cells are fungal cells;
  • the fungal cells are yeast cells;
  • the yeast The cells are Pichia pastoris cells, etc.;
  • the cells are mammalian cells; preferably, the mammalian cells are Chinese hamster ovary cells (CHO), human embryonic kidney cells (293), B cells, T cells, DC cells or NK cells, etc.
  • the fifth aspect of the present invention provides an anti-CD228 antibody conjugate, which is characterized in that the anti-CD228 antibody conjugate includes (a) the CD228 antibody or its antigen-binding fragment, and (b) the The antibody moiety is coupled to a coupling moiety, and the coupling moiety is selected from one or more of detectable markers, drugs, toxins, cytokines, radionuclides and enzymes.
  • the antibody drug conjugate is represented by the following formula 1:
  • Ab is the anti-CD228 antibody of the present invention or its antigen-binding fragment
  • LU is the linker (also known as the linker)
  • D is the drug
  • the subscript p corresponds to the average DAR value of the antibody-drug conjugate, p It is a value selected from 1-10, preferably 1-8, preferably 1-4 or 4-8, and more preferably p is 4.
  • the drug is selected from chemotherapy drugs, radiotherapy drugs, hormone therapy drugs or immunotherapy drugs.
  • the drug is selected from the group consisting of taxanes, maytansinoid, camptothecin, tubulysin, auristatin ( auristatin, calicheamicin, anthracycline, docetaxel, cathepsin, ricin, gelonin, Pseudomonas aeruginosa Pseudomonas exotoxin, diphtheria toxin, RNase or radioisotope.
  • linker LU is composed of the general formula R’-L1-L2-L3;
  • the L3 is:
  • R1 is hydrogen, carboxyl, ester, nitro, sulfonyl, halogen group; or R1 is R 2 to R 6 are each independently hydrogen, n is 0-8;
  • the L2 is: Where A is independently a phenylalanine residue, a glycine residue, an alanine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a histidine residue, Lysine residue, proline residue, or valine, guanidine residue, ⁇ -glycine residue, ⁇ -alanine residue; X is: n is 0-8;
  • the L1 is: and / or
  • R' is: Among them, the c end of R' is connected to L1, and the d end is connected to A;
  • LU-D in formula 1 of the antibody drug conjugate (ADC) is VcMMAE, wherein LU is Vc (valine-citrulline linker), and D is MMAE (monomethyl auristatin E ); VcMMAE can also be written as MC-Val-Cit-PAB-MMAE or mc-vc-PAB-MMAE.
  • LU-D in formula 1 of the antibody drug conjugate (ADC) is BNLD11, where LU is MC- ⁇ -Ala-(glucuronide)PAB, D is MMAE, and the structure of BNLD11 is as follows Show:
  • the Exact Mass of BNLD11 is 1322.690; the BNLD11 is synthesized using conventional methods in the prior art; in a preferred example, BNLD11 is obtained using the synthesis route shown in Figure 30.
  • the three light chain complementarity determining regions of the anti-CD228 antibody or its antigen-binding fragment (Ab) in the ADC formula 1 include the LCDR1 shown in SEQ ID NO: 24, the LCDR1 shown in SEQ ID NO: 25
  • the LCDR2 shown in SEQ ID NO:26 and the LCDR3 shown in SEQ ID NO:26, and the three heavy chain complementarity determining regions of the antibody or its antigen-binding fragment include the HCDR1 shown in SEQ ID NO:16, the HCDR1 shown in SEQ ID NO:27 HCDR2 and HCDR3 shown in SEQ ID NO:28;
  • the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO:7, and the heavy chain shown in SEQ ID NO:8 may Variable region; more preferably, the sequence of the heavy chain constant region of the antibody or its antigen-binding fragment is SEQ ID NO: 33 and/or the sequence of the light chain constant region is SEQ ID NO: 34.
  • LU-D in the ADC formula 1 is VcMMAE
  • p is 4
  • Ab is an anti-CD228 antibody or an antigen-binding fragment thereof
  • the 3 light residues of the anti-CD228 antibody or an antigen-binding fragment thereof are
  • the chain complementarity determining region includes LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25 and LCDR3 shown in SEQ ID NO:26, and is complementary to the three heavy chains of the antibody or its antigen-binding fragment.
  • the determining region includes HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:27 and HCDR3 shown in SEQ ID NO:28; more preferably, the antibody or antigen-binding fragment thereof includes SEQ ID NO:
  • LU-D in the ADC formula 1 is a BNLD11 structure
  • p is 4
  • the BNLD11 structure is as follows:
  • Ab is an anti-CD228 antibody or an antigen-binding fragment thereof.
  • the three light chain complementarity determining regions of the anti-CD228 antibody or an antigen-binding fragment thereof include LCDR1 shown in SEQ ID NO: 24, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO:26, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:27 and SEQ ID HCDR3 shown in NO:28; more preferably, the antibody or antigen-binding fragment thereof includes the light chain variable region shown in SEQ ID NO:7, and the heavy chain variable region shown in SEQ ID NO:8.
  • the sixth aspect of the present invention provides a pharmaceutical composition, which includes the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or antibody-drug conjugate.
  • the pharmaceutical composition further comprises a pharmaceutical Acceptable carrier, preferably, the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, shaping agents or other pharmaceutical excipients.
  • the seventh aspect of the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof, or a nucleic acid encoding an antibody or an antigen-binding fragment thereof, including the pharmaceutical composition or the antibody drug. conjugate.
  • the eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or antibody-drug conjugate in the preparation of a pharmaceutical composition for treating or preventing diseases.
  • the ninth aspect of the present invention provides the use of the antibody or its antigen-binding fragment or nucleic acid in the preparation of diagnostic and detection kits.
  • the tenth aspect of the present invention provides a method for treating or preventing diseases, comprising administering the antibody or antigen-binding fragment, nucleic acid, vector, cell, pharmaceutical composition or antibody-drug conjugate of the present invention to a subject in need. tester.
  • the eleventh aspect of the present invention provides a method for diagnosis and detection, which includes administering the antibody or antigen-binding fragment, nucleic acid, kit or pharmaceutical composition of the present invention to a subject or sample in need.
  • the twelfth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, pharmaceutical composition or antibody-drug conjugate for treating and preventing diseases.
  • a thirteenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, kit, or pharmaceutical composition for detection and diagnosis.
  • the fourteenth aspect of the present invention provides the antibody or antigen-binding fragment thereof, the nucleic acid, the pharmaceutical composition or antibody drug conjugate for preparation of prevention, treatment, detection or diagnosis of diseases related to CD228 application in preparations.
  • the CD228-related diseases include one or more of melanoma, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, colon adenocarcinoma, mesothelioma, pancreatic cancer, and breast cancer. .
  • the fifteenth aspect of the present invention also provides the use of the CD228 antibody or antigen-binding fragment thereof of the present invention in the preparation of antibody drug conjugates (ADC).
  • ADC antibody drug conjugates
  • anti-CD228 antibodies and antibody-drug conjugates thereof provided by the present invention have one or more of the following advantages:
  • the anti-CD228 antibody or antigen-binding fragment thereof provided by the present invention has good affinity for CD228 protein and cells expressing CD228 protein.
  • the anti-CD228 antibody drug conjugate provided by the present invention has good killing ability against human melanoma SK-MEL-5 cells.
  • the anti-CD228 antibody-drug conjugate provided by the present invention has good tumor suppressor effects on SK-MEL-5, NCI-H226, CALU-1, and NUGC4 animal models, and exhibits stable efficacy against CD228-related diseases. data.
  • the anti-CD228 antibody drug conjugate provided by the present invention has good pharmacokinetics in mice.
  • the anti-CD228 antibody drug conjugate provided by the present invention has small side effects and toxicity, and is highly safe.
  • the anti-CD228 antibody of the present invention has better internalization and internal killing effects.
  • Figure 1 shows the expression level of CD228 on the TMA chip in Example 1.
  • Figure 2 shows the serum titer of CD228 immunized mice in Example 2.
  • Figure 3 shows the binding of each anti-CD228 antibody to human CD228 protein in Example 4.
  • Figure 4 shows the binding activity of each anti-CD228 antibody in Example 5 to human melanoma SK-MEL-5 cells expressing CD228 protein.
  • Figure 5A shows the results of the internalization experiment of each anti-CD228 antibody based on human melanoma SK-MEL-5 cells in Example 6.
  • Figure 5B shows the internalization of each anti-CD228 antibody based on human lung cancer cell line A549-CD228 cells in Example 6. Experimental results.
  • Figure 6 shows the ADCC experiment results of each anti-CD228 antibody in Example 7 based on human melanoma SK-MEL-5 cells.
  • Figure 7 is a schematic diagram of the sequence alignment results of full-length hCD228 and soluble antigen sMFI2 in Example 8.
  • Figure 8 is the CA149-BNLD11 HIC-HPLC spectrum in Example 9.
  • Figure 9A shows the results of the killing experiment of CA13-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.
  • Figure 9B shows the results of the killing experiment of CA67-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.
  • Figure 9C is the result of the killing experiment of CA149-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.
  • Figure 9D is the result of the killing experiment of BA352-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.
  • Figure 9E shows the results of the killing experiment of CA518-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.
  • Figure 9F shows the results of the killing experiment of CA185-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.
  • Figure 10 shows the efficacy data (3 mg/kg) of each anti-CD228 ADC on the SK-MEL-5 animal model.
  • Figure 11 shows the efficacy data (5 mg/kg) of each anti-CD228 ADC on the SK-MEL-5 animal model.
  • Figure 12 shows the efficacy data of each anti-CD228 ADC (3 mg/kg) in the NCI-H226 animal model in Example 11.
  • Figure 13 shows the efficacy data of each anti-CD228 ADC (5 mg/kg) on the NCI-H226 animal model in Example 11.
  • Figure 14 is the pharmacokinetic curve in mice of each anti-CD228 ADC in Example 12.
  • Figure 15A is the evaluation of the inhibitory activity of CA149-BNLD11 on MC38-CD228 cells in Example 13.
  • Figure 15B is the evaluation of the inhibitory activity of CA149-BNLD11 on A375-CD228 cells in Example 13.
  • Figure 15C is the evaluation of the inhibitory activity of CA149-BNLD11 on A375-CD228 cells in Example 13. Evaluation of the inhibitory activity of SK-MEL-5 cell proliferation.
  • Figure 15D shows the evaluation of the inhibitory activity of CA149-BNLD11 on A549-CD228 cell proliferation in Example 13.
  • Figure 15E shows the inhibitory activity of CA149-BNLD11 on A375-CD228 cells in Example 13. evaluate.
  • Figure 16 is a tumor growth inhibition curve of the antibody drug conjugate in Example 14 on the Calu-1 lung cancer model.
  • Figure 17 is a histogram of tumor weight in the Calu-1 lung cancer model of the antibody drug conjugate group in Example 14.
  • Figure 18 is a tumor growth inhibition curve of the antibody drug conjugate group in Example 15 on the human melanoma cell SK-MEL-5 nude mouse transplant tumor model.
  • Figure 19 is a histogram of the tumor weight of the antibody drug conjugate group in Example 15 on the human melanoma cell SK-MEL-5 nude mouse transplant tumor model.
  • Figure 20 is a tumor growth inhibition curve of the antibody drug conjugate group in Example 16 on the human gastric cancer cell NUGC4 Balb/c nude mouse transplanted tumor model.
  • Figure 21 is a histogram of the tumor weight of the antibody drug conjugate group in Example 16 on the human gastric cancer cell NUGC4 Balb/c nude mouse transplanted tumor model.
  • Figure 22 is a tumor volume growth curve of human lung squamous cell carcinoma NCI-H226 Balb/c nude mice transplanted tumors in Example 17.
  • Figure 23 is a tumor volume growth curve of human melanoma cell SK-MEL-5 Balb/c nude mice transplanted tumors in Example 18.
  • Figure 24 is a tumor weight growth curve of human melanoma cell SK-MEL-5 Balb/c nude mice transplanted tumors in Example 18.
  • Figure 25 is the metabolism curve of the antibody drug conjugate CA149-BNLD11 in Example 19 in mice.
  • Figure 26 shows the toxicity study of the antibody drug conjugate CA149-BNLD11 in male mice in Example 20.
  • Figure 27 is a toxicity study on female mice of the antibody drug conjugate CA149-BNLD11 in Example 20.
  • Figure 28 shows the body weight changes of cynomolgus monkeys after administration of 6 mg/kg in the medium dose group and 10 mg/kg in the high dose group of Example 21.
  • Figure 29 is the toxicity generation test after the first administration of the medium-dose group 6 mg/kg and the high-dose group 10 mg/kg in Example 21.
  • Figure 30 is a schematic diagram of the BNLD11 synthesis circuit.
  • PDX sample chips for gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, cholangiocarcinoma, pancreatic cancer, cervical cancer, sarcoma, esophageal cancer, and gastric cancer were purchased from Crown Biotechnology Co., Ltd.
  • Tissue white films of 12 mesothelioma patients were purchased from Shanghai Lidi Biotechnology Co., Ltd. (4 cases) and Shanghai Xinchao Biotechnology Co., Ltd. (8 cases).
  • Sino-US Crown Biotechnology Co., Ltd. was entrusted to perform CD228 immunohistochemical staining on all PDX sample tissue chips and 8 cases of mesothelioma tissue white slides purchased from Shanghai Xinchao Biotechnology Co., Ltd.
  • CD228 immunohistochemistry antibody was purchased from Novus Biologicals (catalog number: NBP1-85777), and the primary antibody dilution concentration was 1:200.
  • Immunohistochemical experiments were completed by an IHC automatic staining machine (Bond RX automatic IHC&ISH system, Leica). Immunohistochemical staining results were evaluated using H-score values. pi represents the percentage of positive cells, and i represents the staining intensity (0: negative; 1: weak staining; 2: moderate staining; 3: strong staining). Each sample was independently scored three times, and the final H-score value was the average of the three times. The IHC scoring results of all samples are shown in Figure 1.
  • CD228 plays an important role in gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, cholangiocarcinoma, pancreatic cancer and cervical cancer.
  • the proportion of medium and high expression is relatively high.
  • mice used in the immunization experiment were fully human antibody transgenic mice independently developed by Shandong Boan Biotechnology Co., Ltd. (10 mice were immunized in total).
  • CD228 (0.23 mg/mL, Boan, 20200924, SEQ ID NO: 41) antigen protein independently produced by Shandong Boan Biotechnology Co., Ltd. was used for immunization.
  • the immunization methods used multiple injections under the skin of the abdomen and in the groin, and the antigen protein immunization dose was 20 ⁇ g/animal.
  • the antigen was emulsified in Freund's complete adjuvant
  • the antigen was emulsified in Freund's incomplete adjuvant.
  • the first batch of mice underwent 3 immunizations and a booster immunization; the second batch of mice underwent 4 immunizations and a booster immunization.
  • the interval between each immunization was 14 days. Starting from the second immunization, peripheral blood serum was collected on the 7th day after each immunization to detect antibody titers and mice with unqualified titers were eliminated.
  • mice After immunizing mice, the results of detecting mouse serum titers are shown in Figure 2. 2500X, 12500X, and 62500X represent dilution ratios. Three days after the boosted immunization, the mice were euthanized, and the spleens were harvested to prepare single cells for bank construction.
  • mice were killed, the spleens were removed by dissection, and the spleens were ground and broken with a syringe stopper and filtered with a filter.
  • the filtered spleen cells were frozen, and RNA was extracted to obtain cDNA.
  • the phage library was established according to the usual method. The storage capacity data of the built library is shown in Table 3.
  • variable regions of 8 lead antibodies are as follows in Table 5: (CDR regions are underlined, and the analysis system is the IMGT system).
  • the variable region sequences of each antibody in the examples of this application are shown in Table 5, and the heavy chain and light chain constant region sequences are shown in Table 6.
  • the antibody variable region genes were amplified by conventional molecular biology technology PCR (2 ⁇ Phanta Max Master Mix Manufacturer: Vazyme Catalog No.: P515-P1-AA Batch No.: 7E512E1), and the antibody heavy chain variable region genes were separated through homologous recombination.
  • the vector pCDNA3.4 (Life Technology) containing the nucleic acid sequence of the antibody heavy chain constant region sequence is connected, and the antibody light chain variable region gene is connected to the vector pCDNA3.4 containing the nucleic acid sequence of the antibody light chain constant region sequence.
  • sequenced positive clones were extracted with plasmids and co-transfected into HEK293 cells, cultured in a shaker at 37°C ⁇ 8% CO2 ⁇ 125rpm. After transient expression for 7 days, the supernatant was passed through Protein A affinity chromatography, and the antibody was purified and passed through UV280 combined with theoretical extinction coefficient determines antibody concentration.
  • control antibody H149 sequence was synthesized according to the sequence in patent US20200246479A1.
  • the amino acid sequence is shown in Table 7 below.
  • CBS pH 9.6 carbonate buffer
  • the starting concentration is 0.1ug/mL, three-fold dilution (8 gradients); after washing the plate, add goat anti-human IgG (H+L)/HRP (1:5000 dilution, KPL, 474-1006) , 100 ⁇ L/well, incubate at 37°C for 1 hour. After washing the plate, add 100 ⁇ L TMB (Beijing Meikewande, 1001) to each well for color development. After 10 min, add 50 ⁇ L 2M H 2 SO 4 to each well to terminate color development, and read the OD450 with a microplate reader.
  • Seagen's CD228-targeting antibody hL49 was used as a control antibody.
  • Figure 3 is the binding curve of each antibody and human CD228 protein. Table 8 shows the EC50 values of the antibodies calculated based on Figure 3. It can be seen that the six antibodies showed similar binding activities at the ELISA level and were better than the control antibody HL49.
  • Table 9 shows the source of CD228 protein used in the detection.
  • the binding kinetics of the antibody to each CD228 protein was measured using the BIAcore 8K instrument based on surface plasmon resonance (SRP) technology.
  • the ProA chip captures 2 ⁇ g/mL of CD228 antibodies, and analyzes the binding activity of each CD228 antibody to human, monkey, and mouse CD228.
  • Human, monkey, and mouse CD228 proteins were diluted 2-fold to 5 concentrations using HBS-EP+ buffer, with a starting concentration of 50 nM.
  • Biacore performed CD228 protein binding kinetic analysis and fitted to calculate the affinity activity KD value.
  • the antibody shown has similar affinity to human CD228 protein and monkey CD228 protein, but does not bind to mouse CD228.
  • His chip captures CD228 protein with a threshold of 0.5 nm at 10 ⁇ g/mL. First bind the first CD228 antibody (30ug/ml), and then analyze the competitive binding of the second CD228 antibody (30ug/ml). Octet 8K analyzes the response of antibody 2 and analyzes whether antibody 1 and antibody 2 have a competitive relationship. Table 11 shows Octet antibody epitope response values.
  • the final competition analysis uses the following calculation method: 1-response value/blank value.
  • the results are shown in Table 12. It can be seen that BA352 and hL49 epitopes are similar, and the other antibodies compete with each other and have similar epitopes (values are higher than 75 %, the epitope is relevant).
  • Example 5 Detection of cellular level binding activity of anti-hCD228 antibody by flow cytometry
  • a 96-well round bottom plate add 50 ⁇ L of human melanoma SK-MEL-5 cells (ATCC, HTB-70), the number of cells is 7E4/well, and use FACS buffer (PBS, Boster Biotechnology, Cat. No. PYG0021) to gradiently dilute each antibody , add 50 ⁇ L/well into a 96-well round bottom plate, and incubate at 4°C for 1 hour.
  • FACS buffer PBS, Boster Biotechnology, Cat. No. PYG0021
  • FIG. 4 shows the binding of each anti-CD228 antibody to human melanoma SK-MEL-5 cells (cells that express CD228 protein). It can be seen that the six antibodies have relatively high affinity with SK-MEL-5 cells. High binding activity, and better than hL49; the isotype control antibody in Figure 4 is an unrelated antibody directed against other targets that has the same constant region and different variable regions as the anti-CD228 antibody in the experimental group.
  • a 96-well round-bottom plate add 50 ⁇ L/well of human melanoma SK-MEL-5 cells diluted with buffer (PBS, Boster Biotechnology, Cat. No. PYG0021), and the number of cells is 5E4/well. Then dilute the antibody with buffer to a final concentration of 20 ⁇ g/mL. Add 50 ⁇ L/well of 20 ⁇ g/mL antibody to the round-bottom plate containing 50 ⁇ L/well of cells. After incubation for 30 min, centrifuge at 400 g for 4 min and remove the supernatant.
  • buffer PBS, Boster Biotechnology, Cat. No. PYG0021
  • ADCC working solution (RPMI1640 medium containing 1% FBS); collect Bioassay Effector Cells (Promega, G7011), use ADCC working solution to adjust the cell density to 2.4 ⁇ 10 6 ; collect Target Cells SK-MEL-5 (ATCC, HTB -70), respectively adjust the cell density to 8 ⁇ 10 5 with ADCC working solution; dilute the sample to be tested with ADCC working solution, starting from a starting concentration of 5ug/mL, dilute 4 times to 8 concentrations; mix effector cells, target cells and 25uL of each sample to be tested was added to the reaction white plate (Costar, 3917). The total reaction volume was 75uL.
  • FIG. 6 shows the ADCC experimental results of each anti-CD228 antibody based on human melanoma SK-MEL-5 cells. It can be seen from Figure 6 that as the concentration of the antibody sample increases, the signal gradually increases, indicating that its It has ADCC effect on SK-MEL-5 cells.
  • CA149-Fab full-length antigen CD228 and antibody Fab complex
  • the experimenters analyzed the interaction between the amino acid type and the side chain at the three-dimensional model epitope, and the results showed that the CA149 antibody Fab binds to the antigen through 7 hydrogen bonds and 1 salt bridge.
  • the specific interaction sites are shown in Table 13 shown, where the superscript * represents the amino acid on the light chain of CA149Fab, and italics without superscript represent the amino acid on the heavy chain of CA149Fab.
  • the affinity results show that the binding activity of the mutated antigen to the CA149 antibody is reduced or the binding is lost. It also shows that the above four sites E312A, L313A, R282A, and R275A are the key sites for the binding of the antibody to the antigen.
  • the affinity test results are reported in Table 14.
  • Ab is any of the above-mentioned anti-CD228 antibodies or antigen-binding fragments thereof
  • LU is the linker (also known as the linker)
  • D is the drug
  • subscript p is the average DAR value of the antibody-drug conjugate.
  • an ADC composition with a homogeneous drug load (DAR) of about 4 is prepared.
  • Phosphate buffer (pH 7.5, containing 11mM DTPA) containing antibody (5-10mg/mL) was treated with 2 equivalents of TCEP and incubated at 25°C for approximately 2h.
  • HIC-HPLC analysis to quantify drug loading of antibody drug conjugates. The analysis results are shown in Figure 8 and Table 16. It can be seen that the average DAR value of the antibody drug conjugate CA149-BNLD11 prepared in this example is approximately 4.
  • a 96-well flat bottom plate (Corning, Cat. No. 3917), add 50 ⁇ L/well SK-MEL-5 cells, the number of cells is 1E4/well, and the culture medium is 10% FBS/EMEM. Dilute each anti-CD228-ADC with the above culture medium, with a starting concentration of 1 ⁇ g/mL, and 4-fold concentration gradient dilution, for a total of 6 concentrations. Add 50 ⁇ L/well of the diluted ADC into a 96-well flat-bottomed plate and incubate it at 37°C and 5% CO2 for 4 days. The CellTiter-Glo kit (Promega, G7571, must be protected from light during use) is equilibrated to room temperature.
  • Figure 9A- Figure 9F shows the killing experimental results of each anti-CD228 ADC based on human melanoma SK-MEL-5 cells. It can be seen that 2 types of anti-hCD228 ADC: CA13-vcMMAE and CA67-vcMMAE, showing similar in vitro cell killing activity as hL49-vcMMAE; 4 anti-hCD228 ADCs CA149-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, CA185-vcMMAE showed better than control group hL49-vcMMAE similar In vitro cell killing activity; the isotype control antibodies in Figures 9A to 9F are unrelated antibodies against other targets that have the same constant region as the six antibodies but different variable regions.
  • Human melanoma cells SK-MEL-5 were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% CO2 . The medium composition was EMEM medium containing 10% FBS. NOD/SCID mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The concentration of SK-MEL-5 cells was adjusted to 2.5 ⁇ 10 7 cells/mL using EMEM medium containing 50% Matrigel, and the cells were inoculated subcutaneously into the right side of NOD/SCID mice at a volume of 0.1 mL/mouse. When the average tumor volume reached approximately 76mm3 , the mice were divided into 8 experimental groups according to their tumor volume and body weight, with 5 animals in each group.
  • Figure 10 shows the efficacy data of each anti-CD228 ADC on the SK-MEL-5 animal model (3 mg/kg), that is, the changes in mouse tumor volume after administration. It can be seen that after the administration of 6 antibodies, the tumor volume all become smaller.
  • Figure 10 is based on Table 17 A graph of volume change data. As shown in Table 17, the tumor volume growth inhibition rates TGI% of each group of CA13-vcMMAE, CA67-vcMMAE, CA149-vcMMAE, CA185-vcMMAE, BA352-Vc-MMAE, CA518-vcMMAE, and hL49-vcMMAE were 55.4% and 60.3 respectively.
  • tumor weight inhibition rate IR% were 59.8%, 65.9%, 61.4%, 55.4%, 68.2%, 65.7%, 56.1% respectively; visible human melanoma Cell SK-MEL-5 mouse transplanted tumor model, under the condition of 3 mg/kg administration, both the tumor volume growth inhibition rate TGI% and the tumor weight inhibition rate IR%, the six anti-CD228- vcMMAE was better than the control group hL49-vcMMAE.
  • Figure 11 shows the efficacy data (5 mg/kg) of each anti-CD228 ADC on the SK-MEL-5 animal model.
  • NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of SK-MEL-5 cells was adjusted to 3 ⁇ 10 7 cells/mL using EMEM medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of NCG mice at a volume of 0.1 mL/mouse.
  • the mice were divided into 8 experimental groups according to their tumor volume and body weight, with 6 mice in each group. Administration was started on the day of grouping, and the dosage was 5 mg/kg.
  • FIG. 11 is a graph based on the volume change data in Table 18. As shown in Table 18, the experimental endpoints of the tumor volume growth inhibition rates TGI of the CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE groups were 82%, 84%, 81%, respectively.
  • each anti-CD228-vcMMAE prepared in this project was better than the control group hL49-vcMMAE in terms of tumor volume growth inhibition rate TGI% and tumor weight inhibition rate IR%.
  • Human lung cancer cells NCI-H226 were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% CO2 .
  • the medium composition was RPMI 1640 medium containing 10% FBS.
  • CB-17/SCID mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
  • NCI-H226 cells The concentration of NCI-H226 cells was adjusted to 5.0 ⁇ 10 7 cells/mL using RPMI 1640 medium containing 50% Matrigel, and the cells were inoculated subcutaneously into the right side of CB-17/SCID mice at a volume of 0.1 mL/mouse.
  • the mice When the average tumor volume reached approximately 156mm3 , the mice were divided into 8 experimental groups according to their tumor volume and body weight, with 5 mice in each group. Administration began on the day of grouping, and the dosage was 3 mg/kg.
  • Figure 12 shows the efficacy data of each anti-CD228 ADC on the NCI-H226 animal model (3 mg/kg).
  • FIG. 12 is a graph based on the volume change data in Table 20.
  • Table 19 the experimental endpoints of the tumor volume growth inhibition rates TGI of each group of CA13-vcMMAE, CA67-vcMMAE, CA149-vcMMAE, CA185-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and hL49-vcMMAE were 46.7% and 55.2% respectively. , 45.3%, 50.5%, 47.7%, 63.4%, 55.1%; the tumor weight inhibition rates were 39.7%, 44.5%, 46.9%, 43.1%, 41.6%, 47.8%, 49.8% respectively.
  • Figure 13 shows the efficacy data of each anti-CD228 ADC on the NCI-H226 animal model (5 mg/kg).
  • the NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of NCI-H226 cells was adjusted to 5 ⁇ 10 7 cells/mL using RPMI 1640 medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of NCG mice at a volume of 0.1 mL/mouse.
  • the mice were divided into 8 experimental groups according to their tumor volume and body weight, with 6 mice in each group. Administration was started on the day of grouping, and the dosage was 5 mg/kg.
  • FIG. 13 is a graph based on the volume change data in Table 20.
  • the experimental endpoints of the tumor volume growth inhibition rates TGI of the CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE groups were 94.4%, 92.3%, 94.6%, respectively. 98.3%, 103.9%, 75.6%; tumor weight inhibition rates were 73.1%, 69.2%, 72.1%, 75.5%, 77.4%, 56.7% respectively.
  • Each ADC was administered subcutaneously to 3 Balb/c mice at a dose of 10 mg/kg at 0 h before administration, 1 h, 4 h, 10 h and 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 10 d, Serum was collected on the 14th day to detect the antibody concentration, and the serum detection method was the Elisa method.
  • Figure 14 records the pharmacokinetic curves of CA13-vcMMAE, CA67-vcMMAE, and CA149-vcMMAE. The specific test results are shown in Figure 14. It can be seen that CA149-vcMMAE has a better pharmacokinetic level than CA13 and CA67 antibody construction. CA13-vcMMAE, CA67-vcMMAE.
  • CA149-BNLD11 inhibits the proliferation of MC38-CD228 cells and A375-CD228 cells
  • the starting concentrations were 60 and 12 ⁇ g/mL respectively, and the 5-fold concentration gradient dilution was performed; the antibody ID in CN202180003751.7 was selected as CA521 antibody usage nCov-CA521-vcMMAE prepared by the same method as Example 8 was used as the control group (Isotype).
  • the diluted CA149-BNLD11 was added to the above-mentioned 96-well flat-bottomed cell culture plate at 50 ⁇ L/well, and cultured at 37°C and 5% CO2 for 96 hours.
  • the kit (Norwezan, DD1101-01,) must be protected from light during use, equilibrated to room temperature, inverted and mixed, added to a 96-well plate at 100 ⁇ L/well, shaken horizontally at 300 rpm for 2 minutes, and then left to stand for 10 minutes. Cell viability was detected with a microplate reader (BioTek, SYNERGY neo, USA).
  • CA149-BNLD11 had excellent proliferation inhibitory activity on MC38-CD228 and A375-CD228 cells.
  • the IC 50 was 101.5ng/mL and 61.8ng/mL respectively.
  • the ordinate of Figures 15A-15B is the viable cell rate %, that is, the ratio of the number of viable cells in the drug group to the number of viable cells in the blank group.
  • CA149-BNLD11 has significant proliferation inhibitory activity on cells expressing CD228 compared to the control group (Isotype).
  • CA149-BNLD11 inhibits the proliferation of SK-MEL-5 cells, A549-CD228 cells, and A375-CD228 cells
  • CA149-BNLD11 had excellent proliferation inhibitory activity on SK-MEL-5, A549-CD228, and A375-CD228 cells (IC50 were 11.48ng/mL, 15.25ng/mL, and 13.86ng/mL, respectively); from Figure 15C -15E It can be seen that CA149-BNLD11 has significant proliferation inhibitory activity on cells expressing CD228 compared to the control group (Isotype).
  • Example 14 Evaluation of the efficacy of the antibody drug conjugate CA149-BNLD11 on transplanted tumors of human lung cancer cells CaLu-1 in nude mice
  • CaLu-1 human lung cancer cells were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% CO2 .
  • the medium composition was McCoy's 5A medium containing 10% FBS.
  • Balb/c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of CaLu-1 cells was adjusted to 5.0 ⁇ 10 7 cells/mL using serum-free McCoy's 5A medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of Balb/c nude mice at a volume of 0.1 mL/mouse. When the average tumor volume reached 135 mm3, the mice were divided into 5 experimental groups according to the tumor volume, with 5 animals in each group. Administration began on the day of the grouping. This experiment adopts single dose administration and is observed for 28 days after administration.
  • Figure 16 is the tumor growth inhibition curve of the antibody drug conjugate group on the Calu-1 lung cancer model.
  • Figure 17 is the tumor weight histogram of the antibody drug conjugate group on the Calu-1 lung cancer model. .
  • each antibody drug conjugate administration group can significantly inhibit the growth of tumor volume, and all have statistical differences (P less than 0.05); CA149 -The tumor inhibitory effects of the -BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly better than those of the nCov-CA521-vccMMAE group (P values were 0.0001, 0.0016, and 0.0006 respectively); CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd There were no significant differences among the three groups (P were all greater than 0.05); the tumor volume growth inhibition rates TGI (%) of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 103.1% and 103.1%, respectively. 93.9%, 97.3%, 48.5%.
  • Example 15 Evaluation of the efficacy of the antibody drug conjugate CA149-BNLD11 on transplanted tumors of human melanoma cells SK-MEL-5 in nude mice
  • SK-MEL-5 human melanoma cells were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% CO2 .
  • the medium composition was EMEM medium containing 10% FBS.
  • Balb/c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of SK-MEL-5 cells was adjusted to 5.0 ⁇ 10 7 cells/mL using serum-free EMEM medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of Balb/c nude mice at a volume of 0.1 mL/cell. When the average tumor volume reached 98 mm3, the mice were divided into 4 experimental groups according to the tumor volume, with 5 animals in each group. Administration began on the day of grouping. This experiment adopts single dose administration and is observed for 28 days after administration.
  • Figure 18 shows the tumor growth inhibition curve of the antibody drug conjugate group in the human melanoma cell SK-MEL-5 nude mouse transplant tumor model.
  • Figure 19 shows the antibody drug conjugate group in the human melanoma cell SK-MEL-5 nude mouse transplant tumor model. Histogram of tumor weight on melanoma cell SK-MEL-5 nude mouse transplanted tumor model.
  • each administration group could significantly inhibit the growth of tumor volume, and all had statistical differences (P was less than ⁇ 0.0001); the tumor inhibitory effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly better than those of the nCov-CA521-vcMMAE group (P were all less than ⁇ 0.0001), and 2/5 mice in the CA149-BNLD11 group had complete tumors.
  • the average tumor weight of all administration groups was significantly lower than that of the vehicle control group (PBS phosphate buffer saline), with statistical differences (all P less than 0.05); CA149-BNLD11, CA149-vcMMAE, There was no significant difference between the four groups of CA149-GGFG-Dxd and nCov-CA521-vcMMAE (P were all greater than 0.05); the tumor weight inhibition rate of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups They are 88.3%, 90.7%, 90.5%, and 58.7% respectively.
  • NUGC4 human gastric cancer cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. The cells were cultured in an incubator at 37°C and 5% CO2 .
  • the medium composition was RMPI-1640 medium containing 10% FBS.
  • Balb/c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of NUGC4 cells was adjusted to 1.8 ⁇ 10 7 cells/mL using serum-free RMPI-1640 medium containing 50% Matrigel, and inoculated subcutaneously into the right side of Balb/c nude mice at a volume of 0.1 mL/mouse. When the average tumor volume reached 108 mm3, the mice were divided into 4 experimental groups according to the tumor volume, with 5 animals in each group. Administration began on the day of grouping. This experiment uses a single dose and is observed for 23 days after administration.
  • Figure 20 is the tumor growth inhibition curve of the antibody drug conjugate group on the human gastric cancer cell NUGC4 Balb/c nude mouse transplanted tumor model.
  • Figure 21 is the tumor growth inhibition curve of the antibody drug conjugate group on the human gastric cancer cell NUGC4 Balb/c nude mouse model. Tumor weight histogram on human gastric cancer cell NUGC4 Balb/c nude mouse xenograft tumor model.
  • the tumor growth curve at the end of the experiment, compared with the vehicle control group (PBS phosphate buffer saline), the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups could significantly inhibit the growth of tumor volume.
  • the tumor weight inhibition rates of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 95.7%, 87.1%, 79.4%, and 14.8% respectively.
  • NCI-H226 human lung squamous cell carcinoma cells were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% CO2 .
  • the medium composition was RMPI-1640 medium containing 10% FBS.
  • Balb/c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of NCI-H226 cells was adjusted to 4.0 ⁇ 10 7 cells/mL using serum-free RMPI-1640 medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of Balb/c nude mice at a volume of 0.1 mL/mouse.
  • mice When the average tumor volume reached 145mm3 , the mice were divided into 4 experimental groups according to the tumor volume, with 6 animals in each group. Administration began on the day of grouping. The dosage is 3.3mg/kg, single administration. The results were analyzed by means and standard errors (Mean ⁇ SEM), and Graphpad 8.0 software was used for data analysis and processing. Tumor volume and weight were analyzed using two-factor analysis of variance to compare the differences between groups at each time point; tumor weight was measured using a single factor. Analysis of variance was used to perform statistical difference analysis; T-test was used to compare between the two groups; P ⁇ 0.05 indicated that the difference was statistically significant.
  • the tumor volume growth curve of human lung squamous cell carcinoma NCI-H226 Balb/c nude mice transplanted tumors is shown: compared with the vehicle control group (PBS phosphate buffer saline), CA149-vcMMAE, CA149-BNLD11 , the CA149-GGFG-Dxd group can significantly inhibit the growth of tumor volume, with statistical differences (all P less than 0.05); the nCov-CA521-vcMMAE group has no tumor inhibitory effect (P4>0.05); CA149-vcMMAE, CA149-BNLD11 There was no statistical difference between the , CA149-GGFG-Dxd groups (P>0.05); the tumor volume growth inhibition rate TGI (%) of the CA149-vcMMAE, CA149-BNLD11, CA149-GGFG-Dxd, nCov-CA521-vcMMAE groups They are 86.6%, 81.1%, 93.5%, and 19.1%
  • SK-MEL-5 human melanoma cells were purchased from ATCC. The cells were cultured in an incubator at 37°C and 5% CO2 .
  • the medium composition was EMEM medium containing 10% FBS.
  • Balb/c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the concentration of SK-MEL-5 cells was adjusted to 3.0 ⁇ 10 7 cells/mL using serum-free EMEM medium containing 50% Matrigel, and then inoculated subcutaneously into the right side of Balb/c nude mice at a volume of 0.1 mL/mouse.
  • the mice were divided into 4 experimental groups according to the tumor volume, with 6 mice in each group.
  • the tumor weight inhibition rates in the 1.0 mg/kg, 2.5 mg/kg, and 5.0 mg/kg groups were 21.9%, 66.7%, and 76.6% respectively.
  • Three ICR mice were each selected and administered CA149-BNLD11 and CA149-vcMMAE by tail vein injection at a dose of 10 mg/kg before and 1h, 6h, 24h, 3d, 5d, 7d, and 10d after administration. , 14d, 21d, and 28d, collect serum, and use ELISA method to detect serum antibody concentration. The specific test results are shown in the table below.
  • CA149-BNLD11 has a longer half-life than CA149-vcMMAE.
  • the total antibody exposure of CA149-BNLD11 was 1.4 times that of CA149-vcMMAE, and the exposure of total ADC of CA149-BNLD11 was 2.3 times that of CA149-vcMMAE.
  • the metabolic curve ( Figure 25) that the shedding rate of BNLD11 in mice is much lower than that of mc-vcMMAE.
  • CA149-BNLD11 is metabolized more stably in mice.
  • mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were divided into 6 experimental groups according to their gender and weight, with 3 mice in each group. Their body weight and food weight were measured on the day of grouping, and administration began two days later. The dosing regimen is shown in Table 24.
  • the trial was terminated on the 14th day of administration.
  • the result analysis is expressed as mean and standard error (Mean ⁇ SEM), and Graphpad 8.0 software is used for data analysis and processing. Body weight and food intake are compared using T-test; P ⁇ 0.05 indicates that the difference is statistically significant.
  • CA149-vcMMAE and CA149-BNLD11 can significantly reduce the body weight of mice.
  • CA149-vcMMAE was significantly more toxic than CA149-BNLD11 (females: P less than 0.05; males: P less than 0.05).
  • vehicle control group PBS phosphate buffer
  • 50mg/kg of CA149-vcMMAE, CA149-BNLD11 Both can significantly reduce the food intake of mice; there is no significant difference between the two groups of CA149-vcMMAE and CA149-BNLD11.
  • CA149-BNLD11 low-dose group 2mg/kg
  • CA149-BNLD11 medium dose group (6mg/kg)
  • CA149-BNLD11 high dose group (10mg/kg)
  • CA149-BNLD11 There is one male animal in the medium-dose group (6 mg/kg), and one female animal in the CA149-BNLD11 high-dose group (10 mg/kg).
  • each group is 5 mg/mL, and the corresponding administration concentrations are 0.4, 1.2, and 2 mg/mL respectively; the low-dose group and the high-dose group are administered twice each, and the medium-dose group is administered once, with intravenous infusion of 30 min/ animal.
  • the other hematological indicators of animals in each group at each measurement time point were basically within the normal range, and there were no changes in dose-effect and time-dependent correlation. No abnormalities related to the administration of the test article were found.
  • CA149-BNLD11 at a dose of 10 mg/kg can cause an increase in serum AST in cynomolgus monkeys 5 days after administration, which can be restored 8 days after administration.
  • the remaining blood biochemical indicators of animals in each group at each measurement time point were basically within the normal range, and no abnormalities related to the administration of the test product were found.
  • the accompanying toxicokinetic test results showed that the toxin shedding rate in cynomolgus monkeys in the 10 mg/kg dose group was significantly lower than that in the 2 mg/kg dose group.
  • the toxin shedding rate in the 10 mg/kg dose group was similar to that in the 6 mg/kg dose group.

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Abstract

一种结合CD228的抗体或其抗原结合片段，及含有所述抗体或其抗原结合片段的抗体药物偶联物，所述抗体或其抗原结合片段对CD228蛋白有强的亲和力和介导ADCC的能力，所述抗体药物偶联物对多种肿瘤展示出抑制效果。编码所述抗体或其抗原结合片段的核酸；含有所述核酸的细胞；含有所述抗体或其抗原结合片段、所述核酸、所述细胞或所述抗体药物偶联物的药物组合物、试剂盒。所述抗体或其抗原结合片段、所述核酸、所述抗体药物偶联物或所述药物组合物在预防、治疗、检测或诊断与 CD228 相关的疾病中的应用。

Description

抗CD228抗体及其药物偶联物 技术领域
本发明涉及生物医学或生物制药技术领域，尤其涉及一种抗CD228抗体及其药物偶联物。
背景技术
CD228(也称为黑色素转铁蛋白、MTF、黑素瘤相关抗原p97、MFI2或MAP97)，是转铁蛋白家族中一个90-97kDa的唾液糖蛋白成员。通常发现CD228通过糖基磷脂酰肌醇锚定物拴在细胞膜上，只有少量可溶蛋白被检测到。
CD228对细胞增殖、迁移和肿瘤发生具有一定作用，CD228表达的增加可导致黑色素瘤肿瘤生长加速；细胞模型中，CD228高表达可增加细胞增殖，而CD228下调导致细胞增殖减少。
CD228在多种瘤中均有表达，如黑色素瘤、间皮瘤、胰腺癌、非小细胞肺癌、乳腺癌、结肠癌等，适应症广。CD228在72％的黑色瘤和79％的胰腺癌中表达，同时在83％的间皮瘤和100％的结肠癌、57％的乳腺癌、69％的鳞癌中表达，有较大的临床需求。
抗体-药物偶联物，通过连接子将抗体与小分子化疗药物偶联在一起，拥有抗体的高度靶向性，又充分发挥化疗药物的细胞毒性，达到高效杀伤肿瘤细胞的作用，目前多个抗体-药物偶联物成功上市、在研产品逐渐增多，技术已成熟。CD228在许多肿瘤组织中高表达，在正常组织中低表达或不表达，基于其表达的差异性，CD228可能是一个理想的抗体-药物偶联物靶点。
因此，提供一种新的CD228抗体-药物偶联物作为有效的抗癌药物，在医药领域具有广泛的应用价值。
发明内容
本发明提供一种抗CD228抗体或其抗原结合片段,能结合CD228蛋白；本发明还提供了编码所述抗体或其抗原结合片段的核酸；含有所述核酸的细胞；含有所述抗体或其抗原结合片段、所述核酸、所述细胞的药物组合物；含有所述抗体或其抗原结合片段、所述核酸、所述药物组合物的试剂盒；所述抗体或其抗原结合片段、所述核酸、所述药物组合物在预防、治疗、检测或诊断与CD228相关的疾病的应用,CD228抗体或其抗原结合片段在制备抗体药物偶联物(ADC)中的应用，以及抗CD228抗体偶联物。
本发明的一个方面提供一种抗CD228抗体或其抗原结合片段，所述抗体或其抗原结合片段包含下述3个轻链互补决定区和/或3个重链互补决定区，
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:13所示的LCDR1、SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:18所示的HCDR3；
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:19所示的LCDR1、SEQ ID NO:20所示的LCDR2和SEQ ID NO:21所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:22所示的HCDR3；
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:23所示的LCDR1、SEQ ID NO:20所示的LCDR2和SEQ ID NO:21所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:22所示的HCDR3；
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:24所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:26所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:27所示的HCDR2和SEQ ID NO:28所示的HCDR3；
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:29所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:30所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:28所示的HCDR3；或者
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:31所示的LCDR1、SEQ ID NO:14所示的LCDR2和SEQ ID NO:21所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:32所示的HCDR3；或者
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:19所示的LCDR1、SEQ ID NO:44所示的LCDR2和SEQ ID NO:45所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:49所示的HCDR3；或者，所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:46所示的LCDR1 SEQ ID NO:47所示的LCDR2和SEQ ID NO:48所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:28所示的HCDR3。
在本发明的一个具体的实施方式中，本发明提供一种抗CD228抗体或其抗原结合片段，所述抗体或其抗原结合片段当与CD228结合时与SEQ ID NO:41所示的以下残基的至少一个结合：E312A,L313A，R282A,R275A。
在本发明的一个具体实施方式中，本发明提供一种抗CD228抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
所述抗体或其抗原结合片段包含SEQ ID NO:1所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区；
所述抗体或其抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:4所示的重链可变区；
所述抗体或其抗原结合片段包含SEQ ID NO:5所示的轻链可变区，和/或SEQ ID NO:6所示的重链可变区；
所述抗体或其抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和/或SEQ ID NO:8所示的重链可变区；
所述抗体或其抗原结合片段包含SEQ ID NO:9所示的轻链可变区，和/或SEQ ID NO:10所示的重链可变区；所述抗体或其抗原结合片段包含SEQ ID NO:11所示的轻链可变区，和/或SEQ ID NO:12所示的重链可变区；所述抗体或其抗原结合片段包含SEQ ID NO:38所示的轻链可变区，和/或SEQ ID NO:37所示的重链可变区；或者
所述抗体或其抗原结合片段包含SEQ ID NO:40所示的轻链可变区，和/或SEQ ID NO:39所示的重链可变区。
在本发明的一个具体实施方式中，所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:33。
进一步的，所述抗体或其抗原结合片段的轻链恒定区的序列为SEQ ID NO:34。
在本发明的方案中，本发明的所述抗体或其抗原结合片段包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
本发明的第二方面提供了一种核酸，编码所述抗体或其抗原结合片段。
本发明的第三方面提供了一种载体，其包含编码所述抗体或其抗原结合片段的所述核酸。所述载体可用于表达所述抗体或其抗原结合片段。优选地，所述载体可以是病毒载体；优选地，所述病毒载体包含但不限于慢病毒载体、腺病毒载体、腺相关病毒载体或逆转录病毒载体等；优选地，所述载体可以是非病毒载体；优选地，所述载体可以是哺乳细胞表达载体；优选地，所述表达载体可以是细菌表达载体；优 选地，所述表达载体可以是真菌表达载体。
本发明的第四方面提供了一种细胞，所述细胞包括所述核酸或所述载体，所述细胞可表达所述抗体或其抗原结合片段。优选地，所述细胞为细菌细胞；优选地，所述细菌细胞为大肠杆菌细胞等；优选地，所述细胞为真菌细胞；优选地，所述真菌细胞为酵母细胞；优选地，所述酵母细胞为毕赤酵母细胞等；优选地，所述细胞为哺乳动物细胞；优选地，所述哺乳动物细胞为中国仓鼠卵巢细胞(CHO)、人胚胎肾细胞(293)、B细胞、T细胞、DC细胞或NK细胞等。
本发明第五个方面提供了一种抗CD228抗体偶联物，其特征在于，所述抗CD228抗体偶联物包括(a)所述的CD228抗体或其抗原结合片段,和(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自可检测标记物、药物、毒素、细胞因子、放射性核素和酶中的一种或多种。
在另一优选例中，所述抗体药物偶联物(ADC)如下式1所示：
其中，式1中的：Ab是本发明抗CD228抗体或其抗原结合片段，LU是接头(又称连接子)，D是药物；下标p对应抗体-药物偶联物的平均DAR值，p是选自1-10，优选的1-8，优选的1-4或4-8的值，更有优选的p为4。
其中，所述药物选自化疗药物、放疗药物、激素治疗药物或免疫治疗药物。可选地，所述药物选自下组：紫杉烷类(taxane)、美登素类(maytansinoid)、喜树碱类(camptothecin)、微管素类(tubulysin)、奥利斯他汀类(auristatin)、卡奇霉素(calicheamicin)、蒽环类药物(anthracycline)、多西他赛(docetaxel)、组织蛋白酶(cathepsin)、蓖麻毒素(ricin)、白树毒素(gelonin)、绿脓杆菌外毒素(Pseudomonas exotoxin)、白喉毒素(diphtheria toxin)、核糖核酸酶(RNase)或放射性同位素(radioisotope)。
进一步的，连接子LU由R’-L1-L2-L3通式组成；
在该通式中，所述L3为：
其中L3的a端与所述药物D相连，b端与所述L2相连；
R1为氢、羧基、酯基、硝基、磺酰基、卤素基团；或R1为R2-R6各自独立的为氢、n为0-8；
在该通式中，所述L2为：其中A各自独立的为苯丙氨酸残基、甘氨酸残基、丙氨酸残基、谷氨酸残基、天冬氨酸残基、半胱氨酸残基、组氨酸残基、 赖氨酸残基、脯氨酸残基、或缬氨酸、胍氨酸残基、β-甘氨酸残基、β丙氨酸残基；X为：n为0-8；
在该通式中，所述L1为：和/或
在该通式中,R’为：其中，R’的c端与L1相连，d端与A相连；
在一优选例中，所述抗体药物偶联物(ADC)式1中的LU-D为VcMMAE,其中LU为Vc(缬氨酸-瓜氨酸接头)，D为MMAE(单甲基auristatin E)；VcMMAE也可以写成MC-Val-Cit-PAB-MMAE或mc-vc-PAB-MMAE。
在一优选例中，所述抗体药物偶联物(ADC)式1中的LU-D为BNLD11，其中LU为MC-β-Ala-(glucuronide)PAB，D为MMAE，所述BNLD11结构如下所示：
所述BNLD11的Exact Mass为1322.690；所述BNLD11采用现有技术常规的方法合成获得；在一个优选的示例中，BNLD11采用图30所示合成路线获得。
在一优选实施例中，所述ADC式1中的抗CD228抗体或其抗原结合片段(Ab)的3个轻链互补决定区包含SEQ ID NO:24所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:26所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:27所示的HCDR2和SEQ ID NO:28所示的HCDR3；优选的，所述抗体或其抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和SEQ ID NO:8所示的重链可变区；更优选的，所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:33和/或轻链恒定区的序列为SEQ ID NO:34。
在一优选的实施例中，所述ADC式1中的LU-D为VcMMAE，p为4，Ab为抗CD228抗体或其抗原结合片段，所述抗CD228抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:24所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:26所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:27所示的HCDR2和SEQ ID NO:28所示的HCDR3；更优选的，所述抗体或其抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和SEQ ID NO:8所示的重链可变区；更优选的，所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:33和/或轻链恒定区的序列为SEQ ID NO:34。
在一优选的实施例中，所述ADC式1中的LU-D为BNLD11结构，p为4，所述BNLD11结构如下所示：
Ab为抗CD228抗体或其抗原结合片段，所述抗CD228抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:24所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:26所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:27所示的HCDR2和SEQ ID NO:28所示的HCDR3；更优选的，所述抗体或其抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和SEQ ID NO:8所示的重链可变区。
本发明的第六方面提供了一种药物组合物，其包含所述的抗体或其抗原结合片段、核酸、载体、细胞或抗体药物偶联物，优选地，所述药物组合物还包含药学上可接受的载体，优选地，所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂或其它药用辅料。
本发明的第七方面提供了一种试剂盒，其包含本发明所述抗体或其抗原结合片段，或包含编码抗体或其抗原结合片段的核酸，包括所述药物组合物或包括所述抗体药物偶联物。
本发明的第八方面提供了所述抗体或其抗原结合片段、核酸、载体、细胞或抗体药物偶联物在制备治疗或预防疾病的药物组合物中的应用。
本发明的第九方面提供了所述抗体或其抗原结合片段或核酸在制备诊断、检测试剂盒中的应用。
本发明的第十方面提供了一种治疗或预防疾病的方法，包括将本发明的所述抗体或抗原结合片段、核酸、载体、细胞、药物组合物或抗体药物偶联物给予有需要的受试者。
本发明的第十一方面提供了一种诊断、检测的方法，包括将本发明的所述抗体或抗原结合片段、核酸、试剂盒或药物组合物给予有需要的受试者或样本。
本发明的第十二方面提供了所述的抗体或其抗原结合片段、核酸、载体、细胞、药物组合物或抗体药物偶联物用于治疗、预防疾病的用途。
本发明的第十三方面提供了所述的抗体或其抗原结合片段、核酸、试剂盒、或药物组合物用于检测、诊断的用途。
本发明的第十四方面提供了所述抗体或其抗原结合片段，所述的核酸，所述的药物组合物或抗体药物偶联物用于制备预防、治疗、检测或诊断与CD228相关的疾病的制剂中的应用。
在本发明的方案中，所述与CD228相关的疾病包括黑色素瘤、肺癌、非小细胞肺癌、胃癌、结肠癌、结肠腺癌、间皮瘤、胰腺癌、乳腺癌中的一种或多种。
本发明的第十五方面还提供了本发明的CD228抗体或其抗原结合片段在制备抗体药物偶联物(ADC)中的应用。
本发明提供的抗CD228抗体及其抗体药物偶联物具有以下的一种或多种优势：
1、本发明提供的抗CD228抗体或其抗原结合片段对CD228蛋白，以及表达CD228蛋白的细胞具有良好的亲和力。
2、本发明提供的抗CD228抗体药物偶联物对人黑色素瘤SK-MEL-5细胞具有良好的杀伤能力。
3、本发明提供的抗CD228抗体药物偶联物对SK-MEL-5、NCI-H226、CALU-1,NUGC4动物模型中均具有良好的抑癌效果，对CD228相关疾病展示出稳定的药效数据。
4、本发明提供的抗CD228抗体药物偶联物在小鼠体内具有良好的药代动力学。
5、本发明提供的抗CD228抗体药物偶联物副作用和毒性小，安全性高。
6.本发明的抗CD228抗体相比于市面已知抗体，如Seagen公司的靶向CD228的抗体hL49有更好的内化作用和内杀效果。
附图说明
图1为实施例1中CD228在TMA芯片上的表达水平。
图2为实施例2中CD228免疫小鼠血清滴度。
图3为实施例4中各抗CD228抗体与人CD228蛋白的结合。
图4为实施例5中各抗CD228抗体与表达CD228蛋白的人黑色素瘤SK-MEL-5细胞结合活性。
图5A为实施例6中各抗CD228抗体基于人黑色素瘤SK-MEL-5细胞的内化实验结果，图5B为实施例6中各抗CD228抗体基于人肺癌细胞系A549-CD228细胞的内化实验结果。
图6为实施例7中各抗CD228抗体基于人黑色素瘤SK-MEL-5细胞的ADCC实验结果。
图7为实施例8中全长hCD228与可溶性抗原sMFI2序列比对结果示意图。
图8为实施例9中CA149-BNLD11 HIC-HPLC图谱。
图9A为实施例10中CA13-VcMMAE基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果,图9B为实施例10中CA67-VcMMAE基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果,图9C为实施例10中CA149-VcMMAE基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果,图9D为实施例10中BA352-VcMMAE基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果,图9E为实施例10中CA518-VcMMAE基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果,图9F为实施例10中CA185-VcMMAE基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果。
图10示出了各抗CD228 ADC在SK-MEL-5动物模型上的药效数据(3mg/kg)。
图11示出了各抗CD228 ADC在SK-MEL-5动物模型上的药效数据(5mg/kg)。
图12为实施例11中各抗CD228 ADC(3mg/kg)在NCI-H226动物模型上的药效数据。
图13为实施例11中各抗CD228 ADC(5mg/kg)在NCI-H226动物模型上的药效数据。
图14为实施例12中各抗-CD228 ADC小鼠体内药代动力学曲线。
图15A为实施例13中CA149-BNLD11对MC38-CD228细胞增殖抑制活性评价,图15B为实施例13中CA149-BNLD11对A375-CD228细胞增殖抑制活性评价,图15C为实施例13中CA149-BNLD11对SK-MEL-5细胞增殖抑制活性评价，图15D为实施例13中CA149-BNLD11对A549-CD228细胞增殖抑制活性评价，图15E为实施例13中CA149-BNLD11对A375-CD228细胞增殖抑制活性评价。
图16为实施例14中抗体药物偶联物在Calu-1肺癌模型上肿瘤生长抑制曲线。
图17为实施例14中抗体药物偶联物在抗体药物偶联物组在Calu-1肺癌模型上肿瘤重量柱状图。
图18为为实施例15中抗体药物偶联物组在人黑色素瘤细胞SK-MEL-5裸鼠移植瘤模型上肿瘤生长抑制曲线。
图19为实施例15中抗体药物偶联物组在人黑色素瘤细胞SK-MEL-5裸鼠移植瘤模型上肿瘤重量柱状图。
图20为实施例16中抗体药物偶联物组在对人胃癌细胞NUGC4 Balb/c nude裸鼠移植瘤模型上肿瘤生长抑制曲线。
图21为实施例16中抗体药物偶联物组在对人胃癌细胞NUGC4 Balb/c nude裸鼠移植瘤模型上肿瘤重量柱状图。
图22为实施例17中人肺鳞癌细胞NCI-H226 Balb/c nude裸鼠移植瘤的肿瘤体积生长曲线。
图23为实施例18中人黑色素瘤细胞SK-MEL-5 Balb/c nude裸鼠移植瘤的肿瘤体积生长曲线。
图24为实施例18中人黑色素瘤细胞SK-MEL-5 Balb/c nude裸鼠移植瘤的肿瘤重量增长曲线。
图25为实施例19中的抗体药物偶联物CA149-BNLD11在小鼠体内的代谢曲线。
图26为实施例20中抗体药物偶联物CA149-BNLD11在雄鼠的毒性研究。
图27为实施例20中抗体药物偶联物CA149-BNLD11在雌鼠的毒性研究。
图28为实施例21的中剂量组6mg/kg和高剂量组10mg/kg食蟹猴给药后体重变化。
图29为实施例21的中剂量组6mg/kg和高剂量组10mg/kg首次给药后毒代检测。
图30为BNLD11合成线路示意图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 CD228表达实验
胆囊癌，结肠癌，乳腺癌，黑色素瘤，肺癌，胆管癌，胰腺癌，宫颈癌，肉瘤，食管癌和胃癌的PDX样本芯片均购买自中美冠科生物技术有限公司。12例间皮瘤病人组织白片分别购买自上海立迪生物技术股份有限公司(4例)和上海芯超生物科技有限公司(8例)。委托中美冠科生物技术有限公司进行所有PDX样本组织芯片和8例购买自上海芯超生物科技有限公司的间皮瘤组织白片的CD228免疫组化染色。委托上海立迪生物技术股份有限公司进行4例间皮瘤组织白片的CD228免疫组化染色。CD228免疫组化抗体购买自Novus Biologicals(货号：NBP1-85777)，一抗稀释浓度为1:200。
免疫组化实验由IHC自动染色机完成(Bond RX automatic IHC&ISH system,Leica)。免疫组化染色结果利用H-score值进行评估。pi代表阳性细胞百分比，i表示染色强度(0：阴性；1：弱染色；2：中等染色；3：强染色)。每例样本进行三次独立评分，最终H-score值取三次的平均数。所有样本IHC评分结果如图1所示。从图1展示的CD228在TMA芯片上的表达水平和表1展示的CD228表达率可以看出CD228在胆囊癌、结肠癌、乳腺癌、黑色素瘤、肺癌、胆管癌、胰腺癌和宫颈癌中，中高表达所占比例较高。
表1.CD228在多种肿瘤中表达水平统计
实施例2.抗CD228单克隆抗体的产生
1.1蛋白生产
将三种蛋白的氨基酸序列(见表2)发至江苏金斯瑞生物科技公司，合成基因。然后转染到CHO细胞中，在37℃\8％CO2\125rpm摇床中培养，瞬时表达10天后收集上清。表达上清用Ni(金斯瑞，L00250)柱纯化，然后用SP柱(GE，17-1087-01)进行精纯，获得人CD228、鼠CD228、猴子CD228蛋白。
表2.三种蛋白的氨基酸序列
1.2小鼠免疫方法
免疫实验用小鼠为山东博安生物技术股份有限公司自主研发的全人抗体转基因小鼠(共免疫10只)。使用山东博安生物技术股份有限公司自主生产的CD228(0.23mg/mL，Boan，20200924,SEQ ID NO:41)抗原蛋白进行免疫。
免疫方式均采用腹部皮下及腹股沟多点注射，抗原蛋白免疫剂量均为20μg/只。首免使用弗氏完全佐剂乳化抗原，二免至四免使用弗氏不完全佐剂乳化抗原。第一批小鼠进行3次免疫和一次加强免疫；第二批小鼠进行4次免疫和一次加强免疫。每次免疫间隔14天，从第二次免疫开始，每次免疫后第7天取外周血血清检测抗体滴度并剔除滴度不合格小鼠。免疫小鼠后，检测小鼠血清滴度结果如图2所示，2500X，12500X，以及62500X代表稀释比例。加强免疫3天后，安乐死小鼠，取脾脏制成单细胞进行建库。
1.3噬菌体库的建立
处死小鼠，解剖取出脾脏，把脾脏用注射器胶塞研磨破碎并用滤网过滤，把滤过的脾细胞冷冻，提取RNA后获得cDNA，噬菌体库的建立依据通常方法进行。构建库的库容数据如表3所示。
表3.各免疫小鼠构建噬菌体库库容
1.4以两种方法进行筛选
1.4.1平板筛选，用CD228-His蛋白(自制)包被平板。第二天加入噬菌体库孵育2h，洗涤4-10次后用洗脱缓冲液洗脱特异性结合的噬菌体。
1.44.2磁珠筛选，将CD228-His蛋白按照试剂盒步骤进行生物素化，再与Thermo的磁珠结合，经BSA封闭后与噬菌体库孵育2h，洗涤4-10次后用洗脱缓冲液洗脱特异性结合的噬菌体。筛选获得抗体克隆及来源见表4。
表4.筛选得到抗CD228抗体来源表
实施例3.完整抗体的分子构建与生产
构建133个阳性克隆IgG1并测序，其中8个lead抗体可变区氨基酸序列如下表5：(CDR区以下划线标识，分析系统为IMGT系统)。本申请实施例中各抗体的可变区序列参见表5，重链和轻链恒定区序列见表6。
表5. 8个抗体的可变区氨基酸序列

通过常规的分子生物学技术PCR(2×Phanta Max Master Mix厂家：Vazyme货号：P515-P1-AA批号：7E512E1)扩增抗体可变区基因，通过同源重组分别将抗体重链可变区基因连接入带有抗体重链恒定区序列的核酸序列的载体pCDNA3.4(Life Technology)，将抗体轻链可变区基因连接入带有抗体轻链恒定区序列的核酸序列的载体pCDNA3.4。
表6.抗体的重链和轻链恒定区氨基酸序列

将测序后的阳性克隆提取质粒后共转染进入HEK293细胞在37℃\8％CO2\125rpm摇床中培养，瞬时表达7天后上清通过Protein A亲和层析，纯化获得抗体，并通过UV280结合理论消光系数确定抗体浓度。
对照抗体Hl49序列根据专利US20200246479A1中序列合成，氨基酸序列见下表7.
表7.对照抗体hL49的氨基酸序列
实施例4.抗CD228单克隆抗体分子的表征
4.1抗-hCD228抗体与人CD228蛋白的结合
用pH 9.6碳酸盐缓冲液(以下简称CBS)稀释人CD228(博安自制，20201014，SEQ ID NO:41)蛋白至0.1μg/mL，包被酶标板，100μL/孔，4℃孵育过夜；洗板后用脱脂奶粉封闭。洗板后每孔加入PBST(磷酸盐缓冲液，索莱宝P1010，+0.05％tween20)稀释好的抗体100μL(稀释好的抗体为用PBST梯度稀释的完整抗体，完整抗体包含Fc和Fab区，是具有恒定区的抗体，起始浓度0.1ug/mL，三倍稀释8个梯度)；洗板后加入羊抗人IgG(H+L)/HRP(1:5000稀释，KPL,474-1006)，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB(北京梅科万德，1001)显色，10min后每孔加入50μL 2M H2SO4终止显色，用酶标仪读取OD450。本实验及以下实验均采用Seagen公司的靶向CD228的抗体hL49作为对照抗体。图3为各抗体与人CD228蛋白的结合曲线。表8显示了根据图3算出的抗体的EC50值，可以看出ELISA水平上6个抗体显示出相似的结合活性，并好于对照抗体HL49。
表8.抗体的EC50值
4.2检测抗体与CD228人、猴、鼠蛋白的亲和力
表9为检测所用CD228蛋白来源
抗体与各CD228蛋白的结合动力学使用基于表面等离振子共振(surface plasmon resonance，SRP)技术的BIAcore 8K仪器测量。ProA芯片捕获CD228抗体2μg/mL，分析各CD228抗体与人、猴、鼠CD228的结合活性。人、猴、鼠CD228蛋白用HBS-EP+缓冲液2倍梯度稀释5个浓度，起始浓度为50nM。Biacore进行CD228蛋白结合动力学分析，拟合计算亲和活性KD值。
表10 Biacore检测抗CD228抗体与人、猴、鼠蛋白的结合
由表10可以看出，所示抗体与人CD228蛋白和猴CD228蛋白有相似的亲和力，与鼠CD228不结合。
4.3抗-hCD228抗体表位分析
His芯片捕获CD228蛋白10μg/mL阈值0.5nm。先结合第一个CD228抗体(30ug/ml)，再分析第二个CD228抗体(30ug/ml)的竞争结合情况。Octet 8K分析抗体2的应答响应情况，分析抗体1和抗体2是否有竞争关系。表11显示了Octet抗体表位应答值。
表11 Octet抗体表位应答值
最终的竞争分析采用以下计算方法：1-应答值/空白值，结果如表12所示，可以看出，BA352和hL49表位相似，其余抗体相互都有竞争，表位相似(数值高于75％，表位具有相关性)。
表12最终的竞争分析结果
实施例5.流式细胞法检测anti-hCD228抗体细胞水平结合活性
96孔圆底板中，加入50μL人黑色素瘤SK-MEL-5细胞(ATCC，HTB-70)，细胞数为7E4/孔，用FACS缓冲液(PBS，博士德生物，货号PYG0021)梯度稀释各抗体，按50μL/孔加入96孔圆底板中，4℃孵育1h。400g离心4min后弃上清，用FACS缓冲液洗1次，加入100μL/孔荧光二抗(Jackson,109545-008)，4℃避光孵育30min，400g离心4min后弃上清，用FACS缓冲液洗1次，再用100μL/孔FACS缓冲液重悬，用流式细胞仪(艾森，NovoCyte 2060)进行检测。结果见图4，图4示出了各抗CD228抗体与人黑色素瘤SK-MEL-5细胞(表达CD228蛋白的细胞)的结合，可以看出6种抗体与SK-MEL-5细胞均有较高的结合活性，且优于hL49；图4中的同型对照抗体为与所述实验组抗CD228抗体具有相同的恒定区、不同的可变区的针对其他靶点的无关抗体。
实施例6.抗-hCD228单克隆抗体分子的内化实验
1.SK-MEL-5细胞
96孔圆底板中，加入50μL/孔用缓冲液(PBS，博士德生物，货号PYG0021)稀释好的人黑色素瘤SK-MEL-5细胞，细胞数为5E4/孔。再用缓冲液稀释抗体，终浓度20μg/mL。将50μL/孔20μg/mL的抗体加入含50μL/孔细胞的圆底板中，孵育30min后，400g、4min离心，去上清。用预冷的缓冲液洗涤2次，再加入100μL/孔缓冲液，分别于37℃、4℃两种温度条件下放置孵育，不同时间点终止，400g离心4min，去上清。再加4℃预冷的荧光二抗(Jackson，109-545-008)，100μL/孔，4℃、避光孵育30min。用预冷FACS缓冲液洗1次，再用100μL/孔FACS缓冲液重悬，用流式细胞仪(艾森，NovoCyte 2060)进行检测。结果见图5A,图5A示出了各抗CD228抗体基于人黑色素瘤SK-MEL-5细胞的内化实验结果，由图5A可以看出与对照hL49相比，CA13、CA149和BA352内化率较高。
2.A549-CD228细胞
将对数生长期的稳定表达外源CD228基因的人肺癌细胞系A549-CD228细胞(康源博创，KC-2150)消化处理后，用含血清培养基终止消化并稀释，加入96孔圆底板(NEST,货号701111)中，50μL/孔，1E5/孔。用含血清培养基稀释抗体，将抗体与标记试剂(invitrogen，Z25611)按1:3摩尔比混合(抗体浓度为40nM，标记试剂为120nM)，室温孵育5min后，将标记抗体混合物加入含细胞的孔板中，每孔加入50μL。置于37℃条件分别孵育0h、2h、6h、24h后，PBS洗涤一次，再用100μL/孔PBS重悬细胞，通过流式细胞仪(艾森，NovoCyte 2060)读取MFI值。结果显示，随时间的增加，CA149抗体内化增加；由图5B可以看出，CA149与对照抗体hL49相比，有更优的内化活性。
实施例7.抗体依赖细胞介导的细胞毒作用(荧光素酶报告基因效应细胞)
准备ADCC工作液(含1％FBS的RPMI1640培养基)；收集Bioassay Effector Cells(Promega，G7011)，用ADCC工作液调整细胞密度为2.4×106；收集Target Cells SK-MEL-5(ATCC，HTB-70)，分别用ADCC工作液调整细胞密度为8×105；用ADCC工作液稀释待检样品，从5ug/mL起始浓度开始，4倍稀释8个浓度；将效应细胞，靶细胞和待检样品各25uL，分别加到反应白板中(Costar，3917)，总反应体积75uL，将该反应体系于37度孵育6h；每孔加75uL Bio-Glo Luciferase System(Promega，G7940)，反应15min后用酶标仪读取化学发光值(BioTek，synergy neo2)。结果见图6，图6示出了各抗CD228抗体基于人黑色素瘤SK-MEL-5细胞的ADCC实验结果，由图6可以看出，随着抗体样品浓度的增加，信号逐渐增强，表明其对SK-MEL-5细胞有ADCC效应。
实施例8 CA149抗体与CD228结合表位研究
我们分别制备了全长抗原CD228和抗体Fab复合物(CA149-Fab)，并委托水木未来(杭州)科技有限公司冷冻电镜中心进行了抗原抗体结构分析。实验人员通过对三维模型表位处氨基酸类型与侧链相互作用进行分析，结果表明CA149抗体Fab通过7个氢键和1个盐桥与抗原结合。具体互作位点如表13所 示，其中上标*为CA149Fab轻链上氨基酸，无上标斜体为CA149Fab重链上氨基酸。
表13互作位点
同时，我们根据表位结果对山东博安生物技术股份有限公司自主生产的CD228(序列如SEQ ID NO:41所示)抗原进行特定位点突变验证，分别构建了hCD228(R275A)、hCD228(R282A)、hCD228(E312A)、hCD228(L313A)单点突变以及hCD228(E312A,L313A)、hCD228(R282A,E312A)、hCD228(R275A,R282A)双点突变hCD228抗原，并进行了亲和力分析。亲和力结果显示突变后的抗原与CA149抗体结合活性降低或者失去结合，也表明上述E312A,L313A，R282A,R275A 4个位点为抗体与抗原结合的关键位点。亲和力检测结果记载在表14。
表14亲和力检测
另外，由于人体内同时存在另一可变剪切产物可溶性MFI2，即sMFI2，并且有文献(J Neurochem.2002Nov；83(4):924-33.doi:10.1046/j.1471-4159.2002.01201.x.；J Cereb Blood Flow Metab.2019 Oct；39(10):2074-2088.doi:10.1177/0271678X18772998)表明sMFI可通过LRP蛋白穿过血脑屏障。所以为了降低可能的脱靶风险，靶向CD228抗体不应该结合sMFI2。根据表位结果以及全长hCD228与可溶性抗原sMFI2序列比对结果(如图7)，我们可以看出上述4个结合关键位点不在sMFI2抗原上，也就是CA149抗体不会与sMFI2结合。
实施例9.抗-hCD228抗体与药物的偶联实验
制备如下分子式所示的抗体药物偶联物，
其中：Ab是上述任意一种抗CD228抗体或其抗原结合片段，LU是接头(又称连接子)，D是药物，下标p为抗体-药物偶联物的平均DAR值。
9.1抗-hCD228抗体-vcMMAE抗体药物偶联物制备
将含抗体(5-10mg/mL)的磷酸盐缓冲液(pH 7.5，含有11mM DTPA)用2当量TCEP处理后在25℃下孵育约2h；将5当量的vcMMAE的DMSO溶液添加至经还原的抗体的PBS溶液中，25℃下孵育约1h；而后将10当量的n-乙酰基半胱氨酸(NAC)添加至混合物25℃下孵育5min以淬灭所有未反应的连接子-药物；超滤置换缓冲液，去除游离小分子。并送HIC-HPLC分析。分析结果见表15，可以看出，本申请中所用到的抗体-药物偶联物的平均DAR值在4.01-4.42之间。
表15 DC HIC-HPLC结果
9.2 CA149-BNLD11抗体药物偶联物制备
以抗CD228抗体CA149为例，制备均质药物负荷(DAR)为4左右的ADC组合物。将含抗体(5-10mg/mL)的磷酸盐缓冲液(pH 7.5，含有11mM DTPA)用2当量TCEP处理后在25℃下孵育约2h。将5当量的BNLD-11的DMSO溶液添加至经还原的抗体的PBS溶液中，25℃下孵育约1h；而后将10当量的n-乙酰基半胱氨酸(NAC)添加至混合物25℃下孵育5min以淬灭所有未反应的连接子-药物。HIC-HPLC分析来量化抗体药物偶联物的药物荷载。分析结果见图8和表16，可以看出，本实施例制备得到的抗体药物偶联物CA149-BNLD11的平均DAR值约为4。
表16经积分分析后DAR值如下表所示：
实施例10.抗-hCD228 ADC的体外细胞杀伤实验
96孔平底板(Corning，货号3917)中，加入50μL/孔SK-MEL-5细胞，细胞数为1E4/孔，培养基为10％FBS/EMEM。用上述培养基稀释各抗CD228-ADC，起始浓度为1μg/mL，4倍浓度梯度稀释，共6个浓度。稀释后的ADC按50μL/孔加入96孔平底板中，置于37℃、5％CO2条件培养4天后，CellTiter-Glo试剂盒(Promega，G7571，使用过程中须避光)平衡至室温，并将试剂盒中缓冲液与Substrate混匀，静置1h。将细胞活力检测试剂加入96孔板中，每孔100μL，300rpm水平震荡2min后，静置10min。酶标仪检测(BioTek,SYNERGY neo,USA)。
结果如图9A-图9F所示，图9A-图9F示出了各抗CD228 ADC基于人黑色素瘤SK-MEL-5细胞的杀伤实验结果，可以看出2种抗-hCD228 ADC：CA13-vcMMAE和CA67-vcMMAE，显示出与hL49-vcMMAE相似的体外细胞杀伤活性；4种抗-hCD228 ADC CA149-vcMMAE、CA352-vcMMAE、CA518-vcMMAE、CA185-vcMMAE显示出优于对照组hL49-vcMMAE相似的体外细胞杀伤活性；图9A-图9F中同型对照抗体为与所述6种抗体恒定区相同，可变区不同的针对其他靶点的无关抗体。
实施例11.抗-CD228 ADC在小鼠移植瘤上的体内药效实验
11.1人黑色素瘤细胞SK-MEL-5小鼠移植瘤模型上的药效数据
人黑色素瘤细胞SK-MEL-5购买自ATCC，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的EMEM培养基。NOD/SCID小鼠购买自北京维通利华实验动物技术有限公司。用含有50％Matrigel的EMEM培养基将SK-MEL-5细胞浓度调整为2.5×107个/mL，以0.1mL/只体积接种于NOD/SCID小鼠的右侧皮下。当平均肿瘤体积达到约76mm3时，根据小鼠肿瘤体积和体重将小鼠分为8个实验组，每组5只，分组当天开始给药，给药剂量为3mg/kg。图10示出了各抗CD228 ADC在SK-MEL-5动物模型上的药效数据(3mg/kg)，即给药后小鼠肿瘤体积变化，可以看出6种抗体给药后，肿瘤体积均变小。图10是根据表17 的体积变化数据作的图。如表17所示，CA13-vcMMAE、CA67-vcMMAE、CA149-vcMMAE、CA185-vcMMAE、BA352-Vc-MMAE、CA518-vcMMAE、hL49-vcMMAE各组肿瘤体积生长抑制率TGI％分别为55.4％、60.3％、56.9％、56.1％、69.8％、63.9％、49.0％；瘤重抑制率IR％分别为59.8％、65.9％、61.4％、55.4％、68.2％、65.7％、56.1％；可见人黑色素瘤细胞SK-MEL-5小鼠移植瘤模型，在3mg/kg给药条件下，无论是肿瘤体积生长抑制率TGI％、还是瘤重抑制率IR％方面，本项目制备的6个抗-CD228-vcMMAE均优于对照组hL49-vcMMAE。
表17. 3mg/kg给药肿瘤体积变化
图11示出了各抗CD228 ADC在SK-MEL-5动物模型上的药效数据(5mg/kg)，NCG小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的EMEM培养基将SK-MEL-5细胞浓度调整为3×107个/mL，以0.1mL/只体积接种于NCG小鼠的右侧皮下。当平均肿瘤体积达到约83mm3时，根据小鼠肿瘤体积和体重将小鼠分为8个实验组，每组6只，分组当天开始给药，给药剂量为5mg/kg。筛选的抗体与对照hL49相比，具有比较好的药效。图11是根据表18的体积变化数据作的图。如表18所示，实验终点CA13-vcMMAE、CA149-vcMMAE、CA518-vcMMAE、CA523-vcMMAE、CA579-vcMMAE、hL49-vcMMAE各组肿瘤体积生长抑制率TGI分别为82％、84％、81％、80％、81％、74％；瘤重抑制率分别为77.7％、79.0％、75.5％、75.2％、72.0％、69.1％；可见人黑色素瘤细胞SK-MEL-5小鼠移植瘤模型，在5mg/kg给药条件下，无论是肿瘤体积生长抑制率TGI％、还是瘤重抑制率IR％方面，本项目制备的各个抗-CD228-vcMMAE均优于对照组hL49-vcMMAE。
表18. 5mg/kg给药肿瘤体积变化
11.2人NCI-H226肺癌细胞/小鼠移植瘤模型上的药效数据
人肺癌细胞NCI-H226购买自ATCC，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的RPMI 1640培养基。CB-17/SCID小鼠购买自北京维通利华实验动物技术有限公司。
用含有50％Matrigel的RPMI 1640培养基将NCI-H226细胞浓度调整为5.0×107个/mL，以0.1mL/只体积接种于CB-17/SCID小鼠的右侧皮下。当平均肿瘤体积达到约156mm3时，根据小鼠肿瘤体积和体重将小鼠分为8个实验组，每组5只，分组当天开始给药，给药剂量为3mg/kg。图12示出了各抗CD228 ADC在NCI-H226动物模型上的药效数据(3mg/kg)。可以看出6种抗体给药后，肿瘤体积均变小，显示出与HL49 相似的体内药效。图12是根据表20的体积变化数据作的图。如表19所示，实验终点CA13-vcMMAE、CA67-vcMMAE、CA149-vcMMAE、CA185-vcMMAE、CA352-vcMMAE、CA518-vcMMAE、hL49-vcMMAE各组肿瘤体积生长抑制率TGI分别为46.7％、55.2％、45.3％、50.5％、47.7％、63.4％、55.1％；瘤重抑制率分别为39.7％、44.5％、46.9％、43.1％、41.6％、47.8％、49.8％。
表19. 3mg/kg给药肿瘤体积变化
图13示出了各抗CD228 ADC在NCI-H226动物模型上的药效数据(5mg/kg)，NCG小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的RPMI 1640培养基将NCI-H226细胞浓度调整为5×107个/mL，以0.1mL/只体积接种于NCG小鼠的右侧皮下。当平均肿瘤体积达到约108mm3时，根据小鼠肿瘤体积和体重将小鼠分为8个实验组，每组6只，分组当天开始给药，给药剂量为5mg/kg。筛选的抗体与对照HL49相比，具有比较好的药效。图13是根据表20的体积变化数据作的图。如表16所示，实验终点CA13-vcMMAE、CA149-vcMMAE、CA518-vcMMAE、CA523-vcMMAE、CA579-vcMMAE、hL49-vcMMAE各组肿瘤体积生长抑制率TGI分别为94.4％、92.3％、94.6％、98.3％、103.9％、75.6％；瘤重抑制率分别为73.1％、69.2％、72.1％、75.5％、77.4％、56.7％。
表20. 5mg/kg给药肿瘤体积变化
实施例12.抗-CD228 ADC小鼠体内药代动力学实验
每个ADC选3只Balb/c小鼠皮下注射给药，剂量为10mg/kg，给药前0h、给药后1h、4h、10h及1d、2d、3d、4d、5d、7d、10d、14d采血清检测抗体浓度，检测血清方法为Elisa法。
图14记载CA13-vcMMAE、CA67-vcMMAE、CA149-vcMMAE的药代动力学曲线，检测具体结果见图14，可以看出CA149-vcMMAE具有较好的药代动力学水平优于CA13、CA67抗体构建的CA13-vcMMAE、CA67-vcMMAE。
实施例13.抗体药物偶联物CA149-BNLD11的细胞增殖抑制活性
13.1 CA149-BNLD11对MC38-CD228细胞、A375-CD228细胞增殖抑制活性
将对数生长期的MC38-CD228(康源博创，KC-2023)、A375-CD228细胞(康源博创，KC-2110)消化处理后，用10％FBS/1640培养基稀释并重悬，加入96孔平底板(SARSTED,货号94.6120.096)中，50μL/孔，1E4/孔。取实施例8的8.2部分制备得到的抗体药物偶联物CA149-BNLD11用含血清培养基稀释，起始浓度分别为60、12μg/mL，5倍浓度梯度稀释；选择CN202180003751.7中抗体ID为CA521的抗体使用 实施例8相同方法制备的nCov-CA521-vcMMAE作为对照组(Isotype)。稀释后的CA149-BNLD11按50μL/孔加入上述96孔平底细胞培养板中，置于37℃、5％CO2条件培养96h，将试剂盒(诺唯赞，DD1101-01，)使用过程中须避光，平衡至室温，颠倒混匀，加入96孔板中，100μL/孔，300rpm水平震荡2min后，静置10min。酶标仪检测(BioTek,SYNERGY neo,USA)细胞活力。
结果显示，CA149-BNLD11对MC38-CD228、A375-CD228细胞有优异的增殖抑制活性，如表21所示IC50分别为101.5ng/mL和61.8ng/mL。图15A-15B其纵坐标为活细胞率％，即用药组活细胞数与空白组活细胞数的比值。由图15A-15B可以看出，CA149-BNLD11相对于对照组(Isotype)对表达CD228的细胞的具有显著的增殖抑制活性。
表21.CA149-BNLD11增殖抑制活性数据
13.2 CA149-BNLD11对SK-MEL-5细胞、A549-CD228细胞、A375-CD228细胞增殖抑制活性
将对数生长期的SK-MEL-5(ATCC，HTB-70)、A549-CD228(康源博创，KC-2150)、A375-CD228细胞(康源博创，KC-2110)消化处理后，用含血清培养基终止消化并稀释，加入96孔平底板(SARSTED,货号94.6120.096)中，50μL/孔，1E4/孔。用含血清培养基稀释CA149-BNLD11，起始浓度分别为1.2、60、6μg/mL，分别5倍、6倍、5倍梯度稀释。稀释后的ADC按50μL/孔加入上述96孔平底细胞培养板中，置于37℃、5％CO2条件培养96h或120h后，将试剂盒(诺唯赞，DD1101-01，使用过程中须避光)平衡至室温，颠倒混匀，加入96孔板中，100μL/孔，300rpm水平震荡2min后，静置10min。酶标仪检测(BioTek,SYNERGY neo,USA)细胞活力。结果显示，CA149-BNLD11对SK-MEL-5、A549-CD228、A375-CD228细胞有优异的增殖抑制活性(IC50分别为11.48ng/mL、15.25ng/mL和13.86ng/mL)；由图15C-15E可以看出，CA149-BNLD11相对于对照组(Isotype)对表达CD228的细胞的具有显著的增殖抑制活性。
表22 CA149-BNLD11对表达CD228的细胞的增殖抑制活性数据
实施例14.抗体药物偶联物CA149-BNLD11对人肺癌细胞CaLu-1裸鼠移植瘤的疗效评价
CaLu-1人肺癌细胞购买自ATCC，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的McCoy's 5A培养基。Balb/c nude裸小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的无血清McCoy's 5A培养基将CaLu-1细胞浓度调整为5.0×107个/mL，以0.1mL/只体积接种于Balb/c nude裸小鼠的右侧皮下。当平均肿瘤体积达到135mm3时，根据小鼠肿瘤体积将小鼠分为5个实验组，每组5只，分组当天开始给药。本实验采用单剂量给药，给药后观察28天。
结果记载在图16-17中，图16为抗体药物偶联物组在Calu-1肺癌模型上肿瘤生长抑制曲线，图17为抗体药物偶联物组在Calu-1肺癌模型上肿瘤重量柱状图。
如图16所示，与溶媒对照组(PBS磷酸盐缓冲液)相比，各抗体药物偶联物给药组均能显著抑制肿瘤体积增长，均具有统计学差异(P均小于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组抑瘤效果显著优于nCov-CA521-vccMMAE组(P值分别为0.0001、0.0016、0.0006)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd三组之间未见显著差异(P均大于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的肿瘤体积生长抑制率TGI(％)分别为103.1％、93.9％、97.3％、48.5％。
如图17所示，实验终点时，CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组平均肿瘤重量显著低于溶媒对照组(PBS磷酸盐缓冲液)，具有统计学差异(P均小于0.05)；nCov-CA521-vcMMAE组与对照 组瘤重无统计学差异(P＝0.3250)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd三组之间未见显著差异(P均大于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的瘤重抑制率分别为96.3％、87.3％、87.6％、44.3％。
实验过程中，实验动物在给药期间活动和进食状态良好，体重均有一定程度的上升，表示动物对受试品耐受性良好。各组之间相比均无显著差异(P>0.05)。
实例15.抗体药物偶联物CA149-BNLD11对人黑色素瘤细胞SK-MEL-5裸鼠移植瘤的疗效评价
SK-MEL-5人黑色素瘤细胞购买自ATCC，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的EMEM培养基。Balb/c nude裸小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的无血清EMEM培养基将SK-MEL-5细胞浓度调整为5.0×107个/mL，以0.1mL/只体积接种于Balb/c nude裸小鼠的右侧皮下。当平均肿瘤体积达到98mm3时，根据小鼠肿瘤体积将小鼠分为4个实验组，每组5只，分组当天开始给药。本实验采用单剂量给药，给药后观察28天。
结果记载在图18-19中，图18为抗体药物偶联物组在人黑色素瘤细胞SK-MEL-5裸鼠移植瘤模型上肿瘤生长抑制曲线，图19为抗体药物偶联物组在人黑色素瘤细胞SK-MEL-5裸鼠移植瘤模型上肿瘤重量柱状图。如图18所示的肿瘤生长曲线：实验终点时，与溶媒对照组(PBS磷酸盐缓冲液)相比，各给药组均能显著抑制肿瘤体积增长，均具有统计学差异(P均小于<0.0001)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组抑瘤效果显著优于nCov-CA521-vcMMAE组(P均小于<0.0001)，且CA149-BNLD11组有2/5小鼠肿瘤完全清除；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd三组之间未见统计学差异(P均大于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的肿瘤体积生长抑制率TGI(％)分别为95.4％、99.5％、99.2％、58.3％。
如图19所示，实验终点，所有给药组平均肿瘤重量均显著低于溶媒对照组(PBS磷酸盐缓冲液)，具有统计学差异(P均小于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE四组之间无显著差异(P均大于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的瘤重抑制率分别为88.3％、90.7％、90.5％、58.7％。
实验过程中，实验动物在给药期间活动和进食状态良好，除溶媒对照组(PBS磷酸盐缓冲液)外，其余各组体重均有一定程度的上升，表示动物对受试品耐受性良好。各组之间相比均无显著差异(P>0.05)。
实例16.抗体药物偶联物CA149-BNLD11对人胃癌细胞NUGC4 Balb/c nude裸鼠移植瘤的疗效评价
NUGC4人胃癌细胞购买自康源博创生物科技(北京)有限公司，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的RMPI-1640培养基。Balb/c nude裸小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的无血清RMPI-1640培养基将NUGC4细胞浓度调整为1.8×107个/mL，以0.1mL/只体积接种于Balb/c nude裸小鼠的右侧皮下。当平均肿瘤体积达到108mm3时，根据小鼠肿瘤体积将小鼠分为4个实验组，每组5只，分组当天开始给药。本实验采用单剂量给药，给药后观察23天。
结果记载在图20-21中，图20为抗体药物偶联物组在对人胃癌细胞NUGC4 Balb/c nude裸鼠移植瘤模型上肿瘤生长抑制曲线，图21为抗体药物偶联物组在对人胃癌细胞NUGC4 Balb/c nude裸鼠移植瘤模型上肿瘤重量柱状图。
如图20肿瘤增长曲线所示：实验终点时，与溶媒对照组(PBS磷酸盐缓冲液)相比，CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组均能显著抑制肿瘤体积增长，均具有统计学差异(P均小于<0.0001)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组抑瘤效果显著优于nCov-CA521-vcMMAE组(P均小于<0.0001)，CA149-BNLD11组抑瘤效果显著优于CA149-GGFG-Dxd组(P＝0.0225)；CA149-vcMMAE组与CA149-GGFG-Dxd组之间无显著差异(P＝0.1902)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的肿瘤体积生长抑制率TGI(％)分别为104.2％、97.7％、81.0％、23.0％。
如图21所示：实验终点时，CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组组平均肿瘤重量显著低于溶媒对照组(PBS磷酸盐缓冲液)，具有统计学差异(P均小于等于0.0001)；nCov-CA521-vcMMAE组与对照组瘤重无统计学差异(P＝0.8144)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd组瘤重均显著小于nCov-CA521-vcMMAE组(P均小于0.05)；CA149-BNLD11、CA149-vcMMAE、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的瘤重抑制率分别为95.7％、87.1％、79.4％、14.8％。
实验过程中，实验动物在给药期间活动和进食状态良好，体重均有一定程度的上升，表示动物对受试品耐受性良好。各组之间相比均无显著差异(P>0.05)。
实施例17抗体药物偶联物CA149-BNLD11对人肺鳞癌细胞NCI-H226 Balb/c nude裸鼠移植瘤的疗效评价
NCI-H226人肺鳞癌细胞购买自ATCC，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的RMPI-1640培养基。Balb/c nude裸小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的无血清RMPI-1640培养基将NCI-H226细胞浓度调整为4.0×107个/mL，以0.1mL/只体积接种于Balb/c nude裸小鼠的右侧皮下。当平均肿瘤体积达到145mm3时，根据小鼠肿瘤体积将小鼠分为4个实验组，每组6只，分组当天开始给药。给药剂量分别为3.3mg/kg，单次给药。结果分析用平均数和标准误表示(Mean±SEM)，采用Graphpad 8.0软件进行数据分析和处理，肿瘤体积、体重采用双因素方差分析比较每个时间点各组间的差异；肿瘤重量使用单因素方差分析进行统计学差异分析；两组之间使用T-test进行比较；P<0.05表示差异具有统计学显著性意义。
在分组给药第24天结束试验。如图22记载的人肺鳞癌细胞NCI-H226 Balb/c nude裸鼠移植瘤的肿瘤体积生长曲线所示：与溶媒对照组(PBS磷酸盐缓冲液)相比，CA149-vcMMAE,CA149-BNLD11,CA149-GGFG-Dxd组均能显著抑制肿瘤体积增长，均具有统计学差异(P均小于0.05)；nCov-CA521-vcMMAE组没有抑瘤效果(P4＞0.05)；CA149-vcMMAE、CA149-BNLD11、CA149-GGFG-Dxd组之间未见统计学差异(P＞0.05)；CA149-vcMMAE、CA149-BNLD11、CA149-GGFG-Dxd、nCov-CA521-vcMMAE组的肿瘤体积生长抑制率TGI(％)分别为86.6％、81.1％、93.5％、19.1％。
实施例18抗体药物偶联物CA149-BNLD11对人黑色素瘤细胞SK-MEL-5 Balb/c nude裸鼠移植瘤的多剂量疗效评价
SK-MEL-5人黑色素瘤细胞购买自ATCC，细胞培养在37℃、5％CO2的培养箱中，培养基成分为含有10％FBS的EMEM培养基。Balb/c nude裸小鼠购买自江苏集萃药康生物科技股份有限公司。用含有50％Matrigel的无血清EMEM培养基将SK-MEL-5细胞浓度调整为3.0×107个/mL，以0.1mL/只体积接种于Balb/c nude裸小鼠的右侧皮下。当平均肿瘤体积达到103mm3时，根据小鼠肿瘤体积将小鼠分为4个实验组，每组6只，分组当天开始给药，给药剂量分别为1.0mg/kg、2.5mg/kg、5.0mg/kg，均为单次给药。结果分析用平均数和标准误表示(Mean±SEM)，采用Graphpad 8.0软件进行数据分析和处理，肿瘤体积、体重采用双因素方差分析比较每个时间点各组间的差异；肿瘤重量使用单因素方差分析进行统计学差异分析；两组之间使用T-test进行比较；P<0.05表示差异具有统计学显著性意义。
分组给药第21天结束试验。如图23人黑色素瘤细胞SK-MEL-5 Balb/c nude裸鼠移植瘤的肿瘤体积生长曲线所示：与溶媒对照组(PBS磷酸盐缓冲液)相比，2.5mg/kg、5.0mg/kg组均能显著抑制肿瘤体积增长，均具有统计学差异(P均小于0.05)；1.0mg/kg组与溶媒对照组(PBS磷酸盐缓冲液)相比无显著差异(P＝0.4030)；CA149-BNLD11 1.0mg/kg、2.5mg/kg、5.0mg/kg组的肿瘤体积生长抑制率TGI(％)分别为25.1％、72.1％、91.1％。，可以看出CA149-BNLD11抑瘤活性呈现剂量依赖性。
试验结束时，如图24为人黑色素瘤细胞SK-MEL-5 Balb/c nude裸鼠移植瘤的肿瘤重量增长曲线所示：与溶媒对照组(PBS磷酸盐缓冲液)相比，2.5mg/kg、5.0mg/kg组肿瘤重量显著降低，具有统计学差异(P均小于0.05)；1.0mg/kg组与溶媒对照组(PBS磷酸盐缓冲液)相比无显著差异(P＝0.7086)；CA149-BNLD11 1.0mg/kg、2.5mg/kg、5.0mg/kg组的瘤重抑制率分别为21.9％、66.7％、76.6％。
实验过程中，实验动物在给药期间活动和进食状态良好，各组体重均有一定程度的上升，表示动物对受试品耐受性良好。各组之间相比均无显著差异(P>0.05)
实施例19抗体药物偶联物CA149-BNLD11在小鼠体内的代谢研究
各选3只ICR小鼠分别尾静脉注射给药CA149-BNLD11和CA149-vcMMAE，给药剂量为10mg/kg，于给药前、给药后1h、6h、24h、3d、5d、7d、10d、14d、21d、28d采集血清，采用ELISA方法检测血清抗体浓度，具体检测结果见下表。
表23 ICR小鼠中CA149-BNLD11和CA149-vcMMAE关键代谢参数
由表23和图25展示的小鼠体内代谢研究，可以看出CA149-BNLD11比CA149-vcMMAE半衰期更长。CA149-BNLD11总抗体的暴露量是CA149-vcMMAE的1.4倍，CA149-BNLD11总ADC的暴露量是CA149-vcMMAE的2.3倍。另外，从代谢曲线(图25)也看得出，小鼠体内BNLD11的脱落率比mc-vcMMAE低得多。综上所述，CA149-BNLD11在小鼠体内代谢更稳定。
实施例20抗体药物偶联物CA149-BNLD11在小鼠体内的毒性研究
Balb/c小鼠购买自济南朋悦实验动物繁育有限公司。根据小鼠性别、体重将小鼠分为6个实验组，每组3只，分组当天称体重和食物重量，两天后开始给药。给药方案如表24所示。
表24小鼠体内给药方案
在给药第14天结束试验。结果分析用平均数和标准误表示(Mean±SEM)，采用Graphpad 8.0软件进行数据分析和处理，体重、摄食均使用T-test进行比较；P<0.05表示差异具有统计学显著性意义。
如图26-27展示，与溶媒对照组(PBS磷酸盐缓冲液)相比，50mg/kg的CA149-vcMMAE、CA149-BNLD11均能显著降低小鼠体重。最大体重下降幅度均出现在给药后第4天。雌性最大体重下降分别为17.44％和10.04％；雄性最大体重下降分别为17.97％和5.24％；CA149-vcMMAE组雌/雄小鼠在给药后第4、6天均出现了不同程度的炸毛、萎靡症状，给药后第8天恢复正常；而CA149-BNLD11组小鼠均正常，无异常反应。在给药后第4、6天，CA149-vcMMAE毒性显著强于CA149-BNLD11(雌性：P均小于0.05；雄性：P均小于0.05)，与溶媒对照组(PBS磷酸盐缓冲液)相比，50mg/kg的CA149-vcMMAE、CA149-BNLD11 均能显著降低小鼠摄食量；CA149-vcMMAE和CA149-BNLD11两组之间没有显著差异。
实施例21抗体药物偶联物CA149-BNLD11在食蟹猴体内的预毒理实验及伴随毒代研究
挑选4只食蟹猴，雌、雄各半，给药开始时雄性体重为3.1～3.7kg，雌性体重为2.9～3.7kg，共分3组，分别为CA149-BNLD11低剂量组(2mg/kg)、CA149-BNLD11中剂量组(6mg/kg)、CA149-BNLD11高剂量组(10mg/kg)，其中CA149-BNLD11低剂量组(2mg/kg)有雌、雄动物各一只，CA149-BNLD11中剂量组(6mg/kg)设1只雄性动物，CA149-BNLD11高剂量组(10mg/kg)设1只雌性动物。每组给药容积为5mg/mL，相应给药浓度分别为0.4、1.2、2mg/mL；低剂量组和高剂量组各给药2次，中剂量组单次给药，静脉输注30min/动物。
给药后，除了进行临床观察、摄食体重检测、血液学和血生化检测外，还进行了伴随毒代动力学研究，采血点包括中剂量6mg/kg和高剂量10mg/kg组首次给药前及给药开始后0.5h、2h、6h、24h、72h、120h、168h、240h、336h、504h。
实验期间，各组别无动物濒死、死亡，各剂量组动物均未见异常。各组别动物体重总体波动幅度不大，未见与供试品给药相关的异常。各组别动物摄食量呈无规律性变化，未见与供试品给药相关的异常。如图28所示：与给药前相比，6mg/kg及以上剂量的CA149-BNLD11给药后5～14天可致食蟹猴血液学指标WBC、#NEUT、％NEUT下降，给药后21天有恢复趋势。除此之外，各组别动物各测定时点其余血液学指标基本在正常范围内，无量效及时效相关性改变。未见与供试品给药相关的异常。与给药前相比，10mg/kg剂量的CA149-BNLD11给药后5天可致食蟹猴血清AST有升高趋势，给药后8天可恢复。除此之外，各组别动物各测定时点其余血生化项指标基本在正常范围内，未见与供试品给药相关的异常。
如图29所示，伴随毒代动力学检测结果显示，在食蟹猴中10mg/kg剂量组毒素脱落率明显低于2mg/kg剂量组。10mg/kg剂量组毒素脱落率与6mg/kg剂量组相近。

Claims (12)

  1. 一种抗CD228抗体或其抗原结合片段，所述抗体或其抗原结合片段包含3个轻链互补决定区和3个重链互补决定区，其中
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:13所示的LCDR1、SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:18所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:19所示的LCDR1、SEQ ID NO:20所示的LCDR2和SEQ ID NO:21所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:22所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:23所示的LCDR1、SEQ ID NO:20所示的LCDR2和SEQ ID NO:21所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:22所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:24所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:26所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:27所示的HCDR2和SEQ ID NO:28所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:29所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:30所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:28所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:31所示的LCDR1、SEQ ID NO:14所示的LCDR2和SEQ ID NO:21所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:32所示的HCDR3；
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:19所示的LCDR1、SEQ ID NO:44所示的LCDR2和SEQ ID NO:45所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:49所示的HCDR3；或者，
    所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:46所示的LCDR1SEQ ID NO:47所示的LCDR2和SEQ ID NO:48所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2和SEQ ID NO:28所示的HCDR3。
  2. 根据权利要求1所述抗体或其抗原结合片段，其特征在于，
    所述抗体或其抗原结合片段包含SEQ ID NO:1所示的轻链可变区，和SEQ ID NO:2所示的重链可变区；
    所述抗体或其抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和SEQ ID NO:4所示的重链可变区；
    所述抗体或其抗原结合片段包含SEQ ID NO:5所示的轻链可变区，和SEQ ID NO:6所示 的重链可变区；
    所述抗体或其抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和SEQ ID NO:8所示的重链可变区；
    所述抗体或其抗原结合片段包含SEQ ID NO:9所示的轻链可变区，和SEQ ID NO:10所示的重链可变区；
    所述抗体或其抗原结合片段包含SEQ ID NO:11所示的轻链可变区，和SEQ ID NO:12所示的重链可变区；
    所述抗体或其抗原结合片段包含SEQ ID NO:38所示的轻链可变区，和SEQ ID NO:37所示的重链可变区；或者
    所述抗体或其抗原结合片段包含SEQ ID NO:40所示的轻链可变区，和SEQ ID NO:39所示的重链可变区。
  3. 根据权利要求1或2所述抗体或其抗原结合片段，其特征在于，所述抗体包含SEQ ID NO:33所示的重链恒定区，和/或包含SEQ ID NO:34所示的轻链恒定区。
  4. 一种核酸，其编码权利要求1-3任一项所述的抗CD228抗体或其抗原结合片段。
  5. 一种细胞，其包含权利要求4所述的核酸。
  6. 一种抗体药物偶联物(ADC)，其特征在于，所述ADC结构如下式1所示：
    其中：Ab是根据权利要求1-3任一项所示的抗CD228抗体或其抗原结合片段；
    LU是连接子；
    D是药物；
    p对应抗体-药物偶联物的平均DAR值，p是选自1-10，优选的1-8，优选的1-4或4-8的值，更有优选的p为4。
  7. 根据权利要求6所述的抗体药物偶联物，其特征在于，所述LU-D结构为VcMMAE，Vc为缬氨酸-瓜氨酸，MMAE为单甲基auristatin E。
  8. 根据权利要求6所述的抗体药物偶联物，其特征在于，所述LU-D结构为如下所示结构：
  9. 根据权利要求7或8所述的抗体药物偶联物，其特征在于，所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:24所示的LCDR1、SEQ ID NO:25所示的LCDR2和SEQ ID NO:26所示的LCDR3，和所述抗体或其抗原结合片段的3个重链互补决定区包含 SEQ ID NO:16所示的HCDR1、SEQ ID NO:27所示的HCDR2和SEQ ID NO:28所示的HCDR3；
    优选的，所述抗体或其抗原结合片段包含SEQ ID NO:7所示的轻链可变区，和SEQ ID NO:8所示的重链可变区；
    更优选的,所述抗体或其抗原结合片段的重链恒定区的序列为SEQ ID NO:33和/或轻链恒定区的序列为SEQ ID NO:34。。
  10. 一种药物组合物，其含有权利要求1-3任一项所述的抗CD228抗体或其抗原结合片段，或权利要求4所述的核酸，权利要求5所述的细胞或权利要求6-9任一所述的抗体药物偶联物。
  11. 一种试剂盒，其含有权利要求1-3任一项所述的抗CD228抗体或其抗原结合片段，权利要求4所述的核酸，权利要求6-9任一所述的抗体药物偶联物或权利要求10所述的药物组合物。
  12. 权利要求1-3任一项所述的抗CD228抗体或其抗原结合片段，权利要求4所述的核酸，权利要求6-9任一所述的抗体药物偶联物，权利要求10所述的药物组合物或权利要求11所述的试剂盒在制备预防、治疗、检测或诊断与CD228相关的疾病的试剂中的应用；优选地，所述CD228相关的疾病包括黑色素瘤、肺癌、胃癌、结肠癌、间皮瘤、胰腺癌和乳腺癌中的一种或多种。
PCT/CN2023/096155 2022-05-31 2023-05-25 抗cd228抗体及其药物偶联物 WO2023231877A1 (zh)

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