Anti-CD228 antibodies and drug conjugates thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that binds to CD228, and an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof has a strong affinity for CD228 protein and the ability to mediate ADCC, and said drug conjugate exhibits an inhibitory effect on various tumors. The present invention further relates to a nucleic acid encoding said antibody or antigen-binding fragment thereof, a cell comprising said nucleic acid, a pharmaceutical composition, a kit comprising said antibody or antigen-binding fragment thereof, said nucleic acid, said cell, or said antibody-drug conjugate, and the application of said antibody or antigen-binding fragment thereof, said nucleic acid, said antibody-drug conjugate, or said pharmaceutical composition in the prevention, treatment, detection, or diagnosis of diseases associated with CD228.

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 called 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, whereas downregulation of CD228 results in decreased cell proliferation.

[0004] CD228 is expressed in a variety of tumors, including 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, while it is expressed in 83% of mesotheliomas and 100% of colon cancers, 57% of breast cancers, and 69% of squamous cell carcinomas, creating a great clinical need.

[0005] Antibody-drug conjugates are formed by coupling antibodies with small molecule chemotherapeutic drugs through linkers, which have high targeting of antibodies and can fully exert the cytotoxicity of chemotherapeutic drugs, achieving the effect of efficiently killing tumor cells. At present, several antibody-drug conjugates have been successfully launched on the market, and more products are under development, and the technology is mature. CD228 is highly expressed in many tumor tissues and low or non-expressed in normal tissues. Based on its expression difference, CD228 may be an ideal antibody-drug conjugate target.

[0006] Therefore, the novel CD228 antibody-drug conjugate serves as an effective anticancer drug and has 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, and 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, 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, application of the CD228 antibody or antigen-binding fragment thereof in the manufacture of antibody-drug conjugates (ADCs), and the anti-CD228 antibody-drug conjugates.

[0008] One aspect of the invention provides an anti-CD228 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising 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 as set forth in SEQ ID NO: 13, LCDR2 as set forth in SEQ ID NO: 14, and LCDR3 as 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 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, and HCDR3 as set forth in SEQ ID NO: 18; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 as set forth in SEQ ID NO: 19, LCDR2 as set forth in SEQ ID NO: 20, and LCDR3 as 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 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, and HCDR3 as set forth in SEQ ID NO: 22; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 as set forth in SEQ ID NO:23, LCDR2 as set forth in SEQ ID NO:20, and LCDR3 as 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 as set forth in SEQ ID NO:16, HCDR2 as set forth in SEQ ID NO:17, and HCDR3 as set forth in SEQ ID NO:22; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 as set forth in SEQ ID NO:24, LCDR2 as set forth in SEQ ID NO:25, and LCDR3 as 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 as set forth in SEQ ID NO:16, HCDR2 as set forth in SEQ ID NO:27, and HCDR3 as set forth in SEQ ID NO:28; The three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise an LCDR1 as set forth in SEQ ID NO:29, an LCDR2 as set forth in SEQ ID NO:25, and an LCDR3 as 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 an HCDR1 as set forth in SEQ ID NO:16, an HCDR2 as set forth in SEQ ID NO:17, and an HCDR3 as set forth in SEQ ID NO:28, or The three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise an LCDR1 as set forth in SEQ ID NO:31, an LCDR2 as set forth in SEQ ID NO:14, and an LCDR3 as 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 an HCDR1 as set forth in SEQ ID NO:16, an HCDR2 as set forth in SEQ ID NO:17, and an HCDR3 as 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, the antibody or antigen-binding fragment thereof comprising: 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 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.

[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, and the like.

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

[0015] The 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, preferably the viral vector includes, but is not limited to, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector, and preferably the vector can be a non-viral vector, preferably the vector can be a mammalian cell expression vector, preferably the expression vector can be a bacterial expression vector, and preferably the expression vector can be a fungal expression vector.

[0016] A fourth aspect of the invention provides a cell, said cell comprising said nucleic acid or said vector, said cell capable of expressing said antibody or antigen-binding fragment thereof. Preferably, said cell is a bacterial cell, preferably said bacterial cell such as an E. coli cell, preferably said cell is a fungal cell, preferably said fungal cell is a yeast cell, preferably said yeast cell such as a Pichia yeast cell, preferably said cell is a mammalian cell, preferably said 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 CD228 antibody or antigen-binding fragment thereof; and (b) a coupling moiety coupled to the 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] TIFF2025504566000002.tif34170 In formula 1, 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] Wherein, the drug is selected from the group consisting of chemotherapy drugs, radiotherapy drugs, hormonal therapy drugs, or immunotherapy drugs. Optionally, the drug is selected from the group consisting of taxanes, maytansinoids, camptothecin, tubulysin, auristatin, calicheamicin, anthracycline, docetaxel, cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, RNase, or radioisotope.

[0021] Furthermore, the linker LU is of the general formula R'-L1-L2-L3, In this general formula, L3 is TIFF2025504566000003.tif17170, wherein L3 is connected at its a-terminus to the drug D and at its b-terminus to L2; R 1 is hydrogen, a carboxyl group, an ester group, a nitro group, a sulfonyl group, a halogen group, or R1 is TIFF2025504566000004.tif29170R 2 ~R 6 are each independently TIFF2025504566000005.tif15170n is 0 to 8, In the general formula, L2 is TIFF2025504566000006.tif16170 wherein 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 TIFF2025504566000007.tif15170n is 0 to 8, In the general formula, L1 is TIFF2025504566000008.tif17170 and / or In the general formula, R' is In the formula TIFF2025504566000009.tif25170, R' is connected to L1 at its c-terminus and to A at its 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 a preferred example, LU-D in formula 1 of the antibody drug conjugate (ADC) is BNLD11, in which LU is MC-β-Ala-(glucuronide)PAB and D is MMAE, and the structure of BNLD11 is: The BNLD11 is represented by TIFF2025504566000010.tif35170, and has an exact mass of 1322.690. The BNLD11 is obtained by synthesis using a conventional method in the prior art. In a preferred example, the BNLD11 is obtained using the synthesis route shown in FIG.

[0023] In one preferred embodiment, 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, 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.

[0024] In a 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, 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 and 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 invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, or antibody-drug conjugate, preferably wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable vector, preferably wherein the pharma- ceutically acceptable vector comprises one or more of a pharma- ceutically acceptable solvent, dispersant, excipient, plasticizer, or other pharmaceutical auxiliary material.

[0027] A seventh aspect of the invention provides a kit comprising the above-mentioned antibody or antigen-binding fragment thereof of the invention, or comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof, comprising the above-mentioned pharmaceutical composition, or comprising the above-mentioned 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 the treatment or prevention of a disease.

[0029] A ninth aspect of the present invention provides an application 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 an 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, detection method comprising administering to a subject or a sample in need thereof the above-mentioned antibody or antigen-binding fragment, nucleic acid, kit, or pharmaceutical composition of the present invention.

[0032] A twelfth aspect of the present invention provides the 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, said 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 manufacture of an antibody drug conjugate (ADC).

[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 CD228 protein and cells expressing 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 on all of the SK-MEL-5, NCI-H226, CALU-1, and NUGC4 animal models, 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 compared with known commercially available antibodies, such as Seagen's CD228-targeting antibody hL49. [Brief description of the drawings]

[0043] [Figure 1] 1 shows the expression level of CD228 on a TMA chip in Example 1. [Diagram 2] 1 shows serum titers of CD228-immunized mice in Example 2. [Diagram 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 of each anti-CD228 antibody in Example 6 based on human melanoma SK-MEL-5 cells. [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 an ADCC experiment using human melanoma SK-MEL-5 cells for each anti-CD228 antibody in Example 7. [Figure 7] FIG. 1 is a schematic diagram showing the sequence alignment results of full-length hCD228 and soluble antigen sMFI2 in Example 8. [Figure 8] This is 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] 1 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] The results of a killing experiment of CA518-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10. [Figure 9F] 13 shows the results of a killing experiment of CA185-VcMMAE on human melanoma SK-MEL-5 cells in Example 10. [Figure 10] Efficacy data (3 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model is shown. [Figure 11] 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 of 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 in vivo pharmacokinetic curves in mice for each anti-CD228 ADC in Example 12. [Figure 15A] Evaluation of the proliferation inhibitory activity of CA149-BNLD11 against MC38-CD228 cells in Example 13. [Figure 15B] Evaluation of the proliferation inhibitory activity of CA149-BNLD11 against A375-CD228 cells in Example 13. [Figure 15C] Evaluation of the proliferation inhibitory activity of CA149-BNLD11 against SK-MEL-5 cells in Example 13. [Figure 15D] Evaluation of the proliferation inhibitory activity of CA149-BNLD11 against A549-CD228 cells in Example 13. [Figure 15E] 13 shows an evaluation of the proliferation inhibitory activity of CA149-BNLD11 against A375-CD228 cells in Example 13. [Figure 16] 1 shows the tumor growth inhibition curves of the antibody-drug conjugates in Example 14 in a Calu-1 lung cancer model. [Figure 17] 13 is a histogram of tumor weight in Calu-1 lung cancer model for antibody drug conjugate groups of antibody drug conjugates in Example 14. [Figure 18] 13 shows tumor growth inhibition curves for the antibody-drug conjugate groups in Example 15 in a human melanoma cell SK-MEL-5 nude mouse transplant tumor model. [Figure 19] 13 is a histogram of tumor weight in a human melanoma cell SK-MEL-5 nude mouse transplant tumor model for the antibody-drug conjugate group in Example 15. [Figure 20]13 shows tumor growth inhibition curves for the antibody-drug conjugate group in Example 16 in a human gastric cancer cell NUGC4 Balb / c nude mouse transplant tumor model. [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 NCI-H226 Balb / c nude mouse transplanted tumor in Example 17. [Figure 23] 13 shows a tumor volume growth curve of human melanoma cell SK-MEL-5 Balb / c nude mice transplanted tumors in Example 18. [Figure 24] 13 shows a tumor weight growth curve of human melanoma cell SK-MEL-5 Balb / c nude mice transplanted tumors in Example 18. [Diagram 25] 1 shows the in vivo metabolic curve of antibody-drug conjugate CA149-BNLD11 in Example 19 in mice. [Figure 26] 20 is a toxicity study in male mice of the antibody drug conjugate CA149-BNLD11 in Example 20. [Figure 27] Example 20: Toxicity study of antibody drug conjugate CA149-BNLD11 in female mice. [Figure 28] 2 shows the change 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 of 6 mg / kg and the high dose group of 10 mg / kg in Example 21. [Diagram 30] FIG. 1 is a schematic diagram of the synthetic circuit of BNLD11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention will be further described below with reference to specific examples. The described examples are only a part of the present invention, and are not all of the examples. It should be understood that the following examples are provided to provide a person skilled in the art to which the present invention pertains with a complete disclosure and explanation of how to utilize the method and composition of the present invention, and are not intended to limit the scope of the present invention. Based on the examples in the present invention, all other examples that a person skilled in the art can obtain without creative effort are within the scope of the claims of the present invention.

[0045] Example 1. CD228 expression experiment PDX sample chips of 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. Twelve cases of mesothelioma were purchased from Shanghai Lidi Biotechnology Co., Ltd. (4 cases) and Shanghai Xinchao Biotechnology Co., Ltd. (8 cases). All PDX sample tissue chips and 8 cases of mesothelioma tissue unstained sections purchased from Shanghai Xinchao Biotechnology Co., Ltd. were commissioned to Crown Biotechnology Co., Ltd. for CD228 immunohistochemical staining. 4 cases of mesothelioma tissue unstained sections were commissioned to 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] Immunohistochemistry experiments were performed using an automated IHC stainer (Bond RX automatic IHC&ISH system, Leica). The results of immunohistochemistry staining were evaluated using the H-score value. TIFF2025504566000012.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 three times independently, and the final H-score value was the average of the three. The IHC score results of all samples are shown in Figure 1. From the expression level of CD228 on the TMA chip shown in Figure 1 and the CD228 expression rate shown in Table 1, it was found that CD228 was moderately and highly expressed 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 to synthesize the genes, which were then transfected into CHO cells and incubated at 37°C, 8% CO 2 The cells were cultured in a shaker at 125 rpm for 10 days, and the supernatant was collected after 10 days of transient expression. The expressed supernatant was purified on a Ni (GenScript, L00250) column and then on a 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 experiment were fully human antibody transgenic mice (a total of 10 mice were immunized) independently developed by Shandong Boan Biotechnology Co., Ltd. Immunization was performed using CD228 (0.23 mg / mL, Boan, 20200924, SEQ ID NO: 41) antigen protein independently produced by Shandong Boan Biotechnology Co., Ltd.

[0051] The immunization method could be either abdominal subcutaneous or inguinal multiple point injection, and the immunization dose of the antigen protein was 20 μg / mouse. In the first immunization, the antigen was emulsified with complete Freund's adjuvant, and in the second to fourth immunizations, the antigen was emulsified with incomplete Freund's adjuvant. The first batch of mice received three immunizations and one booster immunization, and the second batch of mice received four immunizations and one booster immunization. The interval between each immunization was 14 days, and peripheral blood serum was collected on the seventh day after each immunization from the second immunization to detect antibody titers, and mice with unacceptable titers were excluded. After the mice were immunized, the results of detecting mouse serum titers are shown in Figure 2, where 2500X, 12500X, and 62500X represent the dilution rates. Three days after immunization, the mice were euthanized, and the spleens were collected and converted into single cells for library construction.

[0052] 1.3 Construction of phage libraries The mice were killed, the spleens were dissected out, the spleens were crushed with a syringe plug, filtered, the filtered splenocytes were frozen, and cDNA was obtained after RNA extraction. The construction of the phage library was carried out according to the conventional method. The library capacity data of the constructed library is shown in Table 3.

[0053] [Table 3]

[0054] 1.4 Screening by two methods 1.4.1 Plate screening was performed, and the plate was coated 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 procedure, and bound to Thermo magnetic beads, sealed with BSA, incubated with the phage library for 2 h, washed 4-10 times, and then the specifically bound phages were eluted with elution buffer. The clones and sources of the antibodies obtained by screening are shown in Table 4.

[0056] [Table 4]

[0057] Example 3. Molecular construction and production of complete antibodies 133 positive clones IgG1 were constructed and sequenced, and the amino acid sequences of the variable regions of the 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] TIFF2025504566000018.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 linked by homologous recombination to the vector pCDNA3.4 (Life Technology) having a nucleic acid sequence carrying an antibody heavy chain constant region sequence, and the antibody light chain variable region gene was linked to the vector pCDNA3.4 having a nucleic acid sequence carrying an antibody light chain constant region sequence.

[0060] [Table 6]

[0061] Plasmids were extracted from sequenced positive clones and co-transfected into HEK293 cells and incubated at 37°C, 8% CO 2 After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain antibodies, which were then passed through UV280 and combined with the theoretical extinction coefficient to determine the antibody concentration.

[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 (Hakuyasu's own work, 20201014, sequence number 41) protein was diluted to 0.1 μg / mL in pH 9.6 carbonate buffer (hereinafter abbreviated as CBS), coated on the enzyme-labeled plate, and incubated at 4°C overnight at 100 μL / well. After washing the plate, it was sealed with skim milk powder. After washing the plate, 100 μL of antibody diluted in PBST (phosphate buffered saline, Solarbio P1010, +0.05% tween20) was added per well (diluted antibody was gradient diluted complete antibody in PBST, complete antibody contains Fc and Fab regions and has constant region, initial concentration was 0.1 ug / mL, 3-fold dilution 8 gradient), after washing the plate, sheep anti-human IgG(H+L) / HRP (1:5000 dilution, KPL, 474-1006) was added at 100 μL / 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 color, and after 10 min, 50 μL of 2 M H 2 SO 4 The color development was stopped by adding 100% ethanol, and the OD450 was read using a microplate reader. In this experiment and the following experiments, Seagen's antibody hL49 targeting 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, and it was found that the six antibodies showed 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 protein

[0067] [Table 9]

[0068] The binding dynamics of the antibodies to each CD228 protein was measured using a BIAcore 8K instrument based on surface plasmon resonance (SRP) technology. The ProA chip captured 2 μg / mL of CD228 antibodies, 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, and the initial concentration was 50 nM. CD228 protein binding dynamics was performed by Biacore and fitted to calculate the affinity activity KD value.

[0069] [Table 10]

[0070] As can be seen from Table 10, the antibodies shown have similar affinity 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, and then the competitive binding status of the second CD228 antibody (30 μg / mL) was analyzed. The response status of antibody 2 was analyzed by Octet 8K to analyze whether antibody 1 and antibody 2 were in a competitive relationship. Table 11 shows the Octet antibody epitope response values.

[0072] [Table 11]

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

[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, with a cell number of 7E4 / well. Each antibody was gradient diluted with FACS buffer (PBS, BOSTER Biological, Cat. No. PYG0021) and added to a 96-well round bottom plate at 50 μL / well and 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, incubated at 4°C for 30 min in the dark, and after centrifugation at 400 g for 4 min, the supernatant was discarded, washed once with FACS buffer, and resuspended in 100 μL / well of FACS buffer and detected by 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), and all six antibodies were found to have high binding activity to SK-MEL-5 cells and were superior to hL49. The isotype control antibody in Figure 4 was an unrelated antibody against another target, which has the same constant region as the anti-CD228 antibody in the above 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, Cat. No. PYG0021) were added to a 96-well round bottom plate at 50 μL / well, with a cell count of 5E4 / well. Antibodies were then diluted in buffer to a final concentration of 20 μg / mL. 20 μg / mL antibodies were added at 50 μL / well to the round bottom plate containing 50 μL / well of cells, incubated for 30 min, then centrifuged at 400 g for 4 min, and the supernatant was discarded. After washing twice with pre-chilled buffer, 100 μL / well of buffer was added, incubated at 37°C and 4°C, respectively, terminated at different times, centrifuged at 400 g for 4 min, and the supernatant was discarded. 100 μL / well of pre-chilled fluorescent secondary antibody (Jackson, 109-545-008) was added at 4°C at 100 μL / well, and incubated at 4°C for 30 min away from light. The cells were washed once with pre-chilled FACS buffer, resuspended in 100 μL / well of FACS buffer, and detected with a flow cytometer (Eisen, NovoCyte 2060). The results are shown in FIG. 5A, which shows the results of an internalization experiment based on human melanoma SK-MEL-5 cells for each anti-CD228 antibody. FIG. 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 exogenous CD228 gene in logarithmic growth phase were digested, terminated and diluted with serum-containing medium, and added to a 96-well round bottom plate (NEST, Cat. No. 701111) at 50 μL / well, 1E5 / well. Antibodies were diluted in serum-containing medium, and the antibody and labeling reagent (invitrogen, Z25611) were mixed at a molar ratio of 1:3 (antibody concentration 40 nM, labeling reagent 120 nM), incubated at room temperature for 5 min, and then the labeled antibody mixture was added to the well plate containing cells at 50 μL per well. After incubation at 37°C for 0 h, 2 h, 6 h, and 24 h, the cells were washed once with PBS, resuspended in 100 μL / well of PBS, and the MFI value was read by flow cytometry (Eisen, NovoCyte 2060). The results showed that the internalization of CA149 antibody increased over time, and Figure 5B showed that CA149 had better 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 (RPMI1640 medium containing 1% FBS), collect Bioassay Effector Cells (Promega, G7011), and adjust the cell density to 2.4 × 10 using the ADCC working solution. 6 The cell density was adjusted to 8 × 10 using the ADCC working solution. 5The ADCC working solution was used to dilute the test samples, starting from an initial concentration of 5 ug / mL, and diluted to 8 concentrations by 4-fold dilution. 25 uL of effector cells, target cells, and test samples were added to a reaction white plate (Costar, 3917) to make a total reaction volume of 75 uL. The reaction system was incubated at 37°C for 6 h, and 75 uL was added to each well. After reacting for 15 min with Bio-Glo Luciferase System (Promega, G7940), the chemiluminescence value was read using a microplate reader (BioTek, synergy neo2). The results are shown in Figure 6, which shows the ADCC experimental results based on human melanoma SK-MEL-5 cells for each anti-CD228 antibody. From Figure 6, it can be seen that the signal gradually increases as the concentration of the antibody sample increases, indicating that there is an ADCC effect on 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 complex (CA149-Fab) respectively, and commissioned the cryo-electron microscope center of Mizuki Future (Hangzhou) Science and Technology Co., Ltd. to conduct antigen-antibody structural analysis. The experimenters analyzed the types of amino acids in the epitope of the three-dimensional model and the side chain interactions, and the results showed that CA149 antibody Fab binds to the antigen through seven hydrogen bonds and one salt bridge. The specific interaction sites are shown in Table 13, where the superscript * is the amino acid on the CA149 Fab light chain, and the italics without superscript are the amino acids on the CA149 Fab heavy chain.

[0080] [Table 13]

[0081] At the same time, based on the epitope results, the inventors carried out site-specific mutation verification on the CD228 (sequence was as shown in SEQ ID NO:41) antigen independently produced by Shandong Boan Biotechnology Co., Ltd., and constructed the hCD228 antigens of single point mutations of hCD228(R275A), hCD228(R282A), hCD228(E312A), hCD228(L313A) and double point mutations of hCD228(E312A, L313A), hCD228(R282A, E312A), hCD228(R275A, R282A), respectively, and carried out affinity analysis. The affinity results showed that the antigen had a reduced or lost binding activity to the CA149 antibody after mutation, and also showed that the above four sites, 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] In addition, another variable shear product, soluble MFI2, i.e., sMFI2, also exists in the human body at the same time, and furthermore, literature (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) has shown that sMFI can cross the blood-brain barrier via LRP proteins. Therefore, to reduce the risk of possible off-targets, antibodies targeting CD228 should not bind to sMFI2. Based on the epitope results and 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 of anti-hCD228 antibody and drug An antibody-drug conjugate having the following molecular formula is prepared: TIFF2025504566000028.tif30170

[0085] where Ab is any of the anti-CD228 antibodies or antigen-binding fragments thereof described above, LU is the linker (also referred to as the 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 Phosphate buffer (pH 7.5, containing 11 mM DTPA) containing antibody (5-10 mg / mL) was treated with 2 equivalents of TCEP and then incubated at 25°C for about 2 h. 5 equivalents of vcMMAE in DMSO was added to the reduced antibody in PBS and incubated at 25°C for about 1 h. Then, 10 equivalents of n-acetylcysteine ​​(NAC) was added to the mixture and incubated at 25°C for 5 min to quench all unreacted linker-drug, and the buffer was replaced by ultrafiltration to remove free small molecules. Then, the mixture was subjected to HIC-HPLC analysis. The analysis results are shown in Table 15, and the average DAR value of the antibody-drug conjugate used in the present invention was 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 Taking the anti-CD228 antibody CA149 as an example, an ADC composition with a uniform drug loading (DAR) of about 4 was prepared. The 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 about 2 h. Five equivalents of a DMSO solution of BNLD-11 was added to a PBS solution of the reduced antibody and incubated at 25°C for about 1 h, and then 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. HIC-HPLC analysis was used to quantify the drug loading of the antibody-drug conjugate. The analysis results are shown in Figure 8 and Table 16, and it was found that the average DAR value of the antibody-drug conjugate CA149-BNLD11 prepared in this example was about 4.

[0089] [Table 16]

[0090] Example 10. In vitro cell killing experiments of anti-hCD228 ADCs SK-MEL-5 cells were added at 50 μL / well to a 96-well flat-bottom plate (Corning, Cat. No. 3917), with a cell number of 1E4 / well, and the medium was 10% FBS / EMEM. Each anti-CD228-ADC was diluted in the above medium to an initial concentration of 1 μg / mL, with a 4-fold gradient dilution for a total of 6 concentrations. The diluted ADCs were added at 50 μL / well to a 96-well flat-bottom plate and incubated at 37°C, 5% CO 2 After 4 days of culture under the conditions, the CellTiter-Glo kit (Promega, G7571, which needed to be protected from light during use) was equilibrated to room temperature, the buffer in the kit was mixed evenly with the substrate, and left to stand for 1 h. The cell viability detection reagent was added to the 96-well plate at 100 μL / well, shaken at 300 rpm for 2 min, and then left to stand for 10 min. Detection was performed using a microplate reader (BioTek, SYNERGY neo, USA).

[0091] The results are shown in Figures 9A to 9F, which show the results of killing experiments based on human melanoma SK-MEL-5 cells for each anti-CD228 ADC. Two anti-hCD228 ADCs, CA13-vcMMAE and CA67-vcMMAE, showed in vitro cell killing activity similar to that of hL49-vcMMAE, and four anti-hCD228 ADCs, CA149-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and CA185-vcMMAE, showed superior in vitro cell killing activity to the control group hL49-vcMMAE. The isotype control antibodies in Figures 9A to 9F were unrelated antibodies against other targets with the same constant regions as the above 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 human melanoma cell SK-MEL-5 mouse xenograft tumor model Human melanoma cells SK-MEL-5 were purchased from ATCC, and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in an incubator at 37 °C for 1 h in the EMEM medium containing 10% FBS. NOD / SCID mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. SK-MEL-5 cells were cultured at a concentration of 2.5 × 10 7 The tumor volume was adjusted to 0.1 ml / mL and inoculated subcutaneously on 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 100 mg / kg, the mice were divided into eight experimental groups of five mice each according to tumor volume and body weight, and administration was started 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 mouse tumor volume after administration, and it was found that all tumor volumes decreased after administration of the 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 transplanted tumor model showed a 3 Under administration conditions of 100 mg / kg, all six anti-CD228-vcMMAEs prepared in this study were found to be superior to the control group hL49-vcMMAE in terms of 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 incubated at a concentration of 3 × 10 in EMEM medium containing 50% Matrigel. 7 The tumor volume was adjusted to 0.1 ml / mL and inoculated subcutaneously on the right side of NCG mice at a volume of 0.1 mL / mouse. The average tumor volume was approximately 83 mm. 3When the tumor volume reached 1000 mg / kg, the mice were divided into 8 experimental groups of 6 mice each according to tumor volume and body weight, and administration was started on the day of grouping at a dose of 5 mg / kg. The screened antibodies had relatively good efficacy compared to the control hL49. Figure 11 was created based on the volume change data in Table 18. As shown in Table 18, the tumor volume growth inhibition rates (TGI%) of the experimental evaluation items 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 transplant tumor model, under the administration condition of 5 mg / kg, it was found that each of the anti-CD228-vcMMAE prepared in this item 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 the cells were incubated at 37°C, 5% CO 2 The mice were cultured in an incubator at 10 °C, and the medium components were 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 tumor volume was adjusted to 0.1 ml / mouse and inoculated subcutaneously on the right side of CB-17 / SCID mice. The average tumor volume was approximately 156 mm. 3When the tumor volume reached 100 mg / kg, the mice were divided into eight experimental groups of five mice each according to tumor volume and body weight, and administration was started 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, all tumor volumes were reduced, demonstrating in vivo efficacy similar to that of HL49. Figure 12 was created based on the volume change data in Table 20. As shown in Table 19, the tumor volume growth inhibition rates (TGI) for each group of experimental endpoints CA13-vcMMAE, CA67-vcMMAE, CA149-vcMMAE, CA185-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and hL49-vcMMAE 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 Jisui Yaokang Biotechnology Co., Ltd. NCI-H226 cells were incubated at a concentration of 5 × 10 in RPMI 1640 medium containing 50% Matrigel. 7 The solution was adjusted to 0.1 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 the tumor volume reached 100 mg / kg, the mice were divided into 8 experimental groups of 6 mice each according to tumor volume and body weight, and administration was started on the day of grouping, at a dose of 5 mg / kg. The screened antibody had relatively good efficacy compared to the control HL49. Figure 13 was a diagram created based on the volume change data in Table 20. As shown in Table 16, the tumor volume growth inhibition rate TGI of each group of the experimental evaluation items CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE was 94.4%, 92.3%, 94.6%, 98.3%, 103.9%, and 75.6%, respectively, and the tumor weight inhibition rate was 73.1%, 69.2%, 72.1%, 75.5%, 77.4%, and 56.7%, respectively.

[0100] [Table 20]

[0101] Example 12. Mouse in vivo pharmacokinetic studies with anti-CD228 ADC Each ADC was administered to three selected Balb / c mice by subcutaneous injection at a dose of 10 mg / kg. Serum was collected 0 h before administration, and 1 h, 4 h, and 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 the Elisa method.

[0102] Figure 14 shows the pharmacokinetic curves of CA13-vcMMAE, CA67-vcMMAE, and CA149-vcMMAE. The specific detection results are shown in Figure 14, which shows that CA149-vcMMAE has a better pharmacokinetic level than CA13-vcMMAE and CA67-vcMMAE constructed by 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 MC38-CD228 (Kangyuan Bochuang, KC-2023) and A375-CD228 cells (Kangyuan Bochuang, KC-2110) in the logarithmic growth phase were digested, diluted with 10% FBS / 1640 medium, and resuspended, and added to a 96-well flat-bottom plate (SARSTED, catalog number 94.6120.096) at 50 μL / well and 1E4 / well. The antibody-drug conjugate CA149-BNLD11 prepared in part 8.2 of Example 8 was diluted with serum-containing medium to give initial concentrations of 60 μg / mL and 12 μg / mL, respectively, and diluted 5-fold in a gradient manner. The antibody with the antibody ID of CA521 in CN202180003751.7 was selected, and nCov-CA521-vcMMAE prepared in the same manner as in Example 8 was used as the control group (Isotype). The diluted CA149-BNLD11 was added to the above 96-well flat-bottom cell culture plate at 50 μL / well and incubated at 37°C, 5% CO 2 The cells were cultured under the conditions for 96 h, and were kept away from light during the use of the CellCounting-Lite® 2.0 kit (Vazyme, DD1101-01), equilibrated to room temperature, mixed evenly by upside down, added to a 96-well plate at 100 μL / well, shaken at 300 rpm for 2 min, and then left to stand for 10 min. The cell viability was detected using a microplate reader (BioTek, SYNERGY neo, USA).

[0104] As a result, CA149-BNLD11 had excellent proliferation inhibitory activity against MC38-CD228 and A375-CD228 cells, and as shown in Table 21, 50 were 101.5 ng / mL and 61.8 ng / mL, respectively. In Figures 15A to 15B, the vertical axis represents the cell viability %, that is, the ratio of the number of live cells in the administration group to the number of live cells in the blank group. As can be seen from Figures 15A to 15B, CA149-BNLD11 had a significant proliferation inhibitory activity against cells expressing CD228, 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 SK-MEL-5 (ATCC, HTB-70), A549-CD228 (Kangyuan Bochuang, KC-2150), and A375-CD228 cells (Kangyuan Bochuang, KC-2110) in logarithmic growth phase were digested, terminated with serum-containing medium, diluted, and added to a 96-well flat-bottom plate (SARSTED, Cat. No. 94.6120.096) at 50 μL / well and 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, and diluted 5-fold, 6-fold, and 5-fold, respectively. The diluted ADC was added to the above 96-well flat-bottom cell culture plate at 50 μL / well and incubated at 37°C, 5% CO 2 After incubation for 96 or 120 h under the conditions, the CellCounting-Lite(R) 2.0 kit (Vazyme, DD1101-01, avoid light during use) was equilibrated to room temperature, mixed evenly by upside down, added to a 96-well plate at 100 μL / well, shaken at 300 rpm for 2 min, and then left to stand for 10 min. Cell viability was detected using a microplate reader (BioTek, SYNERGY neo, USA). As a result, it was shown that CA149-BNLD11 has excellent proliferation inhibitory activity against SK-MEL-5, A549-CD228, and A375-CD228 cells (IC50 was 11.48 ng / mL, 15.25 ng / mL, and 13.86 ng / mL, respectively). As is clear from Figures 15C to 15E, CA149-BNLD11 had a significant proliferation inhibitory activity against cells expressing CD228 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 carcinoma cells were purchased from ATCC, and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in a 100-mL incubator in McCoy's 5A medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Co., Ltd. CaLu-1 cells were cultured at a cell density of 5.0 × 10 in serum-free McCoy's 5A medium containing 50% Matrigel. 7 The cells were adjusted to 1 / mL and 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 135 mm. 3 When the tumor volume reached 1000 mg / kg, the mice were divided into 5 experimental groups of 5 mice each according to the tumor volume, and administration was started on the day of grouping. This experiment employed a single administration, and the mice were observed for 28 days after administration.

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

[0110] As shown in FIG. 16, compared with the solvent control group (PBS phosphate buffer), all of the antibody-drug conjugate-administered groups could significantly inhibit the increase in tumor volume, all with statistical differences (all P less than 0.05), and the tumor inhibition effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly better than those of the nCov-CA521-vccMMAE group (P values ​​were 0.0001 and 0.0016, respectively). , 0.0006), no significant differences were found among the three groups of CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd (all P greater than 0.05), and 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 FIG. 17, at the experimental evaluation point, the average 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 difference (all P less than 0.05); the tumor weights of the nCov-CA521-vcMMAE group were not statistically different from those of the control group (P=0.3250); no significant difference was found among the three groups of CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd (all P greater than 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 course of the experiment, the experimental animals were active and fed during the administration period, and all gained some weight, indicating that the animals tolerated the test substance 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 SK-MEL-5 tumors transplanted into nude mice SK-MEL-5 human melanoma cells were purchased from ATCC, and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in a 100-mL incubator in EMEM medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Co., Ltd. SK-MEL-5 cells were cultured at a concentration of 5.0 × 10 in serum-free EMEM medium containing 50% Matrigel. 7 The cells were adjusted to 1 / mL and 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 the tumor volume reached 1000 mg / kg, the mice were divided into four experimental groups of five mice each according to the tumor volume, and administration was started on the day of grouping. This experiment employed a single administration, and the mice were observed for 28 days after administration.

[0114] The results are shown in Figures 18 to 19. Figure 18 is a tumor growth inhibition curve in the human melanoma cell SK-MEL-5 nude mouse transplanted tumor model of the antibody-drug conjugate group, and Figure 19 is a histogram of tumor weight in the human melanoma cell SK-MEL-5 nude mouse transplanted tumor model of the antibody-drug conjugate group. The tumor growth curve shown in Figure 18 shows that, at the experimental evaluation point, compared with the solvent control group (PBS phosphate buffer), each administration group can significantly inhibit the increase in tumor volume, and all have statistical differences (all P<0.0001), and the tumor inhibition effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups are significantly better than the nCov-CA521-vcMMAE group (all P<0.0001), and the CA149-BNLD11 group In the nCov-CA521-vcMMAE group, tumors completely disappeared in 2 / 5 mice, and no statistical difference was found 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 FIG. 19, at the time of the experimental evaluation, the average tumor weights of all treatment groups were significantly lower than those of the solvent control group (PBS phosphate buffer), with statistical differences (all P less than 0.05), and there was no significant difference between the four groups of CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE (all P greater than 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 course of the experiment, the experimental animals were active and fed during the administration period, and except for the solvent control group (PBS phosphate buffer), the body weight of each group increased to some extent, 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 Balb / c nude mouse transplanted tumors NUGC4 human gastric cancer cells were purchased from Hongyuan Bochuang Biotechnology (Beijing) Co., Ltd., and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in a 100-mL incubator 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 cell density of 1.8 × 10 in serum-free RMPI-1640 medium containing 50% Matrigel. 7 The cells were adjusted to 1 / mL and 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 108 mm. 3 When the tumor volume reached 1000 mg / kg, the mice were divided into four experimental groups of five mice each according to the tumor volume, and administration was started on the day of grouping. This experiment employed a single administration, and the mice were observed for 23 days after administration.

[0118] The results are shown in Figures 20 and 21. Figure 20 shows the tumor growth inhibition curves in the antibody-drug conjugate group in a tumor model transplanted into human gastric cancer cells NUGC4 Balb / c nude mice, and Figure 21 shows a histogram of tumor weight in the antibody-drug conjugate group in a tumor model transplanted into human gastric cancer cells NUGC4 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 could all significantly inhibit the increase in tumor volume, all with statistical differences (all P<0.0001), and the tumor inhibition effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly better than those of the nCov-CA521-vcMMAE group (all P<0.001). 0001), the tumor inhibition effect of the CA149-BNLD11 group was significantly better than that of the CA149-GGFG-Dxd group (P=0.0225), there was no significant difference between the CA149-vcMMAE group and the CA149-GGFG-Dxd group (P=0.1902), and the tumor volume growth inhibition rate TGI (%) of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups was 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-GGGF-Dxd groups were significantly lower than those of the solvent control group (PBS phosphate buffer), with statistical differences (all P less than 0.0001); the tumor weights of the nCov-CA521-vcMMAE group were not statistically different from those 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 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 course of the experiment, the experimental animals were active and fed during the administration period, and all gained some weight, indicating that the animals tolerated the test substance 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 Balb / c nude mouse transplanted tumors NCI-H226 human lung squamous cell carcinoma cells were purchased from ATCC, and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in a 100-mL incubator in RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical 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 cells were adjusted to 1 / mL and 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 145 mm 3 When the tumor volume reached 100 mg / kg, the mice were divided into four experimental groups of 6 mice each according to tumor volume, and treatment was started on the day of grouping. The dose was 3.3 mg / kg, administered in a single dose. Results were expressed as mean and standard error (Mean ± SEM), and data analysis and processing were performed using Graphpad 8.0 software. Tumor volume and body weight were compared between each group at each time point using two-factor analysis of variance, and tumor weight was compared using single-factor analysis of variance for statistical analysis of difference. Comparisons were made between the two groups using T-test, and P<0.05 indicated that the difference had statistical significance.

[0123] The study was terminated on the 24th day of group administration. As shown in the tumor volume growth curves of nude nude mouse transplanted tumors, compared with the solvent control group (PBS phosphate buffer), the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups all significantly inhibited tumor volume growth, all with statistical differences (all P less than 0.05), the nCov-CA521-vcMMAE group had no tumor inhibition effect (P4>0.05), and no statistical differences were found between the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups (P>0.05), and 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 SK-MEL-5 Balb / c nude mouse transplanted tumors SK-MEL-5 human melanoma cells were purchased from ATCC, and the cells were incubated at 37°C and 5% CO 2 The cells were cultured in a 100-mL incubator in EMEM medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jisui Pharmaceutical Co., Ltd. SK-MEL-5 cells were cultured at a concentration of 3.0 × 10 in serum-free EMEM medium containing 50% Matrigel. 7 The cells were adjusted to 1 / mL and 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 4 experimental groups of 6 mice each according to tumor volume, and treatment was started on the day of grouping, with the doses of 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg, respectively, all administered in a single dose. Results were expressed as mean and standard error (Mean ± SEM), and data analysis and processing were performed using Graphpad 8.0 software. Tumor volume and body weight were compared between each group at each time point using two-factor analysis of variance, and tumor weight was compared using single-factor analysis of variance for statistical analysis of difference, and T-test was used to compare between the two groups, with P<0.05 indicating that the difference had statistical significance.

[0125] The test was terminated on the 21st day of group administration. As shown in the tumor volume growth curve of human melanoma cell SK-MEL-5 Balb / c nude nude mouse transplanted tumor in Figure 23, compared with the solvent control group (PBS phosphate buffer), the 2.5 mg / kg and 5.0 mg / kg groups all significantly inhibited tumor volume growth, all with statistical differences (P less than 0.05), the 1.0 mg / kg group had no significant difference compared with the solvent control group (PBS phosphate buffer) (P = 0.4030), and the tumor volume growth inhibition rates TGI (%) of the 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg groups of CA149-BNLD11 were 25.1%, 72.1%, and 91.1%, respectively. It was found that the tumor inhibitory activity of CA149-BNLD11 showed dose-dependence.

[0126] At the end of the study, as shown in the tumor weight growth curve of human melanoma cell SK-MEL-5 Balb / c nude mice transplanted tumor in FIG. 24, the tumor weights of the 2.5 mg / kg and 5.0 mg / kg groups were significantly reduced and had statistical differences (all P values ​​less than 0.05) compared with the solvent control group (PBS phosphate buffer), while the 1.0 mg / kg group had no significant difference compared with the solvent control group (PBS phosphate buffer) (P=0.7086), and 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 course of the experiment, the experimental animals were active and fed during the administration period, and the body weight of each group increased to a certain extent, 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 mice in vivo Three ICR mice were selected and CA149-BNLD11 and CA149-vcMMAE were administered 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] From the in vivo metabolism study in mice shown in Table 23 and Figure 25, it was found that CA149-BNLD11 had a longer half-life than CA149-vcMMAE. The total antibody exposure of CA149-BNLD11 was 1.4 times that of CA149-vcMMAE, and the total ADC exposure of CA149-BNLD11 was 2.3 times that of CA149-vcMMAE. In addition, as can be seen from the metabolism curve (Figure 25), the excretion rate of BNLD11 was significantly lower than mc-vcMMAE in mice in vivo. Thus, CA149-BNLD11 was more stable in metabolism in mice in vivo.

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

[0132] [Table 24]

[0133] The study was terminated on the 14th day of administration. Results were expressed as mean numbers and standard errors (Mean ± SEM). Data analysis and processing were performed using Graphpad 8.0 software. Volume and food intake were compared using T-test, and P < 0.05 indicated that the difference had statistical significance.

[0134] As shown in Figures 26-27, compared with the solvent control group (PBS phosphate buffer), 50 mg / kg of CA149-vcMMAE and CA149-BNLD11 were all able to significantly reduce mouse body weight. The maximum weight loss occurred on the fourth day after administration in all cases. The female maximum weight loss was 17.44% and 10.04%, respectively, and the male maximum weight loss was 17.97% and 5.24%, respectively. The female and male mice in the CA149-vcMMAE group showed different degrees of hairiness and wilting symptoms on the fourth and sixth days after administration, and returned to normal on the eighth day after administration, while the mice in the CA149-BNLD11 group were all normal and had no abnormal reactions. On the 4th and 6th days after administration, the toxicity of CA149-vcMMAE was significantly stronger than that of CA149-BNLD11 (female: all P less than 0.05, male: all P less than 0.05), and compared with the solvent control group (PBS phosphate buffer), 50 mg / kg of CA149-vcMMAE and CA149-BNLD11 were both able to significantly reduce the food intake of mice, with no significant difference between the two groups of CA149-vcMMAE and CA149-BNLD11.

[0135] Example 21. Preliminary toxicity experiments 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 administration, the males weighed 3.1-3.7 kg, and the females weighed 2.9-3.7 kg. They were divided into three groups: a CA149-BNLD11 low dose group (2 mg / kg), a CA149-BNLD11 medium dose group (6 mg / kg), and a CA149-BNLD11 high dose group (10 mg / kg). The CA149-BNLD11 low dose group (2 mg / kg) contained one female and one male, the CA149-BNLD11 medium dose group (6 mg / kg) contained one male, and the CA149-BNLD11 high dose 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, and the medium-dose group was administered once. The drugs were intravenously injected at 30 min / animal.

[0136] After administration, in addition to clinical observations, food intake 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 mid-dose 6 mg / kg and high-dose 10 mg / kg groups.

[0137] During the experimental period, there were no moribund or deaths in any of the groups, and no abnormalities were observed in any of the animals in any of the dose groups. The overall weight fluctuations in each group of animals were not large, and no abnormalities were observed in the animals in any of the dose groups. The food intake of the animals in each group changed irregularly, and no abnormalities were observed in the animals in any of the dose groups. As shown in FIG. 28, compared to before administration, the hematological indices WBC, #NEUT, and %NEUT of cynomolgus monkeys decreased 5 to 14 days after administration of CA149-BNLD11 at doses of 6 mg / kg and above, and showed a tendency to recover 21 days after administration. Other than that, the other hematological indices of the animals in each group at each measurement time point were almost within the normal range, and there was no change in the dose-effect and time-course correlation. No abnormalities were observed in the animals in any of the dose groups. Compared to before administration, CA149-BNLD11 at a dose of 10 mg / kg could bring about a tendency to improve serum AST in cynomolgus monkeys 5 days after administration, and could recover 8 days after administration. Other than that, other blood biochemistry indices of animals in each group at each measurement time point were almost within the normal range, and no abnormalities related to the administration of the subject were observed.

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

Claims

1. An anti-CD228 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising 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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 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 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 include 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 include 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 an 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 as set forth in SEQ ID NO: 33, and / or a light chain constant region as 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 according to any one of claims 1 to 3. Nucleic acid.

5. The nucleic acid according to 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, where p is a value selected from 1 to 10, preferably 1 to 8, preferably 1 to 4 or 4 to 8, more preferably p is 4; An antibody-drug conjugate comprising:

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 include 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 include 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 as set forth in SEQ ID NO:7 and a heavy chain variable region as 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 or antigen-binding fragment thereof of any one of claims 1 to 3, or the nucleic acid of claim 4, the cell of claim 5, or the antibody drug conjugate of any one of claims 6 to 9. Pharmaceutical compositions.

11. The 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. 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 said disease associated with CD228 comprises one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer and breast cancer. application.

Images