Anti-CD228 antibodies and drug conjugates thereof

Abstract

To provide an anti-CD228 antibody, which can bind to CD228 protein and has wide application potential in the medical field as an effective anti-cancer agent.SOLUTION: The present invention relates to an antibody binding to CD228 or an antigen-binding fragment thereof, and to an antibody-drug conjugate comprising the antibody or the antigen-binding fragment thereof. The antibody or the antigen-binding fragment thereof has strong affinity for CD228 protein and the ability to mediate ADCC, and the drug conjugate exhibits inhibitory effects against various tumors. The present invention also relates to nucleic acids encoding the antibody or the antigen-binding fragment thereof, to cells containing the nucleic acids, to pharmaceutical compositions and kits comprising the antibody or the antigen-binding fragment thereof, nucleic acids, cells, or antibody-drug conjugates, and to applications of the antibody or the antigen-binding fragment thereof, nucleic acids, antibody-drug conjugates, or pharmaceutical compositions in the prevention, treatment, detection, or diagnosis of CD228-related diseases.SELECTED DRAWING: Figure 20

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical or biopharmaceutical technology, in particular to anti-CD228 antibodies and drug conjugates thereof. [Background technology]

[0002] CD228 (also known as melanotransferrin, MTF, melanoma-associated antigen p97, MFI2, or MAP97) is a 90-97 kDa salivary glycoprotein member of the transferrin family. CD228 is usually found bound to the cell membrane via a glycosylphosphatidylinositol anchor, with only small amounts of soluble protein being detected.

[0003] CD228 plays a role in cell proliferation, migration, and tumorigenesis; increased expression of CD228 can accelerate melanoma tumor growth; in cell models, high expression of CD228 can increase cell proliferation, while downregulation of CD228 results in decreased cell proliferation.

[0004] CD228 is expressed in a wide range of tumors, including melanoma, mesothelioma, pancreatic cancer, non-small cell lung cancer, breast cancer, and colon cancer, and has a wide range of indications. CD228 is expressed in 72% of melanomas and 79% of pancreatic cancers, while it is expressed in 83% of mesotheliomas, 100% of colon cancers, 57% of breast cancers, and 69% of squamous cell carcinomas, creating a significant clinical need.

[0005] Antibody-drug conjugates, which couple antibodies to small molecule chemotherapeutic drugs via a linker, achieve high antibody targeting and the full cytotoxicity of the chemotherapy drug, effectively killing tumor cells. Currently, several antibody-drug conjugates have been successfully launched, with more products under development, demonstrating the maturity of the technology. CD228 is highly expressed in many tumor tissues and low or absent in normal tissues. Based on these differential expression patterns, CD228 may be an ideal target for antibody-drug conjugates.

[0006] Therefore, the novel CD228 antibody-drug conjugates serve as effective anti-cancer agents and have broad application value in the medical field. Summary of the Invention

[0007] The present invention provides an anti-CD228 antibody or antigen-binding fragment thereof capable of binding to CD228 protein. The present invention further provides a nucleic acid encoding the antibody or antigen-binding fragment thereof, a cell comprising the nucleic acid, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, the nucleic acid, or the cell, a kit comprising the antibody or antigen-binding fragment thereof, the nucleic acid, or the pharmaceutical composition, the application of the antibody or antigen-binding fragment thereof, the nucleic acid, or the pharmaceutical composition in the prevention, treatment, detection, or diagnosis of diseases associated with CD228, the application of a CD228 antibody or antigen-binding fragment thereof in the production of an antibody-drug conjugate (ADC), and the anti-CD228 antibody-drug conjugate.

[0008] One aspect of the present invention provides an anti-CD228 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises 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 antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 13, LCDR2 set forth in SEQ ID NO: 14, and LCDR3 set forth in SEQ ID NO: 15, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 18; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 20, and LCDR3 set forth in SEQ ID NO: 21, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 22; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 23, LCDR2 set forth in SEQ ID NO: 20, and LCDR3 set forth in SEQ ID NO: 21, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 22; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 24, LCDR2 set forth in SEQ ID NO: 25, and LCDR3 set forth in SEQ ID NO: 26, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 27, and HCDR3 set forth in SEQ ID NO: 28; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:29, LCDR2 set forth in SEQ ID NO:25, and LCDR3 set forth in SEQ ID NO:30, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:16, HCDR2 set forth in SEQ ID NO:17, and HCDR3 set forth in SEQ ID NO:28; or the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 31, LCDR2 set forth in SEQ ID NO: 14, and LCDR3 set forth in SEQ ID NO: 21, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 32; or The three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise an LCDR1 set forth in SEQ ID NO: 19, an LCDR2 set forth in SEQ ID NO: 44, and an LCDR3 set forth in SEQ ID NO: 45, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise an HCDR1 set forth in SEQ ID NO: 16, an HCDR2 set forth in SEQ ID NO: 17, and an HCDR3 set forth in SEQ ID NO: 49; or the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise an LCDR1 set forth in SEQ ID NO: 46, an LCDR2 set forth in SEQ ID NO: 47, and an LCDR3 set forth in SEQ ID NO: 48, and / or the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise an HCDR1 set forth in SEQ ID NO: 16, an HCDR2 set forth in SEQ ID NO: 17, and an HCDR3 set forth in SEQ ID NO: 28.

[0009] In one specific embodiment, the invention provides an anti-CD228 antibody, or antigen-binding fragment thereof, that, when bound to CD228, binds to at least one of the following residues as set forth in SEQ ID NO: 41: E312A, L313A, R282A, R275A.

[0010] In one specific embodiment of the invention, the invention provides an anti-CD228 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is: the antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2; the antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 3 and a heavy chain variable region shown in SEQ ID NO: 4; the antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 5 and a heavy chain variable region shown in SEQ ID NO: 6; the antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 7 and a heavy chain variable region shown in SEQ ID NO: 8; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO:9 and / or a heavy chain variable region set forth in SEQ ID NO:10; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO:11 and / or a heavy chain variable region set forth in SEQ ID NO:12; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO:38 and / or a heavy chain variable region set forth in SEQ ID NO:37; or The above-mentioned antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO:40 and a heavy chain variable region shown in SEQ ID NO:39.

[0011] In one specific embodiment of the invention, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO:33.

[0012] Furthermore, the sequence of the light chain constant region of the above antibody or antigen-binding fragment thereof is SEQ ID NO:34.

[0013] In an embodiment of the present invention, the above-mentioned 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.

[0014] A second aspect of the present invention provides a nucleic acid encoding the above-described antibody or antigen-binding fragment thereof.

[0015] A third aspect of the present invention provides a vector comprising the nucleic acid encoding the antibody or antigen-binding fragment thereof. The vector can be used to express the antibody or antigen-binding fragment thereof. Preferably, the vector can be a viral vector, including, but not limited to, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector. Preferably, the vector can be a non-viral vector, preferably a mammalian cell expression vector, preferably a bacterial expression vector, or preferably a fungal expression vector.

[0016] A fourth aspect of the present invention provides a cell, the cell comprising the nucleic acid or the vector, and the cell capable of expressing the antibody or antigen-binding fragment thereof. Preferably, the cell is a bacterial cell, such as an E. coli cell; preferably, the cell is a fungal cell, such as a yeast cell, such as a Pichia yeast cell; preferably, the cell is a mammalian cell, such as a Chinese hamster ovary cell (CHO), a human embryonic kidney cell (293), a B cell, a T cell, a DC cell, or a NK cell.

[0017] A fifth aspect of the present invention provides an anti-CD228 antibody conjugate comprising: (a) the above-mentioned CD228 antibody or antigen-binding fragment thereof; and (b) a coupling moiety coupled to the above-mentioned antibody moiety, wherein the coupling moiety is selected from one or more of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0018] In another preferred example, the antibody drug conjugate (ADC) is represented by the following formula 1:

[0019] TIFF2025183289000002.tif34170In the formula, Ab in Formula 1 is the anti-CD228 antibody of the present invention or an antigen-binding fragment thereof, LU is a linker (also referred to as a linker), D is a drug, and the subscript p corresponds to the average DAR value of the antibody-drug conjugate, where p is a value selected from 1 to 10, preferably 1 to 8, preferably 1 to 4 or 4 to 8, and more preferably p is 4.

[0020] The drug is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, hormonal agents, and immunotherapeutic agents. Optionally, the drug is selected from the group consisting of taxanes, maytansinoids, camptothecin, tubulysin, auristatin, calicheamicin, anthracyclines, docetaxel, cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, ribonuclease (RNase), and radioisotopes.

[0021] Furthermore, the linker LU is of the general formula R'-L1-L2-L3, In this general formula, L3 is TIFF2025183289000003.tif17170, wherein L3 has an a-terminus connected to the drug D and a b-terminus connected to L2; R1 is hydrogen, a carboxyl group, an ester group, a nitro group, a sulfonyl group, a halogen group, or R1 is TIFF2025183289000004.tif29170R2~R6 are each independently TIFF2025183289000005.tif15170n is 0 to 8, In the general formula, L2 is TIFF2025183289000006.tif16170In the formula, 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, a lysine residue, a proline residue, or a valine, a citrulline residue, a β-glycine residue, or a β-alanine residue; and X is TIFF2025183289000007.tif15170n is 0 to 8, In the general formula, L1 is TIFF2025183289000008.tif17170 and / or In the general formula, R' is In the formula TIFF2025183289000009.tif25170, R' is connected to L1 at the c-terminus and to A at the d-terminus, In one preferred example, LU-D in Formula 1 of the above antibody-drug conjugate (ADC) is VcMMAE, where LU is Vc (valine-citrulline linker) and D is MMAE (monomethyl auristatin E), and VcMMAE may be written as MC-Val-Cit-PAB-MMAE or MC-vc-PAB-MMAE.

[0022] In one preferred example, LU-D in Formula 1 of the antibody drug conjugate (ADC) is BNLD11, wherein LU is MC-β-Ala-(glucuronide)PAB and D is MMAE, and the structure of BNLD11 is: The BNLD11 is represented by TIFF2025183289000010.tif35170, and the exact mass of the BNLD11 is 1322.690. The BNLD11 is obtained by synthesis using a conventional method in the prior art. In one preferred example, the BNLD11 is obtained using the synthesis route shown in FIG.

[0023] In one preferred example, the three light chain complementarity determining regions of the anti-CD228 antibody or antigen-binding fragment thereof (Ab) in Formula 1 of the ADC comprise LCDR1 set forth in SEQ ID NO: 24, LCDR2 set forth in SEQ ID NO: 25, and LCDR3 set forth in SEQ ID NO: 26, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 27, and HCDR3 set forth in SEQ ID NO: 28; preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8; more preferably, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 33, and / or the sequence of the light chain constant region is SEQ ID NO: 34.

[0024] In one preferred embodiment, LU-D in formula 1 of the ADC is VcMMAE, p is 4, Ab is an anti-CD228 antibody or antigen-binding fragment thereof, the three light chain complementarity determining regions of the anti-CD228 antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 24, LCDR2 set forth in SEQ ID NO: 25, and LCDR3 set forth in SEQ ID NO: 26, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 27, and HCDR3 set forth in SEQ ID NO: 28, more preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8, and more preferably, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 33 and / or the sequence of the light chain constant region is SEQ ID NO: 34.

[0025] In one preferred embodiment, LU-D in formula 1 of the ADC is a BNLD11 structure, p is 4, and the BNLD11 structure is The Ab is represented by TIFF2025183289000011.tif35170, and is an anti-CD228 antibody or antigen-binding fragment thereof. The three light chain complementarity determining regions of the anti-CD228 antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 24, LCDR2 set forth in SEQ ID NO: 25, and LCDR3 set forth in SEQ ID NO: 26, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 27, and HCDR3 set forth in SEQ ID NO: 28. More preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8.

[0026] A sixth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, or antibody-drug conjugate, preferably wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vector, preferably wherein the pharmaceutically acceptable vector comprises one or more of a pharmaceutically acceptable solvent, dispersant, additive, plasticizer, or other pharmaceutical auxiliary material.

[0027] A seventh aspect of the present invention provides a kit comprising the above-described antibody or antigen-binding fragment thereof of the present invention, or comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof, comprising the above-described pharmaceutical composition, or comprising the above-described antibody-drug conjugate.

[0028] An eighth aspect of the present invention provides an application of the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, or antibody-drug conjugate in the manufacture of a pharmaceutical composition for treating or preventing a disease.

[0029] A ninth aspect of the present invention provides the use of the above-mentioned antibody or antigen-binding fragment thereof, or nucleic acid in the production of a diagnostic or detection kit.

[0030] A tenth aspect of the present invention provides a method for treating or preventing a disease, the method comprising administering to a subject in need thereof the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, pharmaceutical composition, or antibody-drug conjugate of the present invention.

[0031] An eleventh aspect of the present invention provides a diagnostic or detection method comprising administering to a subject or a sample in need thereof the above-described antibody or antigen-binding fragment, nucleic acid, kit, or pharmaceutical composition of the present invention.

[0032] A twelfth aspect of the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, pharmaceutical composition, or antibody-drug conjugate for the treatment and prevention of diseases.

[0033] A thirteenth aspect of the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid, kit, or pharmaceutical composition for detection or diagnosis.

[0034] A fourteenth aspect of the present invention provides an application of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid, the above-mentioned pharmaceutical composition, or the antibody-drug conjugate in the manufacture of a preparation for the prevention, treatment, detection, or diagnosis of a disease associated with CD228.

[0035] In an embodiment of the present invention, the CD228-associated disease comprises one or more of melanoma, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, colon adenocarcinoma, mesothelioma, pancreatic cancer, breast cancer.

[0036] The fifteenth aspect of the present invention further provides the use of the CD228 antibody or antigen-binding fragment thereof of the present invention in the production of antibody drug conjugates (ADCs).

[0037] The anti-CD228 antibodies and antibody-drug conjugates thereof provided by the present invention have one or more of the following advantages: 1. The anti-CD228 antibody or antigen-binding fragment thereof provided by the present invention has good affinity for the CD228 protein and cells expressing the CD228 protein.

[0038] 2. The anti-CD228 antibody-drug conjugate provided by the present invention has good killing ability against human melanoma SK-MEL-5 cells.

[0039] 3. The anti-CD228 antibody-drug conjugate provided by the present invention has good cancer-suppressing effects in all animal models: SK-MEL-5, NCI-H226, CALU-1, and NUGC4, and shows stable efficacy data for CD228-related diseases.

[0040] 4. The anti-CD228 antibody-drug conjugate provided by the present invention has good pharmacokinetics in vivo in mice.

[0041] 5. The anti-CD228 antibody-drug conjugate provided by the present invention has low side effects and toxicity, and is highly safe.

[0042] 6. The anti-CD228 antibody of the present invention has better internalization and internal killing effects than known commercially available antibodies, such as Seagen's CD228-targeting antibody hL49. [Brief explanation of the drawings]

[0043] [Figure 1] 1 shows the expression level of CD228 on a TMA chip in Example 1. [Figure 2] 1 shows serum titers of CD228-immunized mice in Example 2. [Figure 3] Binding of each anti-CD228 antibody to human CD228 protein in Example 4. [Figure 4] 1 shows the binding activity of each anti-CD228 antibody in Example 5 to human melanoma SK-MEL-5 cells expressing CD228 protein. [Figure 5A]1 shows the results of an internalization experiment using human melanoma SK-MEL-5 cells for each anti-CD228 antibody in Example 6. [Figure 5B] 1 shows the results of an internalization experiment of each anti-CD228 antibody in Example 6 using human lung cancer cell line A549-CD228 cells. [Figure 6] 1 shows the results of ADCC experiments using human melanoma SK-MEL-5 cells for each anti-CD228 antibody in Example 7. [Figure 7] FIG. 10 is a schematic diagram showing the sequence alignment results of full-length hCD228 and the soluble antigen sMFI2 in Example 8. [Figure 8] 1 shows the HIC-HPLC pattern of CA149-BNLD11 in Example 9. [Figure 9A] 1 shows the results of a killing experiment of CA13-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 9B] 10 shows the results of a killing experiment of CA67-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 9C] 1 shows the results of a killing experiment of CA149-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 9D] 1 shows the results of a killing experiment of BA352-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 9E] 1 shows the results of a killing experiment of CA518-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 9F] 10 shows the results of a killing experiment of CA185-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 10] The efficacy data (3 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model is shown. [Figure 11] The efficacy data (5 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model is shown. [Figure 12]1 shows efficacy data for each anti-CD228 ADC (3 mg / kg) in the NCI-H226 animal model in Example 11. [Figure 13] 1 shows efficacy data of each anti-CD228 ADC (5 mg / kg) in the NCI-H226 animal model in Example 11. [Figure 14] 1 shows the in vivo pharmacokinetic curves of each anti-CD228 ADC in mice in Example 12. [Figure 15A] Evaluation of the growth inhibitory activity of CA149-BNLD11 against MC38-CD228 cells in Example 13. [Figure 15B] Evaluation of the growth inhibitory activity of CA149-BNLD11 against A375-CD228 cells in Example 13. [Figure 15C] Evaluation of the growth inhibitory activity of CA149-BNLD11 against SK-MEL-5 cells in Example 13. [Figure 15D] Evaluation of the growth inhibitory activity of CA149-BNLD11 against A549-CD228 cells in Example 13. [Figure 15E] 10 shows an evaluation of the growth inhibitory activity of CA149-BNLD11 on A375-CD228 cells in Example 13. [Figure 16] 1 shows tumor growth inhibition curves of antibody-drug conjugates in Example 14 in a Calu-1 lung cancer model. [Figure 17] 10 is a histogram of tumor weight in a Calu-1 lung cancer model for antibody drug conjugate groups in Example 14. [Figure 18] 10 shows tumor growth inhibition curves for antibody-drug conjugate groups in Example 15 in a human melanoma cell SK-MEL-5 nude mouse transplant tumor model. [Figure 19] 10 is a histogram of tumor weight in a nude mouse transplanted human melanoma cell SK-MEL-5 tumor model in the antibody-drug conjugate group in Example 15. [Figure 20]13 shows tumor growth inhibition curves for antibody-drug conjugate groups in Example 16 in a tumor model transplanted into human gastric cancer cells NUGC4 Balb / c nude mice. [Figure 21] 13 is a histogram of tumor weight in a human gastric cancer cell NUGC4 Balb / c nude mouse transplant tumor model in the antibody-drug conjugate group in Example 16. [Figure 22] 17 shows the tumor volume growth curve of human lung squamous cell carcinoma cell line NCI-H226 transplanted into Balb / c nude mice in Example 17. [Figure 23] 10 shows the tumor volume growth curve of human melanoma cell SK-MEL-5 Balb / c nude mice transplanted tumors in Example 18. [Figure 24] 10 shows a tumor weight growth curve of human melanoma cell SK-MEL-5 Balb / c nude mice transplanted tumors in Example 18. [Figure 25] 10 shows the in vivo metabolic curve of antibody-drug conjugate CA149-BNLD11 in mice in Example 19. [Figure 26] 20 is a toxicity study in male mice of the antibody drug conjugate CA149-BNLD11 in Example 20. [Figure 27] 20 is a toxicity study in female mice of the antibody drug conjugate CA149-BNLD11 in Example 20. [Figure 28] 1 shows the changes in body weight of cynomolgus monkeys after administration of 6 mg / kg in the medium dose group and 10 mg / kg in the high dose group in Example 21. [Figure 29] 2 shows toxicokinetic detection 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] Schematic diagram of the BNLD11 synthesis circuit. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention will be further described below with reference to specific examples. The described examples are only a portion of the present invention, and are not all examples. It should be understood that the following examples are provided to provide a complete disclosure and explanation to those skilled in the art to which the present invention pertains about how to use the method and composition of the present invention, and are not intended to limit the scope of the present invention. All other examples that can be obtained by those skilled in the art based on the examples of the present invention without any creative effort belong to the scope of the claims of the present invention.

[0045] Example 1. CD228 expression experiment PDX sample chips for gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, bile duct cancer, pancreatic cancer, cervical cancer, sarcoma, esophageal cancer, and gastric cancer were all purchased from Crown Biotechnology Co., Ltd. Unstained tissue sections from 12 mesothelioma patients were purchased from Shanghai Lidi Biotechnology Co., Ltd. (four cases) and Shanghai Xinchao Biotechnology Co., Ltd. (eight cases). CD228 immunohistochemical staining was performed on all PDX sample tissue chips and eight unstained mesothelioma tissue sections purchased from Shanghai Xinchao Biotechnology Co., Ltd. by Crown Biotechnology Co., Ltd. Immunohistochemical staining for CD228 on four unstained mesothelioma tissue sections was performed by Shanghai Lidi Biotechnology Co., Ltd. CD228 immunohistochemistry antibody was purchased from Novus Biologicals (catalog number: NBP1-85777), and the dilution of the primary antibody was 1:200.

[0046] Immunohistochemical experiments were performed using an automated IHC stainer (Bond RX automatic IHC&ISH system, Leica). Immunohistochemical staining results were evaluated using H-score values. TIFF2025183289000012.tif5170pi 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 scored independently three times, and the final H-score value was the average of the three. The IHC score results for all samples are shown in Figure 1. Based on the CD228 expression levels on the TMA chip shown in Figure 1 and the CD228 expression rates shown in Table 1, we found that CD228 was expressed at high rates in gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, bile duct cancer, pancreatic cancer, and cervical cancer.

[0047] [Table 1]

[0048] Example 2. Production of anti-CD228 monoclonal antibodies 1.1 Protein production The amino acid sequences of the three proteins (see Table 2) were sent to Jiangsu GenScript Biotechnology Company for gene synthesis. They were then transfected into CHO cells and cultured at 37°C, 8% CO2, and 125 rpm in a shaker. After 10 days of transient expression, the supernatant was collected. The expressed supernatant was purified on a Ni (GenScript, L00250) column and then on an SP column (GE, 17-1087-01) to obtain human CD228, mouse CD228, and monkey CD228 proteins.

[0049] [Table 2]

[0050] 1.2 Mouse immunization method The mice used in the immunization experiments were fully human antibody transgenic mice (a total of 10 mice were immunized) independently developed by Shandong Boan Biotechnology Co., Ltd. The mice were immunized with CD228 (0.23 mg / mL, Boan, 20200924, SEQ ID NO: 41) antigen protein independently produced by Shandong Boan Biotechnology Co., Ltd.

[0051] Immunizations were either subcutaneous abdominal or multipoint inguinal injections, with the antigen protein immunization dose being 20 μg per mouse. For the first immunization, the antigen was emulsified in complete Freund's adjuvant, while for the second through fourth immunizations, the antigen was emulsified in incomplete Freund's adjuvant. The first batch of mice received three immunizations and one booster immunization, while the second batch received four immunizations and one booster immunization. The interval between immunizations was 14 days. Peripheral blood serum was collected on day 7 after the second immunization and antibody titers were detected. Mice with failing titers were excluded. The serum titer results after immunization are shown in Figure 2, with dilutions of 2500X, 12500X, and 62500X representing the dilution rates. Three days after immunization, the mice were euthanized, and their spleens were harvested and purified into single cells for library construction.

[0052] 1.3 Construction of phage libraries The mice were sacrificed, and the spleens were dissected and crushed with a syringe and filtered. The filtered splenocytes were frozen, and RNA was extracted and cDNA was obtained. Phage libraries were constructed according to standard procedures. The library volume data for the constructed libraries are shown in Table 3.

[0053] [Table 3]

[0054] 1.4 Two-way screening 1.4.1 Plate screening was performed by coating the plate with CD228-His protein (self-made). The next day, the phage library was added and incubated for 2 h. After washing 4 to 10 times, the specifically bound phages were eluted with elution buffer.

[0055] 1.44.2 Magnetic bead screening was performed. CD228-His protein was biotinylated according to the kit's instructions and bound to Thermo Scientific magnetic beads. The beads were sealed with BSA and then incubated with the phage library for 2 hours. After 4-10 washes, the specifically bound phages were eluted with elution buffer. The clones and sources of the antibodies obtained by screening are listed in Table 4.

[0056] [Table 4]

[0057] Example 3. Molecular construction and production of complete antibodies 133 positive IgG1 clones were constructed and sequenced, and the amino acid sequences of the variable regions of eight lead antibodies are shown in Table 5 below (CDR regions are underlined, and the analysis system was the IMGT system). The variable region sequences of each antibody in the Examples of this application are shown in Table 5, and the heavy and light chain constant region sequences are shown in Table 6.

[0058] [Table 5] TIFF2025183289000018.tif132170

[0059] The antibody variable region genes were amplified by a standard molecular biology technique, PCR (2× Phanta Max Master Mix, manufacturer: Vazyme, catalog number: P515-P1-AA, lot number: 7E512E1), and the antibody heavy chain variable region gene was ligated by homologous recombination into the vector pCDNA3.4 (Life Technology), which contains a nucleic acid sequence carrying an antibody heavy chain constant region sequence, and the antibody light chain variable region gene was ligated into the vector pCDNA3.4, which contains a nucleic acid sequence carrying an antibody light chain constant region sequence.

[0060] [Table 6]

[0061] Plasmids were extracted from the sequenced positive clones, then co-transfected into HEK293 cells and cultured at 37°C, 8% CO2, and 125 rpm in a shaker. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain antibodies. The antibody concentrations were determined by passing UV280 light through the cells and combining the theoretical extinction coefficients.

[0062] The control antibody Hl49 sequence was synthesized according to the sequence described in patent US20200246479A1, and the amino acid sequence is shown in Table 7 below.

[0063] [Table 7]

[0064] Example 4. Characterization of anti-CD228 monoclonal antibody molecules 4.1 Binding of anti-hCD228 antibodies to human CD228 protein Human CD228 protein (Hakuyasu's original work, 20201014, sequence number 41) was diluted to 0.1 μg / mL in pH 9.6 carbonate buffer (hereinafter abbreviated as CBS), coated onto the enzyme-labeled plate, and incubated at 4°C overnight at 100 μL / well. After washing the plate, it was sealed with nonfat dry milk. After washing the plate, 100 μL of antibody diluted in PBST (phosphate-buffered saline, Solarbio P1010, +0.05% Tween 20) was added per well (the diluted antibody was a complete antibody gradient diluted in PBST. The complete antibody contained the Fc and Fab regions and had a constant region. The initial concentration was 0.1 μg / mL, with eight 3-fold dilutions). After washing the plate, 100 μL of sheep anti-human IgG (H+L) / HRP (1:5000 dilution, KPL, 474-1006) was added per well and incubated at 37°C for 1 h. After washing the plate, 100 μL of TMB (Beijing Meike Wande, 1001) was added per well to develop the color. After 10 min, 50 μL of 2 M H2SO4 was added per well to stop the color development. The OD450 was read using a microplate reader. In this and the following experiments, Seagen's antibody hL49, which targets CD228, was used as a control antibody. Figure 3 shows the binding curves of each antibody to human CD228 protein. Table 8 shows the EC50 values ​​of the antibodies calculated from Figure 3. It was found that the six antibodies exhibited similar binding activity at the ELISA level and were superior to the control antibody HL49.

[0065] [Table 8]

[0066] 4.2 Detection of antibody affinity to CD228 human, monkey, and mouse proteins

[0067] [Table 9]

[0068] The binding dynamics of antibodies to each CD228 protein were measured using a BIAcore 8K instrument based on surface plasmon resonance (SRP) technology. CD228 antibodies were captured at 2 μg / mL on a ProA chip, and the binding activity of each CD228 antibody to human, monkey, and mouse CD228 was analyzed. Human, monkey, and mouse CD228 proteins were diluted 5 times in a 2-fold gradient with HBS-EP+ buffer, with the initial concentration being 50 nM. CD228 protein binding dynamics were analyzed using Biacore and fitted to calculate affinity activity KD values.

[0069] [Table 10]

[0070] As can be seen from Table 10, the antibodies shown have similar affinities for human and monkey CD228 proteins and do not bind to mouse CD228.

[0071] 4.3 Anti-hCD228 antibody epitope analysis The His chip captured 10 μg / mL of CD228 protein at a threshold of 0.5 nm. The first CD228 antibody (30 μg / mL) was first bound to the chip, followed by the competitive binding of the second CD228 antibody (30 μg / mL). The response of antibody 2 was analyzed using Octet 8K to determine whether antibody 1 and antibody 2 competed with each other. Table 11 shows the Octet antibody epitope response values.

[0072] [Table 11]

[0073] The final competitive analysis was performed using the 1-response value / blank value calculation method. The results, as shown in Table 12, indicated that the epitopes of BA352 and hL49 were similar, and the remaining antibodies competed with each other and had similar epitopes (values ​​higher than 75%, indicating epitope relatedness).

[0074] [Table 12]

[0075] Example 5. Detection of cellular binding activity of anti-hCD228 antibody by flow cytometry 50 μL of human melanoma SK-MEL-5 cells (ATCC, HTB-70) were added to a 96-well round-bottom plate at 7E4 cells / well. Each antibody was diluted in FACS buffer (PBS, BOSTER Biological, catalog no. PYG0021) and added at 50 μL / well. The plate was incubated at 4°C for 1 h. After centrifugation at 400×g for 4 min, the supernatant was discarded, washed once with FACS buffer, and 100 μL / well of fluorescent secondary antibody (Jackson, 109545-008) was added. The plate was incubated at 4°C for 30 min in the dark, centrifuged at 400×g for 4 min, discarded, washed once with FACS buffer, and resuspended in 100 μL / well of FACS buffer. The cells were then detected using a flow cytometer (Eisen, NovoCyte 2060). The results are shown in Figure 4, which shows the binding of each anti-CD228 antibody to human melanoma SK-MEL-5 cells (cells expressing CD228 protein). All six antibodies had high binding activity to SK-MEL-5 cells and were found to be superior to hL49. The isotype control antibody in Figure 4 was an unrelated antibody against a different target, which had the same constant region as the anti-CD228 antibody in the experimental group but a different variable region.

[0076] Example 6. Internalization experiment of anti-hCD228 monoclonal antibody molecules 1. SK-MEL-5 cells Human melanoma SK-MEL-5 cells diluted in buffer (PBS, BOSTER Biological, catalog no. PYG0021) were added to a 96-well round-bottom plate at 50 μL / well, resulting in a cell count of 5E4 cells / well. Antibodies were then diluted with buffer to a final concentration of 20 μg / mL. 50 μL / well of 20 μg / mL antibody was added to the round-bottom plate containing 50 μL / well of cells. After incubation for 30 min, the plate was centrifuged at 400 g for 4 min and the supernatant discarded. After washing twice with pre-chilled buffer, 100 μL / well of buffer was added. The plate was incubated at 37°C and 4°C, respectively. At different time points, the plate was terminated by centrifugation at 400 g for 4 min and the supernatant discarded. 100 μL / well of pre-chilled fluorescent secondary antibody (Jackson, 109-545-008) was then added at 4°C for 30 min, protected from light. The cells were washed once with pre-chilled FACS buffer, resuspended in 100 μL / well of FACS buffer, and detected using a flow cytometer (Eisen, NovoCyte 2060). The results are shown in Figure 5A, which shows the results of an internalization experiment using human melanoma SK-MEL-5 cells for each anti-CD228 antibody. Figure 5A shows that the internalization rates of CA13, CA149, and BA352 were higher than those of the control hL49.

[0077] 2. A549-CD228 cells Human lung cancer cell line A549-CD228 cells (Kangyuan Bochuang, KC-2150) stably expressing the exogenous CD228 gene in logarithmic growth phase were digested and then terminated with serum-containing medium. The cells were diluted and added to a 96-well round-bottom plate (NEST, catalog no. 701111) at 50 μL / well (1E5 / well). The antibody was diluted in serum-containing medium and mixed with labeling reagent (Invitrogen, Z25611) at a 1:3 molar ratio (40 nM antibody, 120 nM labeling reagent). After 5 min of incubation at room temperature, 50 μL of the labeled antibody mixture was added to each well of the plate containing the cells. After incubation at 37°C for 0, 2, 6, and 24 hours, the cells were washed once with PBS and resuspended in 100 μL / well of PBS. MFI values ​​were measured by flow cytometry (Eisen, NovoCyte 2060). The results showed that internalization of the CA149 antibody increased over time. Figure 5B shows that CA149 has superior internalization activity compared to the control antibody, hL49.

[0078] Example 7. Antibody-dependent cell-mediated cytotoxicity (luciferase reporter gene effector cells) Prepare the ADCC working solution (RPMI 1640 medium containing 1% FBS), collect Bioassay Effector Cells (Promega, G7011), and incubate them in the ADCC working solution at a cell density of 2.4 × 10 6 The cell density was adjusted to 8 × 10 using the ADCC working solution. 5The test samples were diluted with ADCC working solution, starting at an initial concentration of 5 μg / mL and then diluted 4-fold to eight concentrations. Effector cells, target cells, and test samples were added in 25 μL aliquots to a white reaction plate (Costar, 3917) for a total reaction volume of 75 μL. The reaction mixture was incubated at 37°C for 6 hours, and 75 μL of each aliquot was added to each well. After 15 minutes of incubation using the Bio-Glo Luciferase System (Promega, G7940), chemiluminescence readings were read using a microplate reader (BioTek, Synergy Neo2). Figure 6 shows the results of an ADCC experiment using each anti-CD228 antibody on human melanoma SK-MEL-5 cells. Figure 6 shows that the signal gradually increased with increasing antibody sample concentration, indicating ADCC activity against SK-MEL-5 cells.

[0079] Example 8. Epitope study of CA149 antibody binding to CD228 The inventors prepared full-length antigen CD228 and antibody Fab complexes (CA149-Fab) and commissioned the Cryo-Electron Microscopy Center at Mizuki Mirai (Hangzhou) Science and Technology Co., Ltd. to conduct structural analysis of the antigen-antibody complex. The researchers analyzed the amino acid types and side chain interactions in the epitope of a 3D model, and found that CA149 antibody Fab binds to the antigen via seven hydrogen bonds and one salt bridge. The specific interaction sites are shown in Table 13. The superscript * indicates the amino acid on the CA149 Fab light chain, and the italicized amino acids without superscripts indicate the amino acids on the CA149 Fab heavy chain.

[0080] [Table 13]

[0081] At the same time, based on the epitope results, the inventors conducted site-specific mutation testing on the CD228 antigen (the sequence of which is shown in SEQ ID NO: 41) independently produced by Shandong Boan Biotechnology Co., Ltd., and constructed hCD228 antigens with single-point mutations of hCD228 (R275A), hCD228 (R282A), hCD228 (E312A), and hCD228 (L313A), and with double-point mutations of hCD228 (E312A, L313A), hCD228 (R282A, E312A), and hCD228 (R275A, R282A), respectively, and then performed affinity analysis. The affinity results showed that the antigen binding activity to the CA149 antibody was reduced or lost after the mutations, and also indicated that the four sites mentioned above, E312A, L313A, R282A, and R275A, were the main sites for antibody-antigen binding. The affinity detection results are listed in Table 14.

[0082] [Table 14]

[0083] Another variable shear product, soluble MFI2, also exists in the human body. Furthermore, literature has shown that sMFI can cross the blood-brain barrier via LRP proteins (J Neurochem. 2002 Nov;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). Therefore, to reduce the risk of potential off-targeting, antibodies targeting CD228 should not bind to sMFI2. Based on the epitope results and the sequence alignment of full-length hCD228 with the soluble antigen sMFI2 (Figure 7), the inventors found that the above four major binding sites are not present on the sMFI2 antigen, i.e., the CA149 antibody does not bind to sMFI2.

[0084] Example 9. Coupling experiment between anti-hCD228 antibody and drug An antibody-drug conjugate having the following molecular formula is prepared: TIFF2025183289000028.tif30170

[0085] where Ab is any of the above anti-CD228 antibodies or antigen-binding fragments thereof, LU is the linker (also referred to as linker), D is the drug, and the subscript p was the average DAR value of the antibody-drug conjugate.

[0086] 9.1 Preparation of anti-hCD228 antibody-vcMMAE antibody drug conjugate Antibody (5-10 mg / mL) in phosphate buffer (pH 7.5, containing 11 mM DTPA) was treated with 2 equivalents of TCEP and then incubated at 25°C for approximately 2 h. Five equivalents of vcMMAE in DMSO were added to the reduced antibody in PBS and incubated at 25°C for approximately 1 h. Next, 10 equivalents of n-acetylcysteine ​​(NAC) were added to the mixture and incubated at 25°C for 5 min to quench any unreacted linker-drug. The buffer was then replaced by ultrafiltration to remove free small molecules. The mixture was then subjected to HIC-HPLC analysis. The analytical results are shown in Table 15. The average DAR values ​​of the antibody-drug conjugates used in this invention were found to be in the range of 4.01 to 4.42.

[0087] [Table 15]

[0088] 9.2 Preparation of CA149-BNLD11 Antibody Drug Conjugate Using the anti-CD228 antibody CA149 as an example, an ADC composition with a uniform drug loading (DAR) of approximately 4 was prepared. Antibody (5–10 mg / mL) in phosphate buffer (pH 7.5, containing 11 mM DTPA) was treated with 2 equivalents of TCEP and incubated at 25°C for approximately 2 h. Five equivalents of BNLD-11 in DMSO were added to the reduced antibody in PBS and incubated at 25°C for approximately 1 h. Next, 10 equivalents of n-acetylcysteine ​​(NAC) were added to the mixture and incubated at 25°C for 5 min to quench any unreacted linker-drug. The drug loading of the antibody-drug conjugate was quantified using HIC-HPLC analysis. The analytical results are shown in Figure 8 and Table 16. The average DAR of the antibody-drug conjugate CA149-BNLD11 prepared in this example was approximately 4.

[0089] [Table 16]

[0090] Example 10. In vitro cell killing experiments of anti-hCD228 ADCs SK-MEL-5 cells were added to a 96-well flat-bottom plate (Corning, catalog no. 3917) at 50 μL / well, with a cell count of 1E4 cells / well, in 10% FBS / EMEM medium. Each anti-CD228 ADC was diluted with the above medium at an initial concentration of 1 μg / mL using a 4-fold gradient dilution method for a total of six concentrations. The diluted ADCs were added to the 96-well flat-bottom plate at 50 μL / well and incubated at 37°C and 5% CO2 for 4 days. The CellTiter-Glo kit (Promega, G7571, must be protected from light during use) was then equilibrated to room temperature, and the kit buffer was mixed evenly with the substrate and allowed to stand for 1 hour. Cell viability detection reagent was added to the 96-well plate at 100 μL / well, shaken at 300 rpm for 2 minutes, and then allowed to stand for 10 minutes. Detection was performed using a microplate reader (BioTek, SYNERGY neo, USA).

[0091] The results are shown in Figures 9A-9F, which show the results of killing experiments using human melanoma SK-MEL-5 cells with each anti-CD228 ADC. Two anti-hCD228 ADCs, CA13-vcMMAE and CA67-vcMMAE, exhibited in vitro cell-killing activity similar to that of hL49-vcMMAE. Four anti-hCD228 ADCs, CA149-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and CA185-vcMMAE, exhibited superior in vitro cell-killing activity to the control, hL49-vcMMAE. The isotype control antibodies in Figures 9A-9F were unrelated antibodies against other targets, with the same constant regions as the six antibodies but different variable regions.

[0092] Example 11. In vivo efficacy study of anti-CD228 ADC in mouse xenograft tumors 11.1 Efficacy data in a mouse model of human melanoma cells SK-MEL-5 Human melanoma cells SK-MEL-5 were purchased from ATCC and cultured in an incubator at 37°C with 5% CO2 in EMEM medium containing 10% FBS. NOD / SCID mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. SK-MEL-5 cells were grown at a cell density of 2.5 × 10 in EMEM medium containing 50% Matrigel. 7 The concentration of cells / mL was adjusted and inoculated subcutaneously into the right side of NOD / SCID mice at a volume of 0.1 mL / mouse. The average tumor volume was approximately 76 mm. 3When the tumor volume reached 1000 mcg, the mice were divided into eight experimental groups of five mice each based on tumor volume and body weight, and treatment began on the day of grouping at a dose of 3 mg / kg. Figure 10 shows the efficacy data (3 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model, i.e., the change in tumor volume after treatment. It was found that tumor volume decreased after treatment with all six antibodies. Figure 10 was created based on the volume change data in Table 17. As shown in Table 17, the tumor volume growth inhibition rates (TGI%) of the CA13-vcMMAE, CA67-vcMMAE, CA149-vcMMAE, CA185-vcMMAE, BA352-Vc-MMAE, CA518-vcMMAE, and hL49-vcMMAE groups were 55.4%, 60.3%, 56.9%, 56.1%, 69.8%, 63.9%, and 49.0%, respectively, and the tumor weight inhibition rates (IR%) were 59.8%, 65.9%, 61.4%, 55.4%, 68.2%, 65.7%, and 56.1%, respectively. The human melanoma cell SK-MEL-5 mouse transplant tumor model showed a 3-fold increase in tumor volume growth inhibition rate (TGI%) of 3%. Under the administration conditions of mg / kg, all six anti-CD228-vcMMAE compounds produced in this study were found to be superior to the control group hL49-vcMMAE in both tumor volume growth inhibition rate (TGI%) and tumor weight inhibition rate (IR%).

[0093] [Table 17]

[0094] Figure 11 shows the efficacy data (5 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model. NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. SK-MEL-5 cells were cultured at a concentration of 3 × 10 in EMEM medium containing 50% Matrigel. 7 The concentration of the antibody was adjusted to 1 / mL and inoculated subcutaneously into the right side of NCG mice at a volume of 0.1 mL per mouse. The average tumor volume was approximately 83 mm. 3When tumor volume reached 1000 mcg, mice were divided into eight experimental groups of six mice each based on tumor volume and body weight. Treatment began on the day of grouping at a dose of 5 mg / kg. The screened antibodies demonstrated relatively good efficacy compared to the control hL49. Figure 11 was generated based on the volume change data in Table 18. As shown in Table 18, the tumor volume growth inhibition rates (TGI%) for the CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE groups were 82%, 84%, 81%, 80%, 81%, and 74%, respectively, and the tumor weight inhibition rates were 77.7%, 79.0%, 75.5%, 75.2%, 72.0%, and 69.1%, respectively. In the human melanoma cell SK-MEL-5 mouse xenograft tumor model, at a dose of 5 mg / kg, each of the anti-CD228-vcMMAE compounds produced in this study was superior to the control group, hL49-vcMMAE, in both tumor volume growth inhibition rate (TGI%) and tumor weight inhibition rate (IR%).

[0095] [Table 18]

[0096] 11.2 Drug Efficacy Data in Human NCI-H226 Lung Cancer Cells / Mouse Xenograft Tumor Model Human lung cancer cells NCI-H226 were purchased from ATCC and cultured in an incubator at 37°C with 5% CO2 in RPMI 1640 medium containing 10% FBS. CB-17 / SCID mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0097] NCI-H226 cells were cultured at a concentration of 5.0 × 10 in RPMI 1640 medium containing 50% Matrigel. 7 The resulting solution was adjusted to cells / mL and inoculated subcutaneously into the right side of CB-17 / SCID mice at a volume of 0.1 mL per mouse. The average tumor volume was approximately 156 mm 3When tumor volume reached 1000 mcg, the mice were divided into eight experimental groups of five mice each based on tumor volume and body weight. Treatment began on the day of grouping at a dose of 3 mg / kg. Figure 12 shows the efficacy data (3 mg / kg) of each anti-CD228 ADC in the NCI-H226 animal model. After administration of the six antibodies, tumor volume decreased in all cases, demonstrating in vivo efficacy similar to that of HL49. Figure 12 was generated based on the volume change data in Table 20. As shown in Table 19, the tumor volume growth inhibition rates (TGI) for the experimental endpoints CA13-vcMMAE, CA67-vcMMAE, CA149-vcMMAE, CA185-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and hL49-vcMMAE groups were 46.7%, 55.2%, 45.3%, 50.5%, 47.7%, 63.4%, and 55.1%, respectively, and the tumor weight inhibition rates were 39.7%, 44.5%, 46.9%, 43.1%, 41.6%, 47.8%, and 49.8%, respectively.

[0098] [Table 19]

[0099] Figure 13 shows the efficacy data (5 mg / kg) of each anti-CD228 ADC in the NCI-H226 animal model. NCG mice were purchased from Jiangsu Jisu Yaokang Biotechnology Co., Ltd. NCI-H226 cells were grown at a concentration of 5 × 10 in RPMI 1640 medium containing 50% Matrigel. 7 The solution was adjusted to 1 mL / mL and inoculated subcutaneously into the right side of NCG mice at a volume of 0.1 mL / mouse. The average tumor volume was approximately 108 mm 3When tumor volume reached 1000 mg / kg, the mice were divided into eight experimental groups of six mice each based on tumor volume and body weight. Treatment began on the day of grouping at a dose of 5 mg / kg. The screened antibodies demonstrated relatively good efficacy compared to the control HL49. Figure 13 was generated based on the volume change data in Table 20. As shown in Table 16, the experimental endpoints of tumor volume growth inhibition (TGI) for CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE were 94.4%, 92.3%, 94.6%, 98.3%, 103.9%, and 75.6%, respectively, and the tumor weight inhibition rates were 73.1%, 69.2%, 72.1%, 75.5%, 77.4%, and 56.7%, respectively.

[0100] [Table 20]

[0101] Example 12. In vivo pharmacokinetics experiments with anti-CD228 ADC in mice Each ADC was administered subcutaneously to three selected Balb / c mice at a dose of 10 mg / kg. Serum samples were collected 0 h before administration, and 1 h, 4 h, 10 h, and 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 10 d, and 14 d after administration to detect antibody concentrations. The serum detection method was ELISA.

[0102] Figure 14 shows the pharmacokinetic curves of CA13-vcMMAE, CA67-vcMMAE, and CA149-vcMMAE. Specific detection results are shown in Figure 14, which indicate that CA149-vcMMAE has better pharmacokinetic levels than CA13-vcMMAE and CA67-vcMMAE, which are constructed using CA13 and CA67 antibodies.

[0103] Example 13. Cell proliferation inhibitory activity of antibody-drug conjugate CA149-BNLD11 13.1 Growth inhibitory activity of CA149-BNLD11 against MC38-CD228 and A375-CD228 cells Logarithmic growth phase MC38-CD228 (Kangyuan Bochuang, KC-2023) and A375-CD228 cells (Kangyuan Bochuang, KC-2110) were digested, diluted in 10% FBS / 1640 medium, and resuspended. 50 μL / well and 1E4 cells / well were added to a 96-well flat-bottom plate (SARSTED, catalog number 94.6120.096). The antibody-drug conjugate CA149-BNLD11 prepared in section 8.2 of Example 8 was diluted with serum-containing medium to initial concentrations of 60 μg / mL and 12 μg / mL, respectively. A 5-fold gradient dilution was performed to select the antibody with the antibody ID CA521 in CN202180003751.7. nCov-CA521-vcMMAE was prepared in the same manner as in Example 8 and used as a control (isotype). The diluted CA149-BNLD11 solution was added to the 96-well flat-bottom cell culture plate at 50 μL / well and incubated at 37°C and 5% CO for 96 h. The mixture was equilibrated to room temperature and mixed evenly by inverting. The mixture was then added to the 96-well plate at 100 μL / well, shaken at 300 rpm for 2 min, and then allowed to stand for 10 min. Cell viability was detected using a microplate reader (BioTek, SYNERGY neo, USA).

[0104] As a result, CA149-BNLD11 had excellent growth inhibitory activity against MC38-CD228 and A375-CD228 cells, and as shown in Table 21, 50 The concentrations were 101.5 ng / mL and 61.8 ng / mL, respectively. In Figures 15A-15B, the vertical axis represents the percentage of viable cells, i.e., the ratio of the number of viable cells in the treatment group to the number of viable cells in the blank group. As can be seen from Figures 15A-15B, CA149-BNLD11 had significant growth inhibitory activity against CD228-expressing cells compared to the control group (isotype).

[0105] [Table 21]

[0106] 13.2 Growth inhibitory activity of CA149-BNLD11 against SK-MEL-5, A549-CD228, and A375-CD228 cells Logarithmically growing SK-MEL-5 (ATCC, HTB-70), A549-CD228 (Kangyuan Bochuang, KC-2150), and A375-CD228 cells (Kangyuan Bochuang, KC-2110) were digested and diluted with serum-containing medium. The digestion was terminated and diluted, and 50 μL / well of the solution was added to a 96-well flat-bottom plate (SARSTED, catalog number 94.6120.096) at 1E4 / well. CA149-BNLD11 was diluted in serum-containing medium to initial concentrations of 1.2 μg / mL, 60 μg / mL, and 6 μg / mL, respectively, with a 5-fold, 6-fold, and 5-fold gradient dilution. The diluted ADC was added to the above 96-well flat-bottom cell culture plate at 50 μL / well and cultured at 37°C and 5% CO for 96 or 120 hours. The Cell Counting-Lite® 2.0 kit (Vazyme, DD1101-01, protected from light during use) was equilibrated to room temperature, mixed evenly by inverting, and added to the 96-well plate at 100 μL / well. The plate was shaken at 300 rpm for 2 minutes, then allowed to stand for 10 minutes. Cell viability was detected using a microplate reader (BioTek, SYNERGY neo, USA). The results showed that CA149-BNLD11 had excellent growth inhibitory activity against SK-MEL-5, A549-CD228, and A375-CD228 cells (IC50 values ​​of 11.48 ng / mL, 15.25 ng / mL, and 13.86 ng / mL, respectively). As can be seen from Figures 15C to 15E, CA149-BNLD11 had significant growth inhibitory activity against CD228-expressing cells compared to the control group (isotype).

[0107] [Table 22]

[0108] Example 14. Efficacy evaluation of antibody-drug conjugate CA149-BNLD11 against human lung cancer cell line CaLu-1 tumors transplanted into nude mice CaLu-1 human lung cancer cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in McCoy's 5A medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Biotechnology Co., Ltd. CaLu-1 cells were grown at a density of 5.0 × 10 in serum-free McCoy's 5A medium containing 50% Matrigel. 7 The concentration of cells / mL was adjusted and inoculated subcutaneously into the right side of Balb / c nude mice at a volume of 0.1 mL / mouse. The average tumor volume was 135 mm 3 When tumor volume reached 1000 mcg, the mice were divided into five experimental groups of five mice each according to tumor volume, and treatment began on the day of grouping. This experiment employed a single dose, and observation was conducted for 28 days after administration.

[0109] The results are shown in Figures 16 and 17. Figure 16 shows the tumor growth inhibition curve in the Calu-1 lung cancer model for the antibody-drug conjugate group, and Figure 17 shows the histogram of tumor weight in the Calu-1 lung cancer model for the antibody-drug conjugate group.

[0110] As shown in Figure 16, compared with the solvent control group (PBS phosphate buffer), all antibody-drug conjugate-administered groups were able to significantly inhibit the increase in tumor volume, all with statistical significance (all P less than 0.05). The tumor-inhibiting effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly superior to those of the nCov-CA521-vccMMAE group (P values ​​0.0001 and 0.0016, respectively). , 0.0006), and no significant differences were found among the three groups of CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd (all P 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%, 93.9%, 97.3%, and 48.5%, respectively.

[0111] As shown in Figure 17, at the experimental endpoint, the mean tumor weights of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly lower than that of the solvent control group (PBS phosphate buffer), with statistical significance (all P<0.05). The tumor weight of the nCov-CA521-vcMMAE group was not statistically different from that of the control group (P=0.3250). No significant differences were found among the three groups (CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd) (all P>0.05). The tumor weight inhibition rates of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 96.3%, 87.3%, 87.6%, and 44.3%, respectively.

[0112] During the experiment, the animals were active and fed well during the administration period, and all gained some weight, indicating that the animals tolerated the treatment well. Comparisons between groups showed no significant differences (P>0.05).

[0113] Example 15. Efficacy evaluation of antibody-drug conjugate CA149-BNLD11 against human melanoma cell line SK-MEL-5 tumors transplanted into nude mice SK-MEL-5 human melanoma cells were purchased from ATCC and cultured in an incubator at 37°C with 5% CO2 in EMEM medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Biotechnology Co., Ltd. SK-MEL-5 cells were grown at a concentration of 5.0 × 10 in serum-free EMEM medium containing 50% Matrigel. 7 The concentration was adjusted to 1 / mL and the mixture was inoculated subcutaneously into the right side of Balb / c nude mice at a volume of 0.1 mL per mouse. The average tumor volume was 98 mm 3 When tumor volume reached 1000 mcg, the mice were divided into four experimental groups of five mice each according to tumor volume, and treatment began on the day of grouping. This experiment employed a single dose, and observation was conducted for 28 days after administration.

[0114] The results are shown in Figures 18 and 19. Figure 18 shows the tumor growth inhibition curves of the antibody-drug conjugate group in a human melanoma cell SK-MEL-5 nude mouse tumor model, and Figure 19 shows a histogram of tumor weights of the antibody-drug conjugate group in a human melanoma cell SK-MEL-5 nude mouse tumor model. The tumor growth curves shown in Figure 18 show that, compared with the solvent control group (PBS phosphate buffer), the increase in tumor volume was significantly inhibited in all treatment groups at the experimental endpoint, with all statistically significant differences (P<0.0001). The tumor inhibitory effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly superior to those of the nCov-CA521-vcMMAE group (P<0.0001). The CA149-BNLD11 group In the study, tumors completely disappeared in 2 / 5 mice, and no statistical difference was observed among the three groups (CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd) (all P 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 95.4%, 99.5%, 99.2%, and 58.3%, respectively.

[0115] As shown in Figure 19, at the experimental endpoint, the mean tumor weights of all treatment groups were significantly lower than those of the solvent control group (PBS phosphate buffer), with statistical significance (all P<0.05). There was no significant difference among the four groups, CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE (all P>0.05). The tumor weight inhibition rates of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 88.3%, 90.7%, 90.5%, and 58.7%, respectively.

[0116] During the experiment, the experimental animals were active and fed well during the administration period. Except for the vehicle control group (PBS phosphate buffer), all other groups showed some increase in body weight, indicating that the animals tolerated the test substance well. Comparisons between groups showed no significant differences (P>0.05).

[0117] Example 16. Efficacy evaluation of antibody-drug conjugate CA149-BNLD11 against human gastric cancer cell NUGC4 tumors transplanted into Balb / c nude mice NUGC4 human gastric cancer cells were purchased from Hongyuan Bochuang Biotechnology (Beijing) Co., Ltd. and cultured in an incubator at 37°C with 5% CO2 in RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Co., Ltd. NUGC4 cells were grown at a concentration of 1.8 × 10 in serum-free RMPI-1640 medium containing 50% Matrigel. 7 The solution was adjusted to cells / mL and inoculated subcutaneously into the right side of Balb / c nude mice at a volume of 0.1 mL / mouse. The average tumor volume was 108 mm 3 When tumor volume reached 1000 mcg, the mice were divided into four experimental groups of five mice each according to tumor volume, and treatment began on the day of grouping. This experiment employed a single dose, and observation was conducted for 23 days after administration.

[0118] The results are shown in Figures 20 and 21. Figure 20 shows the tumor growth inhibition curves for the antibody-drug conjugate group in a tumor model in which human gastric cancer cells NUGC4 were transplanted into Balb / c nude mice, and Figure 21 shows a histogram of tumor weight for the antibody-drug conjugate group in a tumor model in which human gastric cancer cells NUGC4 were transplanted into Balb / c nude mice.

[0119] As shown in the tumor growth curves in Figure 20, at the experimental endpoint, compared with the solvent control group (PBS phosphate buffer), the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups all significantly inhibited the increase in tumor volume, with all statistically significant differences (all P<0.0001). The tumor-inhibiting effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly superior to those of the nCov-CA521-vcMMAE group (all P<0.001). 0001), the tumor inhibition effect of the CA149-BNLD11 group was significantly superior to that of the CA149-GGFG-Dxd group (P=0.0225), and there was no significant difference between the CA149-vcMMAE group and the CA149-GGFG-Dxd group (P=0.1902). The tumor volume growth inhibition rates (TGI) (%) of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 104.2%, 97.7%, 81.0%, and 23.0%, respectively.

[0120] As shown in Figure 21, at the experimental endpoint, the mean tumor weights of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly lower than those of the vehicle control group (PBS phosphate buffer), with statistical significance (all P less than 0.0001). The tumor weight of the nCov-CA521-vcMMAE group was not statistically different from that of the control group (P = 0.8144). The tumor weights of the 149-vcMMAE and CA149-GGFG-Dxd groups were all significantly smaller than those of the nCov-CA521-vcMMAE group (all P values ​​less than 0.05), and 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.

[0121] During the experiment, the animals were active and fed well during the administration period, and all gained some weight, indicating that the animals tolerated the treatment well. Comparisons between groups showed no significant differences (P>0.05).

[0122] Example 17. Efficacy evaluation of antibody-drug conjugate CA149-BNLD11 against human lung squamous cell carcinoma cell line NCI-H226 tumors transplanted into Balb / c nude mice NCI-H226 human lung squamous cell carcinoma cells were purchased from ATCC and cultured in an incubator at 37°C with 5% CO2 in RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Biotechnology Co., Ltd. NCI-H226 cells were grown at a cell density of 4.0 × 10 in serum-free RMPI-1640 medium containing 50% Matrigel. 7 The solution was adjusted to cells / mL and inoculated subcutaneously into the right side of Balb / c nude mice at a volume of 0.1 mL / mouse. The average tumor volume was 145 mm 3 When tumor volume reached 1000 mcg, mice were divided into four experimental groups of six mice each according to tumor volume, and treatment began on the day of grouping. Each group received a single dose of 3.3 mg / kg. Results are expressed as mean ± SEM (mean ± SEM). Data analysis and processing were performed using Graphpad 8.0 software. Tumor volume and body weight were compared between groups at each time point using two-way analysis of variance. Tumor weight was compared using single-way analysis of variance for statistical analysis. Groups were compared using t-tests. P<0.05 indicated statistical significance.

[0123] The study was terminated on day 24 of the group administration. As shown in the tumor volume growth curves of nude mice, compared with the vehicle control group (PBS phosphate buffer), the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups all significantly inhibited tumor volume growth, with all statistically significant differences (P<0.05). The nCov-CA521-vcMMAE group had no tumor-inhibitory effect (P>0.05). No statistically significant differences were observed among the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups (P>0.05). The tumor volume growth inhibition rates (TGI) of the CA149-vcMMAE, CA149-BNLD11, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 86.6%, 81.1%, 93.5%, and 19.1%, respectively.

[0124] Example 18. Efficacy evaluation of antibody-drug conjugate CA149-BNLD11 at multiple doses against human melanoma cell line SK-MEL-5 tumors transplanted into Balb / c nude mice SK-MEL-5 human melanoma cells were purchased from ATCC and cultured in an incubator at 37°C with 5% CO2 in EMEM medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Biotechnology Co., Ltd. SK-MEL-5 cells were grown at a concentration of 3.0 × 10 in serum-free EMEM medium containing 50% Matrigel. 7 The concentration of the antibody was adjusted to 1 / mL and the antibody was inoculated subcutaneously into the right side of Balb / c nude mice at a volume of 0.1 mL per mouse. The average tumor volume was approximately 103 mm. 3When tumor volume reached 1000 mg / kg, mice were divided into four experimental groups of six mice each according to tumor volume. Treatment began on the day of grouping. The doses were 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg, respectively, and all were administered as a single dose. Results are expressed as mean ± SEM. Data analysis and processing were performed using Graphpad 8.0 software. Differences between groups at each time point were compared using two-way analysis of variance for tumor volume and body weight. Statistical analysis of tumor weight was performed using single-way analysis of variance. Comparisons between groups were performed using t-tests. P<0.05 indicated statistical significance.

[0125] The study was terminated on Day 21 of group administration. As shown in Figure 23 , tumor volume growth curves for human melanoma cell lines SK-MEL-5 transplanted into Balb / c nude mice showed significant tumor volume growth inhibition in the 2.5 mg / kg and 5.0 mg / kg groups compared with the vehicle control group (PBS phosphate buffer). All were statistically significant (P<0.05). The 1.0 mg / kg group showed no significant difference compared with the vehicle control group (PBS phosphate buffer) (P=0.4030). The tumor volume growth inhibition rates (TGI) (%) of CA149-BNLD11 in the 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg groups were 25.1%, 72.1%, and 91.1%, respectively. The tumor-inhibitory activity of CA149-BNLD11 was dose-dependent.

[0126] At the end of the study, as shown in Figure 24, which shows the tumor weight growth curves of human melanoma cell line SK-MEL-5 Balb / c nude mice transplanted tumors, the tumor weights of the 2.5 mg / kg and 5.0 mg / kg groups were significantly reduced compared to the solvent control group (PBS phosphate buffer), with statistical differences (all P<0.05). There was no significant difference in the 1.0 mg / kg group compared to the solvent control group (PBS phosphate buffer) (P=0.7086). The tumor volume growth inhibition rates of CA149-BNLD11 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.

[0127] During the experiment, the experimental animals were active and fed well during the administration period, and all groups gained some weight, indicating that the animals tolerated the test substance well. Comparisons between groups showed no significant differences (P>0.05).

[0128] Example 19. Metabolic studies of antibody drug conjugate CA149-BNLD11 in vivo in mice Three ICR mice were selected and administered CA149-BNLD11 and CA149-vcMMAE via tail vein injection at a dose of 10 mg / kg. Serum was collected before administration and 1 h, 6 h, 24 h, 3 d, 5 d, 7 d, 10 d, 14 d, 21 d, and 28 d after administration. Serum antibody concentrations were measured by ELISA. The specific detection results are shown in the table below.

[0129] [Table 23]

[0130] In vivo metabolic studies in mice, shown in Table 23 and Figure 25, demonstrated that CA149-BNLD11 had a longer half-life than CA149-vcMMAE. The total antibody exposure of CA149-BNLD11 was 1.4-fold higher than that of CA149-vcMMAE, and the total ADC exposure of CA149-BNLD11 was 2.3-fold higher than that of CA149-vcMMAE. Furthermore, as evidenced by the metabolic curve (Figure 25), the excretion rate of BNLD11 in vivo in mice was significantly lower than that of mc-vcMMAE. These results suggest that CA149-BNLD11 exhibited more stable metabolism in vivo in mice.

[0131] Example 20. Mouse in vivo toxicity study of antibody drug conjugate CA149-BNLD11 Balb / c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. Mice were divided into six experimental groups of three mice each based on sex and weight. Body weight and food weight were measured on the day of grouping, and administration began two days later. The administration regimen is shown in Table 24.

[0132] [Table 24]

[0133] The study was terminated on day 14 of administration. Results were expressed as mean numbers and standard errors (Mean ± SEM). Data analysis and processing were performed using Graphpad 8.0 software. Comparisons of volume and food intake were performed using T-test, with P<0.05 indicating statistical significance.

[0134] As shown in Figures 26-27, compared with the vehicle control group (PBS phosphate buffer), both 50 mg / kg CA149-vcMMAE and CA149-BNLD11 significantly reduced mouse body weight. The maximum weight loss occurred on day 4 after administration. The maximum weight loss in females was 17.44% and 10.04%, respectively, and in males was 17.97% and 5.24%, respectively. Both male and female mice in the CA149-vcMMAE group showed varying degrees of hairiness and baldness on days 4 and 6 after administration, returning to normal on day 8. Meanwhile, all mice in the CA149-BNLD11 group were normal and showed no abnormal reactions. On days 4 and 6 after administration, the toxicity of CA149-vcMMAE was significantly stronger than that of CA149-BNLD11 (female: P<0.05, male: P<0.05). Compared with the solvent control group (PBS phosphate buffer), both CA149-vcMMAE and CA149-BNLD11 at 50 mg / kg significantly reduced food intake in mice, with no significant difference between the two groups.

[0135] Example 21. Preliminary toxicity and toxicokinetic studies of antibody-drug conjugate CA149-BNLD11 in cynomolgus monkeys in vivo Four cynomolgus monkeys were selected, half male and half female. At the start of treatment, the males weighed 3.1-3.7 kg, and the females weighed 2.9-3.7 kg. They were divided into three groups: a low-dose CA149-BNLD11 group (2 mg / kg), a medium-dose CA149-BNLD11 group (6 mg / kg), and a high-dose CA149-BNLD11 group (10 mg / kg). The low-dose CA149-BNLD11 group (2 mg / kg) contained one female and one male, the medium-dose CA149-BNLD11 group (6 mg / kg) contained one male, and the high-dose CA149-BNLD11 group (10 mg / kg) contained one female. The dose was 5 mg / mL in each group, and the corresponding administration concentrations were 0.4 mg / mL, 1.2 mg / mL, and 2 mg / mL, respectively. The low-dose and high-dose groups were administered twice each, while the medium-dose group was administered once. These were administered intravenously at a rate of 30 min per animal.

[0136] After administration, in addition to clinical observations, food intake body weight measurements, hematological and blood biochemistry measurements, toxicokinetic studies were also conducted, and blood sampling times included before the first administration and 0.5 h, 2 h, 6 h, 24 h, 72 h, 120 h, 168 h, 240 h, 336 h, and 504 h after the start of administration in the medium dose 6 mg / kg and high dose 10 mg / kg groups.

[0137] During the experimental period, no animals in any group experienced moribundity or death, and no abnormalities were observed in any of the dose groups. The overall body weight fluctuations in each group were not significant, and no abnormalities related to the administration of the test substance were observed. The food intake of animals in each group varied irregularly, and no abnormalities related to the administration of the test substance were observed. As shown in Figure 28, compared to pre-administration, the hematological indices WBC, #NEUT, and %NEUT in cynomolgus monkeys decreased 5 to 14 days after administration of CA149-BNLD11 at doses of 6 mg / kg or higher, and a tendency toward recovery was observed by day 21 after administration. Other hematological indices in each group at each measurement time point were within the normal range, and there were no changes in the dose-effect or time-course correlation. No abnormalities related to the administration of the test substance were observed. Compared to pre-administration, CA149-BNLD11 at a dose of 10 mg / kg tended to increase serum AST in cynomolgus monkeys 5 days after administration, with recovery by day 8 after administration. Other than that, the other blood biochemistry indices of the animals in each group at each measurement time point were within the normal range, and no abnormalities related to the administration of the test substance were observed.

[0138] As shown in Figure 29, the results of toxicokinetic measurements showed that the toxin excretion rate in the 10 mg / kg dose group was significantly lower than that in the 2 mg / kg dose group in cynomolgus monkeys, and the toxin excretion rate in the 10 mg / kg dose group was similar to that in the 6 mg / kg dose group.

Claims

1. An anti-CD228 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises three light chain complementarity-determining regions and three heavy chain complementarity-determining regions, the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 13, LCDR2 set forth in SEQ ID NO: 14, and LCDR3 set forth in SEQ ID NO: 15, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 18; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 20, and LCDR3 set forth in SEQ ID NO: 21, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 22; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 23, LCDR2 set forth in SEQ ID NO: 20, and LCDR3 set forth in SEQ ID NO: 21, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 22; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 24, LCDR2 set forth in SEQ ID NO: 25, and LCDR3 set forth in SEQ ID NO: 26, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 27, and HCDR3 set forth in SEQ ID NO: 28; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO:29, LCDR2 set forth in SEQ ID NO:25, and LCDR3 set forth in SEQ ID NO:30, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO:16, HCDR2 set forth in SEQ ID NO:17, and HCDR3 set forth in SEQ ID NO:28; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 31, LCDR2 set forth in SEQ ID NO: 14, and LCDR3 set forth in SEQ ID NO: 21, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 32; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 44, and LCDR3 set forth in SEQ ID NO: 45, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 49; or the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 46, LCDR2 set forth in SEQ ID NO: 47, and LCDR3 set forth in SEQ ID NO: 48, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, and HCDR3 set forth in SEQ ID NO: 28; An antibody or antigen-binding fragment thereof.

2. the antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 1 and a heavy chain variable region shown in SEQ ID NO: 2; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 3 and a heavy chain variable region set forth in SEQ ID NO: 4; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 5 and a heavy chain variable region set forth in SEQ ID NO: 6; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 9 and a heavy chain variable region set forth in SEQ ID NO: 10; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 11 and a heavy chain variable region set forth in SEQ ID NO: 12; the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 38 and a heavy chain variable region set forth in SEQ ID NO: 37; or The antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 40 and a heavy chain variable region set forth in SEQ ID NO:

39. The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody comprises a heavy chain constant region set forth in SEQ ID NO: 33 and / or a light chain constant region set forth in SEQ ID NO:

34.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A gene encoding the anti-CD228 antibody or antigen-binding fragment thereof of any one of claims 1 to 3. Nucleic acid.

5. The nucleic acid of claim 4, cell.

6. An antibody drug conjugate (ADC), the structure of which is represented by Formula 1: wherein Ab is an anti-CD228 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; LU is the linker, D is a drug, p corresponds to the average DAR value of the antibody-drug conjugate, and p is a value selected from 1 to 10, preferably 1 to 8, preferably 1 to 4 or 4 to 8, and more preferably p is 4; An antibody-drug conjugate characterized in that:

7. The structure of LU-D is VcMMAE, where Vc is valine-citrulline and MMAE is monomethyl auristatin E.

7. The antibody-drug conjugate of claim 6.

8. The structure of the LU-D is as shown below:

7. The antibody-drug conjugate of claim 6.

9. the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 24, LCDR2 set forth in SEQ ID NO: 25, and LCDR3 set forth in SEQ ID NO: 26, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 27, and HCDR3 set forth in SEQ ID NO: 28; Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region set forth in SEQ ID NO: 7 and a heavy chain variable region set forth in SEQ ID NO: 8; More preferably, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 33 and / or the sequence of the light chain constant region is SEQ ID NO:

34.

9. The antibody-drug conjugate of claim 7 or 8.

10. The antibody-drug conjugate of any one of claims 6 to 9, comprising an anti-CD228 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, or a nucleic acid of claim 4, a cell of claim 5, or an antibody-drug conjugate of any one of claims 6 to 9. Pharmaceutical compositions.

11. The anti-CD228 antibody or antigen-binding fragment thereof of any one of claims 1 to 3, the nucleic acid of claim 4, the antibody-drug conjugate of any one of claims 6 to 9, or the pharmaceutical composition of claim 10. kit.

12. 12. An application of the anti-CD228 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid according to claim 4, the antibody-drug conjugate according to any one of claims 6 to 9, the pharmaceutical composition according to claim 10, or the kit according to claim 11 in the manufacture of a reagent for the prevention, treatment, detection or diagnosis of a disease associated with CD228, preferably wherein the disease associated with CD228 comprises one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer and breast cancer. application.

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