Anti-CD228 antibodies and drug conjugates thereof

Abstract

The invention relates to an antibody binding to CD228 or an antigen binding fragment thereof, and an antibody drug conjugate containing the antibody or the antigen binding fragment thereof, the antibody or the antigen binding fragment thereof has strong affinity and ADCC mediation capability on CD228 protein, and the antibody drug conjugate shows an inhibition effect on various tumors. A nucleic acid encoding the antibody or antigen-binding fragment thereof; a cell containing the nucleic acid; the invention also relates to a pharmaceutical composition and a kit containing the antibody or the antigen binding fragment thereof, the nucleic acid, the cell or the antibody drug conjugate. The invention also discloses application of the antibody or the antigen binding fragment thereof, the nucleic acid, the antibody drug conjugate or the pharmaceutical composition in prevention, treatment, detection or diagnosis of CD228 related diseases.

Description

Anti-CD228 antibody and drug conjugate Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to an anti-CD228 antibody and a drug conjugate thereof. Background Art

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

[0003] CD228 plays a role in cell proliferation, migration and tumorigenesis. Increased expression of CD228 can lead to accelerated growth of melanoma tumors. In cell models, high expression of CD228 can increase cell proliferation, while downregulation of CD228 leads to reduced cell proliferation.

[0004] CD228 is expressed in a variety 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, as well as 83% of mesotheliomas, 100% of colon cancers, 57% of breast cancers, and 69% of squamous cell carcinomas, indicating significant clinical demand.

[0005] Antibody-drug conjugates (ADCs) combine antibodies with small molecule chemotherapy drugs via linkers. They combine the highly targeted properties of antibodies with the cytotoxicity of chemotherapy drugs to effectively kill tumor cells. Currently, several ADCs have been successfully marketed, with a growing number of products under development, demonstrating the maturity of the technology. CD228 is highly expressed in many tumor tissues and low or absent in normal tissues. Given this heterogeneous expression pattern, CD228 may be an ideal target for ADCs.

[0006] Therefore, providing a new CD228 antibody-drug conjugate as an effective anti-cancer drug has broad application value in the medical field.

[0007] Summary of the Invention

[0008] The present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, which can bind to the CD228 protein. The present invention also provides a nucleic acid encoding the antibody or the antigen-binding fragment thereof; a cell containing the nucleic acid; a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof, the nucleic acid, and the cell; a kit containing the antibody or the antigen-binding fragment thereof, the nucleic acid, and the pharmaceutical composition; the use of the antibody or the antigen-binding fragment thereof, the nucleic acid, and the pharmaceutical composition in preventing, treating, detecting, or diagnosing diseases associated with CD228; the use of the CD228 antibody or the antigen-binding fragment thereof in preparing an antibody-drug conjugate (ADC); and an anti-CD228 antibody conjugate.

[0009] One aspect of the present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the following three light chain complementary determining regions and / or three heavy chain complementary determining regions:

[0010] The three light chain complementary 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 complementary 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;

[0011] The three light chain complementary 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 complementary 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;

[0012] The three light chain complementary 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 complementary 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;

[0013] The three light chain complementary 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 complementary 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;

[0014] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:30, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:28; or

[0015] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:31, LCDR2 shown in SEQ ID NO:14, and LCDR3 shown in SEQ ID NO:21, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:32; or

[0016] The three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:44, and LCDR3 shown in SEQ ID NO:45, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:49; alternatively, the three light chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:46, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:48, and / or the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:28.

[0017] In a specific embodiment of the present invention, the present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, which, when bound to CD228, binds to at least one of the following residues shown in SEQ ID NO:41: E312A, L313A, R282A, R275A.

[0018] In one embodiment of the present invention, the present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0019] The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 1, and / or a heavy chain variable region shown in SEQ ID NO: 2;

[0020] The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 3, and / or a heavy chain variable region shown in SEQ ID NO: 4;

[0021] The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 5, and / or a heavy chain variable region shown in SEQ ID NO: 6;

[0022] The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 7, and / or a heavy chain variable region shown in SEQ ID NO: 8;

[0023] The antibody or antigen-binding fragment thereof comprises a light chain variable region as set forth in SEQ ID NO: 9, and / or a heavy chain variable region as set forth in SEQ ID NO: 10; the antibody or antigen-binding fragment thereof comprises a light chain variable region as set forth in SEQ ID NO: 11, and / or a heavy chain variable region as set forth in SEQ ID NO: 12; the antibody or antigen-binding fragment thereof comprises a light chain variable region as set forth in SEQ ID NO: 38, and / or a heavy chain variable region as set forth in SEQ ID NO: 37; or

[0024] The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 40, and / or a heavy chain variable region shown in SEQ ID NO: 39.

[0025] In a specific embodiment of the present invention, the sequence of the heavy chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 33.

[0026] Furthermore, the sequence of the light chain constant region of the antibody or antigen-binding fragment thereof is SEQ ID NO: 34.

[0027] In the embodiments of the present invention, the antibodies or antigen-binding fragments thereof include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, scFv or dsFv fragments, etc.

[0028] The second aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0029] 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; 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; 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; preferably, the expression vector can be a fungal expression vector.

[0030] The fourth aspect of the present invention provides a cell, comprising the nucleic acid or the vector, and capable of expressing the antibody or antigen-binding fragment thereof. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is an Escherichia coli cell, etc.; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cell is a Pichia pastoris cell, etc.; preferably, the cell is a mammalian cell; preferably, the mammalian cell is 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, etc.

[0031] A fifth aspect of the present invention provides an anti-CD228 antibody conjugate, characterized in that the anti-CD228 antibody conjugate comprises (a) the CD228 antibody or its antigen-binding fragment, and (b) a coupling portion coupled to the antibody portion, wherein the coupling portion is selected from one or more of a detectable marker, a drug, a toxin, a cytokine, a radionuclide and an enzyme.

[0032] In another preferred embodiment, the antibody drug conjugate (ADC) is shown in the following formula 1:

[0033] Wherein, in Formula 1: Ab is an anti-CD228 antibody or antigen-binding fragment thereof of the present invention, LU is a linker (also known as a linker), and D is a drug; the subscript p corresponds to the average DAR value of the antibody-drug conjugate, and p is a value selected from 1-10, preferably 1-8, preferably 1-4 or 4-8, and more preferably p is 4.

[0034] Wherein, the drug is selected from chemotherapy drugs, radiotherapy drugs, hormone therapy drugs or immunotherapy drugs. Optionally, the drug is selected from the following group: taxanes, maytansinoids, camptothecins, tubulysins, auristatins, calicheamicins, anthracyclines, docetaxel, cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, RNase or radioisotope.

[0035] Furthermore, the linker LU consists of the general formula R'-L1-L2-L3;

[0036] In this general formula, L3 is:

[0037] Wherein the a end of L3 is connected to the drug D, and the b end is connected to the L2;

[0038] R1 is hydrogen, carboxyl, ester, nitro, sulfonyl, or halogen; or R1 is R2-R6 are each independently hydrogen, n is 0-8;

[0039] In this general formula, L2 is: 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 residue, a guanine residue, a β-glycine residue, or a β-alanine residue; and X is: n is 0-8;

[0040] In this general formula, L1 is: and / or

[0041] In this general formula, R' is: Among them, the c-terminus of R' is connected to L1, and the d-terminus is connected to A;

[0042] In a preferred embodiment, LU-D in the antibody-drug conjugate (ADC) formula 1 is VcMMAE, wherein LU is Vc (valine-citrulline linker) and D is MMAE (monomethyl auristatin E); VcMMAE can also be written as MC-Val-Cit-PAB-MMAE or mc-vc-PAB-MMAE.

[0043] In a preferred embodiment, LU-D in the antibody-drug conjugate (ADC) formula 1 is BNLD11, wherein LU is MC-β-Ala-(glucuronide)PAB, D is MMAE, and the structure of BNLD11 is as follows:

[0044] The Exact Mass of the BNLD11 is 1322.690; the BNLD11 is synthesized using conventional methods in the prior art; in a preferred example, BNLD11 is obtained using the synthesis route shown in FIG30 .

[0045] In a preferred embodiment, the three light chain complementary determining regions of the anti-CD228 antibody or antigen-binding fragment thereof (Ab) in the ADC formula 1 comprise LCDR1 shown in SEQ ID NO: 24, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO: 26, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 27, and HCDR3 shown in SEQ ID NO: 28; preferably, 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; 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.

[0046] In a preferred embodiment, LU-D in the ADC formula 1 is VcMMAE, p is 4, and Ab is an anti-CD228 antibody or an antigen-binding fragment thereof, wherein the three light chain complementary determining regions of the anti-CD228 antibody or the 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 complementary determining regions of the antibody or the 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 the 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 the antigen-binding fragment thereof is SEQ ID NO:33 and / or the sequence of the light chain constant region is SEQ ID NO:34.

[0047] In a preferred embodiment, the LU-D in the ADC formula 1 is a BNLD11 structure, p is 4, and the BNLD11 structure is as follows:

[0048] Ab is an anti-CD228 antibody or an antigen-binding fragment thereof, wherein the three light chain complementary determining regions of the anti-CD228 antibody or the antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:26, and the three heavy chain complementary determining regions of the antibody or the antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28; more preferably, the antibody or the 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.

[0049] The sixth aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or antibody-drug conjugate. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier includes one or more of the following: a pharmaceutically acceptable solvent, dispersant, additive, plasticizer or other pharmaceutical excipient.

[0050] The seventh aspect of the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the present invention, or a nucleic acid encoding the antibody or antigen-binding fragment thereof, the pharmaceutical composition, or the antibody-drug conjugate.

[0051] The eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell or antibody-drug conjugate in the preparation of a pharmaceutical composition for treating or preventing a disease.

[0052] The ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof or nucleic acid in the preparation of a diagnostic or detection kit.

[0053] The tenth aspect of the present invention provides a method for treating or preventing a disease, comprising administering the antibody or antigen-binding fragment, nucleic acid, vector, cell, pharmaceutical composition or antibody-drug conjugate of the present invention to a subject in need.

[0054] The eleventh aspect of the present invention provides a method for diagnosis and detection, comprising administering the antibody or antigen-binding fragment, nucleic acid, kit or pharmaceutical composition of the present invention to a subject or sample in need.

[0055] The twelfth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, pharmaceutical composition or antibody-drug conjugate for treating or preventing diseases.

[0056] The thirteenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof, nucleic acid, kit, or pharmaceutical composition for detection and diagnosis.

[0057] A fourteenth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof, the nucleic acid, the pharmaceutical composition or the antibody-drug conjugate for preparing a preparation for preventing, treating, detecting or diagnosing a disease associated with CD228.

[0058] In the embodiments of the present invention, the diseases associated with CD228 include one or more of melanoma, lung cancer, non-small cell lung cancer, gastric cancer, colon cancer, colon adenocarcinoma, mesothelioma, pancreatic cancer, and breast cancer.

[0059] A fifteenth aspect of the present invention further provides use of the CD228 antibody or antigen-binding fragment thereof of the present invention in the preparation of an antibody-drug conjugate (ADC).

[0060] The anti-CD228 antibodies and antibody-drug conjugates provided by the present invention have one or more of the following advantages:

[0061] 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.

[0062] 2. The anti-CD228 antibody-drug conjugate provided by the present invention has good killing ability against human melanoma SK-MEL-5 cells.

[0063] 3. The anti-CD228 antibody-drug conjugate provided by the present invention has a good anti-cancer effect in SK-MEL-5, NCI-H226, CALU-1, and NUGC4 animal models, and shows stable efficacy data for CD228-related diseases.

[0064] 4. The anti-CD228 antibody-drug conjugate provided by the present invention has good pharmacokinetics in mice.

[0065] 5. The anti-CD228 antibody-drug conjugate provided by the present invention has low side effects and toxicity and high safety.

[0066] 6. The anti-CD228 antibody of the present invention has better internalization and endocytosis effects than known antibodies on the market, such as Seagen's CD228-targeting antibody hL49. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG1 shows the expression level of CD228 on the TMA chip in Example 1.

[0068] FIG2 shows the serum titers of CD228-immunized mice in Example 2.

[0069] FIG3 shows the binding of each anti-CD228 antibody in Example 4 to human CD228 protein.

[0070] FIG4 shows the binding activity of each anti-CD228 antibody in Example 5 to human melanoma SK-MEL-5 cells expressing CD228 protein.

[0071] FIG5A shows the internalization experimental results of each anti-CD228 antibody in Example 6 based on human melanoma SK-MEL-5 cells, and FIG5B shows the internalization experimental results of each anti-CD228 antibody in Example 6 based on human lung cancer cell line A549-CD228 cells.

[0072] FIG6 shows the ADCC experimental results of each anti-CD228 antibody in Example 7 based on human melanoma SK-MEL-5 cells.

[0073] FIG7 is a schematic diagram showing the sequence alignment results of full-length hCD228 and soluble antigen sMFI2 in Example 8.

[0074] FIG8 is a HIC-HPLC spectrum of CA149-BNLD11 in Example 9.

[0075] Figure 9A is the killing experiment result of CA13-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10, Figure 9B is the killing experiment result of CA67-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10, Figure 9C is the killing experiment result of CA149-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10, Figure 9D is the killing experiment result of BA352-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10, Figure 9E is the killing experiment result of CA518-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10, and Figure 9F is the killing experiment result of CA185-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10.

[0076] FIG10 shows the efficacy data of various anti-CD228 ADCs in the SK-MEL-5 animal model (3 mg / kg).

[0077] FIG11 shows the efficacy data of various anti-CD228 ADCs in the SK-MEL-5 animal model (5 mg / kg).

[0078] FIG12 shows the efficacy data of each anti-CD228 ADC (3 mg / kg) in Example 11 in the NCI-H226 animal model.

[0079] FIG13 shows the efficacy data of each anti-CD228 ADC (5 mg / kg) in Example 11 in the NCI-H226 animal model.

[0080] FIG14 is the pharmacokinetic curves of each anti-CD228 ADC in Example 12 in mice.

[0081] Figure 15A is the evaluation of the inhibitory activity of CA149-BNLD11 on MC38-CD228 cells in Example 13, Figure 15B is the evaluation of the inhibitory activity of CA149-BNLD11 on A375-CD228 cells in Example 13, Figure 15C is the evaluation of the inhibitory activity of CA149-BNLD11 on SK-MEL-5 cells in Example 13, Figure 15D is the evaluation of the inhibitory activity of CA149-BNLD11 on A549-CD228 cells in Example 13, and Figure 15E is the evaluation of the inhibitory activity of CA149-BNLD11 on A375-CD228 cells in Example 13.

[0082] FIG16 is a tumor growth inhibition curve of the antibody drug conjugate in Example 14 in the Calu-1 lung cancer model.

[0083] FIG17 is a histogram of tumor weight in the Calu-1 lung cancer model of the antibody drug conjugate group in Example 14.

[0084] FIG18 is a tumor growth inhibition curve of the antibody drug conjugate group in Example 15 in a human melanoma cell SK-MEL-5 nude mouse transplanted tumor model.

[0085] FIG19 is a histogram of tumor weight of the antibody drug conjugate group in Example 15 in a human melanoma cell SK-MEL-5 nude mouse xenograft tumor model.

[0086] FIG20 is a tumor growth inhibition curve of the antibody drug conjugate group in Example 16 in the human gastric cancer cell NUGC4 Balb / c nude mouse transplant tumor model.

[0087] FIG21 is a bar graph showing the tumor weight of the antibody drug conjugate group in Example 16 in a human gastric cancer cell NUGC4 Balb / c nude mouse xenograft tumor model.

[0088] FIG22 is a tumor volume growth curve of the human lung squamous cell carcinoma cell NCI-H226 Balb / c nude mouse xenograft tumor in Example 17.

[0089] FIG23 is a tumor volume growth curve of the human melanoma cell SK-MEL-5 Balb / c nude mouse xenograft tumor in Example 18.

[0090] FIG24 is a tumor weight growth curve of the human melanoma cell SK-MEL-5 Balb / c nude mouse transplanted tumor in Example 18.

[0091] FIG25 is a metabolic curve of the antibody drug conjugate CA149-BNLD11 in Example 19 in mice.

[0092] Figure 26 shows the toxicity study of the antibody drug conjugate CA149-BNLD11 in Example 20 in male mice.

[0093] Figure 27 shows the toxicity study of the antibody drug conjugate CA149-BNLD11 in Example 20 in female mice.

[0094] Figure 28 shows the changes in body weight of cynomolgus monkeys in the middle-dose group (6 mg / kg) and the high-dose group (10 mg / kg) after administration of Example 21.

[0095] Figure 29 is the toxicity test after the first administration of the medium-dose group 6 mg / kg and the high-dose group 10 mg / kg in Example 21.

[0096] Figure 30 is a schematic diagram of the BNLD11 synthesis route. DETAILED DESCRIPTION

[0097] The present invention will be further described below in conjunction with specific examples. The described embodiments are some embodiments of the present invention, rather than all embodiments. It should be understood that the following examples are provided to provide a complete disclosure and description of how to utilize the methods and compositions of the present invention to those skilled in the art, and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0098] Example 1 CD228 expression experiment

[0099] PDX microarrays of gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, bile duct cancer, pancreatic cancer, cervical cancer, sarcoma, esophageal cancer, and gastric cancer were purchased from Crown Biotech Co., Ltd. Tissue slides from 12 mesothelioma patients were purchased from Shanghai Lidi Biotechnology Co., Ltd. (4 slides) and Shanghai Xinchao Biotechnology Co., Ltd. (8 slides). CD228 immunohistochemical staining was performed on all PDX tissue slides and the eight mesothelioma tissue slides purchased from Shanghai Xinchao Biotechnology Co., Ltd. CD228 immunohistochemical staining was performed on four mesothelioma tissue slides at the Shanghai Lidi Biotechnology Co., Ltd. CD228 immunohistochemical staining was performed on the four mesothelioma tissue slides at the Shanghai Lidi Biotechnology Co., Ltd. CD228 immunohistochemical antibody was purchased from Novus Biologicals (Cat. No. NBP1-85777) at a primary antibody dilution of 1:200.

[0100] Immunohistochemistry experiments were performed using an automated IHC staining system (Bond RX automatic IHC&ISH system, Leica). Immunohistochemistry staining results were evaluated using the H-score. pi represents the percentage of positive cells, and i represents the staining intensity (0: negative; 1: weak staining; 2: moderate staining; 3: strong staining). Each sample was scored three times independently, and the final H-score value was the average of the three times. The IHC scoring results of all samples are shown in Figure 1. From the expression levels of CD228 on the TMA chip shown in Figure 1 and the CD228 expression rates shown in Table 1, it can be seen that CD228 has a high proportion of medium and high expression in gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, bile duct cancer, pancreatic cancer, and cervical cancer.

[0101] Table 1. CD228 expression level statistics in various tumors

[0102] Example 2. Generation of anti-CD228 monoclonal antibodies

[0103] 1.1 Protein production

[0104] The amino acid sequences of the three proteins (see Table 2) were sent to Jiangsu GenScript Biotech Co., Ltd. for gene synthesis. The genes were then transfected into CHO cells and cultured at 37°C, 8% CO₂, and shaken at 125 rpm. After 10 days of transient expression, the supernatant was collected. The expression supernatant was purified using a Ni (GenScript, L00250) column and then polished using an SP column (GE, 17-1087-01) to obtain human CD228, mouse CD228, and monkey CD228 proteins.

[0105] Table 2. Amino acid sequences of three proteins

[0106] 1.2 Mouse immunization method

[0107] 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. The CD228 antigen protein (0.23 mg / mL, Boan, 20200924, SEQ ID NO: 41) produced independently by Shandong Boan Biotechnology Co., Ltd. was used for immunization.

[0108] The immunization method adopted multiple injections at the subcutaneous and inguinal points of the abdomen, and the antigen protein immunization dose was 20 μg / mouse. Freund's complete adjuvant was used to emulsify the antigen for the first immunization, and Freund's incomplete adjuvant was used to emulsify the antigen for the second to fourth immunizations. The first batch of mice underwent three immunizations and one booster immunization; the second batch of mice underwent four immunizations and one booster immunization. Each immunization was 14 days apart. Starting from the second immunization, peripheral blood serum was collected on the 7th day after each immunization to detect the antibody titer and mice with unqualified titers were eliminated. After immunization of mice, the results of the mouse serum titer are shown in Figure 2, where 2500X, 12500X, and 62500X represent the dilution ratios. Three days after the booster immunization, the mice were euthanized, and the spleens were taken to make single cells for bank construction.

[0109] 1.3 Establishment of phage library

[0110] Mice were sacrificed, and the spleens were dissected and removed. The spleens were ground with a syringe stopper and filtered through a mesh. The filtered splenocytes were frozen, and RNA was extracted to obtain cDNA. Phage libraries were constructed according to conventional methods. The library capacity data are shown in Table 3.

[0111] Table 3. Phage library capacity of each immunized mouse

[0112] 1.4 Screening in two ways

[0113] 1.4.1 Plate screening: Coat plates with CD228-His protein (homemade). The next day, add the phage library and incubate for 2 hours. Wash 4-10 times and elute the specifically bound phage with elution buffer.

[0114] 1.44.2 Magnetic Bead Screening: Biotinylate CD228-His protein according to the kit instructions, bind to Thermo magnetic beads, block with BSA, and incubate with the phage library for 2 hours. Wash 4-10 times, and elute specifically bound phage with elution buffer. Antibody clones obtained and their sources are shown in Table 4.

[0115] Table 4. Sources of anti-CD228 antibodies obtained through screening

[0116] Example 3. Molecular construction and production of complete antibodies

[0117] 133 positive IgG1 clones were constructed and sequenced, of which 8 lead antibody variable region amino acid sequences are shown in Table 5 (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.

[0118] Table 5. Variable region amino acid sequences of 8 antibodies

[0119] The antibody variable region genes were amplified by conventional molecular biology PCR (2×Phanta Max Master Mix manufacturer: Vazyme product number: P515-P1-AA batch number: 7E512E1). The antibody heavy chain variable region genes were ligated into the vector pCDNA3.4 (Life Technology) containing the nucleic acid sequence of the antibody heavy chain constant region sequence by homologous recombination, and the antibody light chain variable region genes were ligated into the vector pCDNA3.4 containing the nucleic acid sequence of the antibody light chain constant region sequence.

[0120] Table 6. Amino acid sequences of the heavy and light chain constant regions of antibodies

[0121] The plasmids of the positive clones after sequencing were extracted and co-transfected into HEK293 cells and cultured in a shaker at 37°C\8% CO2\125rpm. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain the antibody, and the antibody concentration was determined by UV280 binding theoretical extinction coefficient.

[0122] The control antibody H149 sequence was synthesized based on the sequence in patent US20200246479A1. The amino acid sequence is shown in Table 7 below.

[0123] Table 7. Amino acid sequence of control antibody hL49

[0124] Example 4. Characterization of anti-CD228 monoclonal antibody molecules

[0125] 4.1 Binding of anti-hCD228 antibodies to human CD228 protein

[0126] Human CD228 (Boan homemade, 20201014, SEQ ID NO: 41) protein was diluted to 0.1 μg / mL with pH 9.6 carbonate buffer (hereinafter referred to as CBS), coated with ELISA plates, 100 μL / well, incubated overnight at 4°C; after washing, the plates were blocked with skim milk powder. After washing, 100 μL of antibody diluted with PBST (phosphate buffer, Solebol P1010, +0.05% tween20) was added to each well (the diluted antibody was a complete antibody diluted with PBST gradient. The complete antibody contains Fc and Fab regions and is an antibody with a constant region. The starting concentration was 0.1 ug / mL and the three-fold dilution was 8 gradients). After washing, goat 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 hour. After washing, 100 μL of TMB (Beijing Meikewande, 1001) was added to each well for color development. After 10 minutes, 50 μL of 2M H2SO4 was added to each well to stop color development, and the OD450 was read using a microplate reader. This experiment and the following experiments all used Seagen's CD228-targeting antibody hL49 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 based on Figure 3. It can be seen that the six antibodies showed similar binding activity at the ELISA level and were better than the control antibody HL49.

[0127] Table 8. EC50 values ​​of antibodies

[0128] 4.2 Detecting the affinity of antibodies to CD228 human, monkey, and mouse proteins

[0129] Table 9 shows the sources of CD228 protein used in the assay

[0130] The binding kinetics of the antibodies to each CD228 protein were measured using a BIAcore 8K instrument based on surface plasmon resonance (SRP) technology. 2 μg / mL of CD228 antibody was captured 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 serially diluted two-fold in HBS-EP+ buffer over five concentrations, starting at a 50 nM concentration. Biacore analysis of CD228 protein binding kinetics was performed, and affinity activity KD values ​​were calculated using a fitting method.

[0131] Table 10 Biacore detection of binding of anti-CD228 antibodies to human, monkey, and mouse proteins

[0132] As can be seen from Table 10, the antibodies have similar affinities to human CD228 protein and monkey CD228 protein, and do not bind to mouse CD228.

[0133] 4.3 Anti-hCD228 Antibody Epitope Analysis

[0134] The His chip captured CD228 protein at 10 μg / mL with a threshold of 0.5 nm. The first CD228 antibody (30 μg / ml) was first bound, followed by competitive binding analysis with the second CD228 antibody (30 μg / ml). The Octet 8K assay analyzed the response to Antibody 2 to determine if there was a competitive relationship between Antibodies 1 and 2. Table 11 shows the Octet antibody epitope response values.

[0135] Table 11 Octet antibody epitope response values

[0136] The final competition analysis was calculated using the following method: 1-response value / blank value. The results are shown in Table 12. It can be seen that BA352 and hL49 have similar epitopes, and the remaining antibodies compete with each other and have similar epitopes (values ​​greater than 75%, indicating that the epitopes are related).

[0137] Table 12 Final competition analysis results

[0138] Example 5. Flow cytometry detection of anti-hCD228 antibody cell-level binding activity

[0139] To a 96-well round-bottom plate, 50 μL of human melanoma SK-MEL-5 cells (ATCC, HTB-70) were added at a cell count of 7E4 cells / well. Antibodies were serially diluted in FACS buffer (PBS, Boster Biotechnology, Cat. No. PYG0021) and added to the 96-well round-bottom plate at a rate of 50 μL / well. The cells were incubated at 4°C for 1 hour. After centrifugation at 400 g for 4 minutes, the supernatant was discarded. The cells were washed once with FACS buffer, and 100 μL / well of fluorescent secondary antibody (Jackson, 109545-008) was added. The cells were incubated at 4°C in the dark for 30 minutes. After centrifugation at 400 g for 4 minutes, the supernatant was discarded, the cells were washed once with FACS buffer, and the cells were resuspended in 100 μL / well of FACS buffer. Flow cytometry was performed (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). It can be seen that all six antibodies have high binding activity with SK-MEL-5 cells and are better than hL49; the isotype control antibody in Figure 4 is an unrelated antibody targeting other targets with the same constant region and different variable regions as the anti-CD228 antibody in the experimental group.

[0140] Example 6. Internalization experiment of anti-hCD228 monoclonal antibody molecules

[0141] 1.SK-MEL-5 cells

[0142] To a 96-well round-bottom plate, add 50 μL / well of human melanoma SK-MEL-5 cells diluted in PBS (Boster Biosciences, Cat. No. PYG0021) to a total of 5E4 cells / well. Antibody was then diluted in PBS to a final concentration of 20 μg / mL. Add 50 μL / well of 20 μg / mL antibody to the 50 μL / well round-bottom plate containing cells. Incubate for 30 minutes, centrifuge at 400g for 4 minutes, and remove the supernatant. Wash twice with pre-chilled buffer, then add 100 μL / well of buffer. Incubate at 37°C and 4°C, respectively, at different time points. Centrifuge at 400g for 4 minutes, and remove the supernatant. Add 100 μL / well of 4°C pre-chilled fluorescent secondary antibody (Jackson, 109-545-008) and incubate at 4°C for 30 minutes in the dark. The cells were washed once with pre-cooled FACS buffer, resuspended in 100 μL / well FACS buffer, and analyzed using a flow cytometer (NovoCyte 2060). The results are shown in Figure 5A, which shows the internalization of each anti-CD228 antibody in human melanoma SK-MEL-5 cells. As shown in Figure 5A, CA13, CA149, and BA352 had higher internalization rates than the control hL49.

[0143] 2. A549-CD228 cells

[0144] A549-CD228 cells (Kangyuan Broad, KC-2150), a human lung cancer cell line stably expressing the exogenous CD228 gene, were digested and treated with serum-containing medium. Digestion was terminated and diluted with serum-containing medium. The cells were then added to a 96-well round-bottom plate (NEST, Cat. No. 701111) at a rate of 50 μL / well, 1E5 per well. Antibodies were diluted with serum-containing medium and mixed with a labeling reagent (Invitrogen, Z25611) at a 1:3 molar ratio (antibody concentration: 40 nM, labeling reagent: 120 nM). After incubation at room temperature for 5 minutes, the labeled antibody mixture was added to the cell-containing plate at a rate of 50 μL per well. 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 using a flow cytometer (NovoCyte 2060). The results showed that the internalization of CA149 antibody increased with time; as can be seen in Figure 5B, CA149 has better internalization activity than the control antibody hL49.

[0145] Example 7. Antibody-dependent cell-mediated cytotoxicity (luciferase reporter gene effector cells)

[0146] Prepare ADCC working solution (RPMI1640 medium containing 1% FBS); collect Bioassay Effector Cells (Promega, G7011) and adjust the cell density to 2.4×10 6 Target cells SK-MEL-5 (ATCC, HTB-70) were collected and the cell density was adjusted to 8×10 5 ; Dilute the sample to be tested with ADCC working solution, starting from a starting concentration of 5ug / mL, and dilute 8 concentrations 4 times; 25uL of effector cells, target cells and sample to be tested were added to a reaction white plate (Costar, 3917) with a total reaction volume of 75uL. The reaction system was incubated at 37 degrees for 6h; 75uL Bio-Glo Luciferase System (Promega, G7940) was added to each well, and the chemiluminescence value was read with a microplate reader (BioTek, synergy neo2) after 15min of reaction. The results are shown in Figure 6, which shows the ADCC experimental results of each anti-CD228 antibody based on human melanoma SK-MEL-5 cells. As can be seen from Figure 6, as the concentration of the antibody sample increases, the signal gradually increases, indicating that it has an ADCC effect on SK-MEL-5 cells.

[0147] Example 8 Study on the binding epitope of CA149 antibody to CD228

[0148] We prepared the full-length CD228 antigen and the antibody Fab complex (CA149-Fab) and commissioned the Cryo-Electron Microscopy Center of Mizumu Future (Hangzhou) Technology Co., Ltd. to perform structural analysis of the antigen and antibody. The researchers analyzed the amino acid types and side chain interactions at the epitope in the three-dimensional model. The results showed that the 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 superscript * indicates amino acids on the CA149 Fab light chain, and non-superscript italicized amino acids indicate amino acids on the CA149 Fab heavy chain.

[0149] Table 13 Interaction sites

[0150] Based on the epitope analysis, we also validated specific site mutations in the CD228 antigen (sequence shown in SEQ ID NO:41), produced independently by Shandong Boan Biotechnology Co., Ltd. We constructed single-point mutants of hCD228 (R275A), hCD228 (R282A), hCD228 (E312A), and hCD228 (L313A), as well as double-point mutants of hCD228 (E312A, L313A), hCD228 (R282A, E312A), and hCD228 (R275A, R282A), and performed affinity analysis. Affinity analysis revealed that the mutated antigens exhibited reduced or absent binding activity to the CA149 antibody, indicating that the four sites, E312A, L313A, R282A, and R275A, are key sites for antibody-antigen binding. The affinity test results are summarized in Table 14.

[0151] Table 14 Affinity detection

[0152] In addition, since there is another variable splicing product soluble MFI2, namely sMFI2, in the human body, and there are literatures (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) indicating that sMFI can cross the blood-brain barrier through LRP protein. Therefore, in order to reduce the possible off-target risk, CD228-targeted antibodies should not bind to sMFI2. According to the epitope results and the sequence alignment results of full-length hCD228 and soluble antigen sMFI2 (as shown in Figure 7), we can see that the above four key binding sites are not on the sMFI2 antigen, that is, the CA149 antibody will not bind to sMFI2.

[0153] Example 9. Conjugation experiment of anti-hCD228 antibody and drug

[0154] Prepare an antibody drug conjugate as shown in the following molecular formula:

[0155] Wherein: Ab is any of the above-mentioned anti-CD228 antibodies or antigen-binding fragments thereof, LU is a linker (also known as a connector), D is a drug, and the subscript p is the average DAR value of the antibody-drug conjugate.

[0156] 9.1 Preparation of anti-hCD228 antibody-vcMMAE antibody drug conjugate

[0157] 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 hours. 5 equivalents of vcMMAE in DMSO were added to the reduced antibody in PBS and incubated at 25°C for about 1 hour. 10 equivalents of n-acetylcysteine ​​(NAC) were then added to the mixture and incubated at 25°C for 5 minutes to quench any unreacted linker-drug. The buffer was replaced by ultrafiltration to remove free small molecules. The mixture was then submitted for HIC-HPLC analysis. The analysis results are shown in Table 15. It can be seen that the average DAR values ​​of the antibody-drug conjugates used in this application are between 4.01 and 4.42.

[0158] Table 15 DC HIC-HPLC results

[0159] 9.2 Preparation of CA149-BNLD11 Antibody-Drug Conjugate

[0160] Taking the anti-CD228 antibody CA149 as an example, an ADC composition with a homogeneous drug loading (DAR) of approximately 4 was prepared. A phosphate buffer solution (pH 7.5, containing 11 mM DTPA) containing the antibody (5-10 mg / mL) was treated with 2 equivalents of TCEP and incubated at 25°C for approximately 2 hours. A 5 equivalent solution of BNLD-11 in DMSO was added to the reduced antibody solution in PBS and incubated at 25°C for approximately 1 hour. Ten equivalents of n-acetylcysteine ​​(NAC) were then added to the mixture and incubated at 25°C for 5 minutes 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, showing that the average DAR value of the antibody-drug conjugate CA149-BNLD11 prepared in this example was approximately 4.

[0161] Table 16 The DAR values ​​after integral analysis are shown in the following table:

[0162] Example 10. In vitro cell killing assay of anti-hCD228 ADC

[0163] SK-MEL-5 cells (1E4 cells / well) were added to a 96-well flat-bottom plate (Corning, Cat. No. 3917) in 10% FBS / EMEM medium. Each anti-CD228 ADC was diluted in this medium, starting at 1 μg / mL, and serially diluted fourfold to a total of six concentrations. The diluted ADC was added to the 96-well plate at 50 μL / well and incubated at 37°C, 5% CO₂ for 4 days. The CellTiter-Glo kit (Promega, G7571, protected from light during use) was then equilibrated to room temperature. The kit buffer and substrate were mixed and allowed to stand for 1 hour. Cell viability assay reagent (100 μL per well) was added to the 96-well plate, shaken horizontally 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).

[0164] The results are shown in Figures 9A to 9F, which show the results of killing experiments of various anti-CD228 ADCs based on human melanoma SK-MEL-5 cells. It can be seen that two anti-hCD228 ADCs: CA13-vcMMAE and CA67-vcMMAE, showed in vitro cell killing activity similar to that of hL49-vcMMAE; four anti-hCD228 ADCs CA149-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and CA185-vcMMAE showed in vitro cell killing activity similar to that of the control group hL49-vcMMAE; the isotype control antibodies in Figures 9A to 9F are unrelated antibodies against other targets with the same constant regions as the six antibodies but different variable regions.

[0165] Example 11. In vivo efficacy study of anti-CD228 ADC on mouse transplanted tumors

[0166] 11.1 Efficacy Data in the Human Melanoma Cell SK-MEL-5 Mouse Xenograft Model

[0167] Human melanoma SK-MEL-5 cells were purchased from ATCC and cultured in EMEM medium containing 10% FBS in a 37°C, 5% CO2 incubator. NOD / SCID mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. SK-MEL-5 cells were concentrated to 2.5 × 10 cells / mL in EMEM medium containing 50% Matrigel. 7The cells / mL were inoculated subcutaneously on the right side of NOD / SCID mice at a volume of 0.1 mL / mouse. When the average tumor volume reached about 76 mm 3 At the same time, the mice were divided into 8 experimental groups according to the tumor volume and body weight of the mice, with 5 mice in each group. The drugs were administered on the day of grouping, and the dosage was 3 mg / kg. Figure 10 shows the efficacy data of each anti-CD228 ADC in the SK-MEL-5 animal model (3 mg / kg), that is, the changes in the tumor volume of the mice after administration. It can be seen that after the administration of the six antibodies, the tumor volume was reduced. Figure 10 is a graph based on the volume change data in Table 17. As shown in Table 17, the tumor volume growth inhibition rate TGI% of 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; the tumor weight ... The inhibition rates IR% were 59.8%, 65.9%, 61.4%, 55.4%, 68.2%, 65.7% and 56.1% respectively; it can be seen that in the human melanoma cell SK-MEL-5 mouse transplant tumor model, under the condition of 3 mg / kg administration, both the tumor volume growth inhibition rate TGI% and the tumor weight inhibition rate IR% of the six anti-CD228-vcMMAE prepared in this project were superior to the control group hL49-vcMMAE.

[0168] Table 17. Changes in tumor volume after 3 mg / kg administration

[0169] Figure 11 shows the efficacy data of each anti-CD228 ADC in the SK-MEL-5 animal model (5 mg / kg). NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The SK-MEL-5 cell concentration was adjusted to 3×10 7 The cells / mL were inoculated subcutaneously on the right side of NCG mice at a volume of 0.1 mL / mouse. When the average tumor volume reached about 83 mm 3At 14:00 AM, mice were divided into eight experimental groups of six mice each based on tumor volume and body weight. Dosing began on the same day of grouping at a dose of 5 mg / kg. The screened antibodies demonstrated comparable efficacy compared to the control hL49. Figure 11 is a graph based on the volume change data in Table 18. As shown in Table 18, the experimental endpoints of CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE 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. It can be seen that in the human melanoma cell SK-MEL-5 mouse transplant tumor model, under the condition of 5 mg / kg administration, both the tumor volume growth inhibition rate TGI% and the tumor weight inhibition rate IR% of the anti-CD228-vcMMAE prepared in this project were superior to the control group hL49-vcMMAE.

[0170] Table 18. Changes in tumor volume after 5 mg / kg administration

[0171] 11.2 Efficacy Data in Human NCI-H226 Lung Cancer Cell / Mouse Xenograft Model

[0172] Human lung cancer cells NCI-H226 were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in RPMI 1640 medium containing 10% FBS. CB-17 / SCID mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0173] The concentration of NCI-H226 cells was adjusted to 5.0 × 10 cells / mL using RPMI 1640 medium containing 50% Matrigel. 7 The cells / mL were inoculated subcutaneously on the right side of CB-17 / SCID mice at a volume of 0.1 mL / mouse. When the average tumor volume reached about 156 mm 3At 1:10 PM, mice were divided into eight experimental groups of five mice each based on tumor volume and body weight. Dosing began on the same day of grouping, at a dose of 3 mg / kg. Figure 12 shows the efficacy data (3 mg / kg) of various anti-CD228 ADCs in the NCI-H226 animal model. Tumor volume decreased after administration of all six antibodies, demonstrating similar in vivo efficacy to that of HL49. Figure 12 is a graph based on the volume change data in Table 20. As shown in Table 19, the experimental endpoints of the tumor volume growth inhibition rates TGI of 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; the tumor weight inhibition rates were 39.7%, 44.5%, 46.9%, 43.1%, 41.6%, 47.8%, and 49.8%, respectively.

[0174] Table 19. Changes in tumor volume after 3 mg / kg administration

[0175] Figure 13 shows the efficacy data of each anti-CD228 ADC in the NCI-H226 animal model (5 mg / kg). NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The concentration of NCI-H226 cells was adjusted to 5×10 7 The tumor was inoculated into the right side of NCG mice at a volume of 0.1 mL / mouse. When the average tumor volume reached about 108 mm 3 At the same time, mice were divided into eight experimental groups of six mice each based on tumor volume and body weight. Dosing began on the day of grouping at a dose of 5 mg / kg. The screened antibodies demonstrated comparable efficacy compared to the control HL49. Figure 13 is a graph based on the volume change data in Table 20. As shown in Table 16, at the experimental endpoint, the tumor volume growth inhibition rates (TGI) for the CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE groups 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.

[0176] Table 20. Changes in tumor volume after 5 mg / kg administration

[0177] Example 12. Pharmacokinetics of anti-CD228 ADC in mice

[0178] Three Balb / c mice were selected for each ADC and administered subcutaneously at a dose of 10 mg / kg. Serum was collected at 0 h before administration and at 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 concentration. The serum detection method was ELISA.

[0179] Figure 14 records the pharmacokinetic curves of CA13-vcMMAE, CA67-vcMMAE, and CA149-vcMMAE. The specific test results are shown in Figure 14. It can be seen that CA149-vcMMAE has a better pharmacokinetic level than CA13-vcMMAE and CA67-vcMMAE constructed with CA13 and CA67 antibodies.

[0180] Example 13. Cell proliferation inhibitory activity of antibody drug conjugate CA149-BNLD11

[0181] 13.1 Inhibitory activity of CA149-BNLD11 on MC38-CD228 and A375-CD228 cell proliferation

[0182] 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. They were then added to a 96-well flat-bottom plate (SARSTED, Catalog No. 94.6120.096) at 50 μL / well, 1E4 / well. The antibody-drug conjugate CA149-BNLD11 prepared in Section 8.2 of Example 8 was diluted with serum-containing medium to a starting concentration of 60 and 12 μg / mL, respectively, and then diluted 5-fold. The antibody with the antibody ID CA521 in CN202180003751.7 was selected and prepared using the same method as Example 8 as the control group (Isotype). The diluted CA149-BNLD11 was added into the above 96-well flat-bottom cell culture plate at 50 μL / well and cultured at 37°C and 5% CO2 for 96 h. The kit (Novozyme, DD1101-01) must be protected from light during use. Equilibrate to room temperature, mix thoroughly by inversion, and add 100 μL / well to a 96-well plate. Shake horizontally at 300 rpm for 2 minutes and let stand for 10 minutes. Cell viability was measured using a microplate reader (BioTek, SYNERGY neo, USA).

[0183] The results showed that CA149-BNLD11 had excellent proliferation inhibitory activity against MC38-CD228 and A375-CD228 cells, as shown in Table 21. 50 The ordinates of Figures 15A-15B show the viability percentage (%), which is the ratio of the viable cell count in the treatment group to the viable cell count in the blank group. As can be seen in Figures 15A-15B, CA149-BNLD11 exhibits significant proliferation inhibition activity against CD228-expressing cells relative to the control group (isotype).

[0184] Table 21. CA149-BNLD11 proliferation inhibition activity data

[0185] 13.2 Inhibitory Activity of CA149-BNLD11 on SK-MEL-5, A549-CD228, and A375-CD228 Cell Proliferation

[0186] After digestion of SK-MEL-5 (ATCC, HTB-70), A549-CD228 (Kangyuan Bochuang, KC-2150), and A375-CD228 cells (Kangyuan Bochuang, KC-2110) in the logarithmic growth phase, digestion was terminated and diluted with serum-containing medium, and then added to a 96-well flat-bottom plate (SARSTED, catalog number 94.6120.096), 50 μL / well, 1E4 / well. CA149-BNLD11 was diluted with serum-containing medium, with starting concentrations of 1.2, 60, and 6 μg / mL, respectively, and then diluted 5-fold, 6-fold, and 5-fold, respectively. The diluted ADC was added to the above-mentioned 96-well flat-bottom cell culture plate at 50 μL / well, and cultured at 37°C and 5% CO2 for 96 h or 120 h. The kit (Novozymes, DD1101-01, protected from light during use) was equilibrated to room temperature, mixed by inversion, and added to a 96-well plate at 100 μL / well. After horizontal shaking at 300 rpm for 2 minutes, the plate was allowed to rest for 10 minutes. Cell viability was measured using a microplate reader (BioTek, SYNERGY neo, USA). The results showed that CA149-BNLD11 exhibited excellent anti-proliferation 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 shown in Figures 15C-15E, CA149-BNLD11 exhibited significant anti-proliferation activity against cells expressing CD228 compared to the control (isotype).

[0187] Table 22 Data on the proliferation inhibition activity of CA149-BNLD11 on cells expressing CD228

[0188] Example 14. Evaluation of the therapeutic efficacy of the antibody drug conjugate CA149-BNLD11 on human lung cancer cell CaLu-1 xenografts in nude mice

[0189] 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 Jicui Pharmaceutical Biotechnology Co., Ltd. The CaLu-1 cell concentration was adjusted to 5.0 × 10 cells / mL using serum-free McCoy's 5A medium containing 50% Matrigel. 7 The drug was administered subcutaneously on the right side of Balb / c nude mice at a volume of 0.1 mL per mouse. When the average tumor volume reached 135 mm³, the mice were divided into five experimental groups of 5 mice each. Dosing began on the same day of grouping. This experiment used a single dose, and observation lasted 28 days.

[0190] The results are shown in Figures 16-17. Figure 16 is a tumor growth inhibition curve of the antibody drug conjugate group in the Calu-1 lung cancer model. Figure 17 is a histogram of tumor weight of the antibody drug conjugate group in the Calu-1 lung cancer model.

[0191] As shown in Figure 16, compared with the vehicle control group (PBS phosphate buffer), each antibody drug conjugate administration group was able to significantly inhibit tumor volume growth, with statistically significant differences (P less than 0.05). The CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups had significantly better tumor inhibition effects than the nCov-CA521-vccMMAE group (P values ​​were 0.0001, 0.0016, and 0.0006, respectively). ; No significant differences were found among the three groups of CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd (P were all greater than 0.05); the tumor volume growth inhibition rate 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.

[0192] As shown in Figure 17, at the end point of the experiment, the average 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 statistically significant differences (P less than 0.05); there was no statistical difference in tumor weight between the nCov-CA521-vcMMAE group and the control group (P = 0.3250); there were no significant differences among the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups (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.

[0193] During the experiment, the animals were active and ate well during the dosing period, and their weight increased to a certain extent, indicating that the animals tolerated the test product well. There were no significant differences between the groups (P>0.05).

[0194] Example 15. Evaluation of the therapeutic efficacy of the antibody drug conjugate CA149-BNLD11 on human melanoma cell SK-MEL-5 nude mouse xenografts

[0195] 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 Jicui Yaokang Biotechnology Co., Ltd. The SK-MEL-5 cell concentration was adjusted to 5.0 × 10 cells / mL using serum-free EMEM medium containing 50% Matrigel. 7 The drug was administered subcutaneously on the right side of Balb / c nude mice at a volume of 0.1 mL per mouse. When the average tumor volume reached 98 mm³, the mice were divided into four experimental groups of 5 mice each. Dosing began on the same day of grouping. This experiment used a single dose, and observation lasted 28 days.

[0196] The results are recorded in Figures 18-19. Figure 18 is the tumor growth inhibition curve of the antibody drug conjugate group in the human melanoma cell SK-MEL-5 nude mouse transplant tumor model, and Figure 19 is a bar graph of tumor weight of the antibody drug conjugate group in the human melanoma cell SK-MEL-5 nude mouse transplant tumor model. As shown in the tumor growth curve in Figure 18: at the end of the experiment, compared with the solvent control group (PBS phosphate buffer), each drug-treated group was able to significantly inhibit the growth of tumor volume, and all had statistical differences (P less than <0.0001); the tumor inhibition effect of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups was significantly better than that of the nCov-CA521-vcMMAE group (P less than <0.0001), and the CA149-BNLD11 group had The tumors in 2 / 5 mice were completely cleared; no statistical differences were found among the three groups of CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd (P greater than 0.05); the tumor volume growth inhibition rate 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.

[0197] As shown in Figure 19, at the end of the experiment, the average tumor weights of all drug-treated groups were significantly lower than those of the vehicle control group (PBS phosphate buffer), with statistical differences (P less than 0.05); there were no significant differences among the four groups of CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE (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.

[0198] During the experimental period, the animals were active and ate well. With the exception of the vehicle control group (PBS phosphate buffer), all other groups showed some increase in body weight, indicating that the animals tolerated the test product well. There were no significant differences between the groups (P>0.05).

[0199] Example 16. Evaluation of the therapeutic efficacy of the antibody drug conjugate CA149-BNLD11 on human gastric cancer cell NUGC4 Balb / c nude mouse xenografts

[0200] NUGC4 human gastric cancer cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. and cultured in a 37°C, 5% CO2 incubator in RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The NUGC4 cell concentration was adjusted to 1.8 × 10 cells / mL using serum-free RMPI-1640 medium containing 50% Matrigel. 7 The drug was administered subcutaneously on the right flank of Balb / c nude mice at a volume of 0.1 mL per mouse. When the average tumor volume reached 108 mm³, mice were divided into four experimental groups of 5 mice each. Dosing began on the same day of grouping. This experiment used a single dose, and observation lasted 23 days.

[0201] The results are recorded in Figures 20-21. Figure 20 is a tumor growth inhibition curve of the antibody-drug conjugate group in the human gastric cancer cell NUGC4 Balb / c nude mouse xenograft tumor model, and Figure 21 is a bar graph of tumor weight of the antibody-drug conjugate group in the human gastric cancer cell NUGC4 Balb / c nude mouse xenograft tumor model.

[0202] As shown in the tumor growth curve in Figure 20: At the end of the experiment, compared with the vehicle control group (PBS phosphate buffer), the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were able to significantly inhibit tumor volume growth, with statistically significant differences (all P less than <0.0001); 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 less than <0.0001). 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); the tumor volume growth inhibition rate 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.

[0203] As shown in Figure 21: At the end of the experiment, the average 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 statistically significant differences (P less than or equal to 0.0001); there was no statistical difference in tumor weight between the nCov-CA521-vcMMAE group and the control group (P = 0.8144); the tumor weights of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly lower than those of the nCov-CA521-vcMMAE group (P less than 0.05); 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.

[0204] During the experiment, the animals were active and ate well during the dosing period, and their weight increased to a certain extent, indicating that the animals tolerated the test product well. There were no significant differences between the groups (P>0.05).

[0205] Example 17 Evaluation of the therapeutic efficacy of the antibody drug conjugate CA149-BNLD11 on human lung squamous cell carcinoma NCI-H226 Balb / c nude mouse xenografts

[0206] NCI-H226 human lung squamous cell carcinoma cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The NCI-H226 cell concentration was adjusted to 4.0 × 10 cells / mL using serum-free RMPI-1640 medium containing 50% Matrigel. 7 The cells / mL were inoculated into the right side of Balb / c nude mice at a volume of 0.1 mL / mouse. When the average tumor volume reached 145 mm 3 At 4 hr, mice were divided into four experimental groups of six based on tumor volume. Dosing began on the same day of grouping. The dose was 3.3 mg / kg, administered as a single dose. Results are expressed as mean ± SEM. Data were analyzed and processed 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 analyzed using one-way analysis of variance. T-tests were used to compare two groups. P < 0.05 indicated statistical significance.

[0207] The experiment was terminated on the 24th day after group administration. The tumor volume growth curve of nude mouse transplanted tumors showed that compared with the vehicle control group (PBS phosphate buffer), the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups could significantly inhibit the growth of tumor volume, and the differences were statistically significant (P less than 0.05); the nCov-CA521-vcMMAE group had no tumor inhibition effect (P4>0.05); there was no statistical difference between the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups (P>0.05); the tumor volume growth inhibition rate 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.

[0208] Example 18 Evaluation of the efficacy of multiple doses of the antibody drug conjugate CA149-BNLD11 on human melanoma cell SK-MEL-5 Balb / c nude mouse xenografts

[0209] 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 Jicui Yaokang Biotechnology Co., Ltd. The SK-MEL-5 cell concentration was adjusted to 3.0 × 10 cells / mL using serum-free EMEM medium containing 50% Matrigel. 7 The cells / mL were inoculated subcutaneously on the right side of Balb / c nude mice at a volume of 0.1 mL / mouse. When the average tumor volume reached 103 mm 3 At 4 pm, mice were divided into four experimental groups based on tumor volume, with six mice in each group. Drug administration began on the day of grouping, with doses of 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg, respectively, all administered as a single dose. Results are expressed as mean ± SEM. Data were analyzed and processed using Graphpad 8.0 software. Tumor volume and body weight were compared between groups at each time point using two-way ANOVA. Tumor weight was analyzed using one-way ANOVA. T-tests were used to compare two groups. P < 0.05 indicated statistical significance.

[0210] The experiment was terminated on day 21 of group dosing. Figure 23 shows the tumor volume growth curve of human melanoma cell SK-MEL-5 Balb / c nude mice xenografts: Compared with the vehicle control group (PBS phosphate buffer), the 2.5 mg / kg and 5.0 mg / kg groups significantly inhibited tumor volume growth, with statistically significant differences (P < 0.05). There was no significant difference between the 1.0 mg / kg group and the vehicle control group (PBS phosphate buffer) (P = 0.4030). The tumor volume growth inhibition rates (TGI) (%) of the CA149-BNLD11 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg groups were 25.1%, 72.1%, and 91.1%, respectively. This indicates that the antitumor activity of CA149-BNLD11 is dose-dependent.

[0211] At the end of the experiment, as shown in the tumor weight growth curve of human melanoma cell SK-MEL-5 Balb / c nude mouse xenografts in Figure 24: compared with the vehicle control group (PBS phosphate buffer), the tumor weights of the 2.5 mg / kg and 5.0 mg / kg groups were significantly reduced, with statistical differences (P less than 0.05); there was no significant difference between the 1.0 mg / kg group and the vehicle control group (PBS phosphate buffer) (P=0.7086); the tumor weight inhibition rates of the CA149-BNLD11 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg groups were 21.9%, 66.7%, and 76.6%, respectively.

[0212] During the experiment, the animals were active and ate well during the administration period, and the weight of each group increased to a certain extent, indicating that the animals tolerated the test product well. There was no significant difference between the groups (P>0.05).

[0213] Example 19 Metabolic Study of Antibody Drug Conjugate CA149-BNLD11 in Mice

[0214] Three ICR mice were selected and administered CA149-BNLD11 and CA149-vcMMAE by tail vein injection at a dose of 10 mg / kg. Serum was collected before administration and at 1h, 6h, 24h, 3d, 5d, 7d, 10d, 14d, 21d, and 28d after administration. The serum antibody concentration was detected by ELISA. The specific test results are shown in the table below.

[0215] Table 23 Key metabolic parameters of CA149-BNLD11 and CA149-vcMMAE in ICR mice

[0216] From the in vivo metabolism studies in mice shown in Table 23 and Figure 25, it can be seen that CA149-BNLD11 has a longer half-life than CA149-vcMMAE. The exposure of CA149-BNLD11 total antibody is 1.4 times that of CA149-vcMMAE, and the exposure of CA149-BNLD11 total ADC is 2.3 times that of CA149-vcMMAE. In addition, it can be seen from the metabolic curve (Figure 25) that the shedding rate of BNLD11 in mice is much lower than that of mc-vcMMAE. In summary, CA149-BNLD11 is more stable in mouse metabolism.

[0217] Example 20 Toxicity Study of Antibody Drug Conjugate CA149-BNLD11 in Mice

[0218] Balb / c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. Mice were divided into six experimental groups of three mice based on sex and weight. Body weight and food were measured on the day of grouping, and dosing began two days later. The dosing schedule is shown in Table 24.

[0219] Table 24 In vivo dosing regimen in mice

[0220] The experiment was terminated on day 14 of drug administration. Results were expressed as mean ± SEM, and data were analyzed and processed using Graphpad 8.0 software. Body weight and food intake were compared using T-test; P < 0.05 indicated statistical significance.

[0221] As shown in Figures 26-27, compared with the vehicle control group (PBS phosphate buffer), 50 mg / kg of CA149-vcMMAE and CA149-BNLD11 can significantly reduce the body weight of mice. The maximum weight loss occurred on the 4th day after administration. The maximum weight loss of females was 17.44% and 10.04%, respectively; the maximum weight loss of males was 17.97% and 5.24%, respectively; the female / male mice in the CA149-vcMMAE group showed varying degrees of frizzy hair and wilting symptoms on the 4th and 6th days after administration, and returned to normal on the 8th day after administration; while the mice in the CA149-BNLD11 group were normal and had no abnormal reactions. On days 4 and 6 after administration, CA149-vcMMAE was significantly more toxic than CA149-BNLD11 (females: P less than 0.05; males: P less than 0.05). Compared with the vehicle control group (PBS phosphate buffer), 50 mg / kg of CA149-vcMMAE and CA149-BNLD11 significantly reduced the food intake of mice; there was no significant difference between the CA149-vcMMAE and CA149-BNLD11 groups.

[0222] Example 21 Pre-toxicological experiment and concomitant toxicological metabolite study of antibody drug conjugate CA149-BNLD11 in cynomolgus monkeys

[0223] Four cynomolgus macaques (half male and half female) were selected. At the start of dosing, males weighed 3.1-3.7 kg, and 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) consisted of one male and one female, the medium-dose CA149-BNLD11 group (6 mg / kg) consisted of one male, and the high-dose CA149-BNLD11 group (10 mg / kg) consisted of one female. Each group received a 5 mg / mL dose, with corresponding concentrations of 0.4, 1.2, and 2 mg / mL, respectively. The low-dose and high-dose groups each received two doses, while the medium-dose group received a single dose, with each animal receiving a 30-minute intravenous infusion.

[0224] After administration, in addition to clinical observations, food intake and weight testing, hematological and blood biochemical tests, concomitant toxicokinetic studies were also conducted. Blood sampling points included before the first administration of the medium-dose 6 mg / kg and high-dose 10 mg / kg groups, as well as 0.5h, 2h, 6h, 24h, 72h, 120h, 168h, 240h, 336h, and 504h after the start of administration.

[0225] During the experiment, no animals were dying or dying in any group, and no abnormalities were observed in any dose group. Overall body weight fluctuations were minimal across all groups, with no abnormalities associated with test article administration observed. Food intake showed irregular changes across all groups, with no abnormalities associated with test article administration observed. As shown in Figure 28, CA149-BNLD11 doses of 6 mg / kg and above resulted in decreases in the hematological parameters WBC, #NEUT, and %NEUT in cynomolgus monkeys 5 to 14 days after administration compared to pre-dose, with a recovery trend observed 21 days after administration. Other hematological parameters were generally within normal ranges across all groups at all time points, with no dose- or time-dependent changes. No abnormalities associated with test article administration were observed. CA149-BNLD11 doses of 10 mg / kg resulted in an increase in serum AST in cynomolgus monkeys 5 days after administration compared to pre-dose, with recovery observed 8 days after administration. In addition, the other blood biochemical indicators of animals in each group at each measurement time point were basically within the normal range, and no abnormalities related to the administration of the test article were found.

[0226] As shown in Figure 29, the toxicokinetic test results showed that the toxin shedding rate in cynomolgus monkeys was significantly lower in the 10 mg / kg dose group than in the 2 mg / kg dose group. The toxin shedding rate in the 10 mg / kg dose group was similar to that in the 6 mg / kg dose group.

Claims

1. An anti-CD228 antibody or antigen-binding fragment thereof, comprising three light chain complementary determining regions and three heavy chain complementary determining regions, wherein The three light chain complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary 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 complementary determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 44, and LCDR3 shown in SEQ ID NO: 45, and the three heavy chain complementary determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, and HCDR3 shown in SEQ ID NO: 49; or, The three light chain complementary determining regions of the antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:46, LCDR2 shown in SEQ ID NO:47, and LCDR3 shown in SEQ ID NO:48, and the three heavy chain complementary determining regions of the antibody or its antigen-binding fragment include HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:

28.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that 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 as shown in SEQ ID NO: 5, and a light chain variable region as shown in SEQ ID NO:

6. the heavy chain variable region of 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 shown in SEQ ID NO: 9 and a heavy chain variable region shown in SEQ ID NO: 10; The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 11 and a heavy chain variable region shown in SEQ ID NO: 12; The antibody or antigen-binding fragment thereof comprises a light chain variable region shown in SEQ ID NO: 38 and a heavy chain variable region shown in SEQ ID NO: 37; or The 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.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody comprises the heavy chain constant region shown in SEQ ID NO: 33, and / or the light chain constant region shown in SEQ ID NO:

34.

4. A nucleic acid encoding the anti-CD228 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. A cell comprising the nucleic acid of claim 4 .

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

7. The antibody-drug conjugate according to claim 6, characterized in that The LU-D structure is VcMMAE, Vc is valine-citrulline, and MMAE is monomethyl auristatin E.

8. The antibody-drug conjugate according to claim 6, characterized in that The LU-D structure is as follows:

9. The antibody-drug conjugate according to claim 7 or 8, characterized in that: The three light chain complementary 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 complementary 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 shown in SEQ ID NO: 7, and a heavy chain variable region shown 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.

10. A pharmaceutical composition comprising the anti-CD228 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the nucleic acid according to claim 4, the cell according to claim 5, or the antibody-drug conjugate according to any one of claims 6 to 9.

11. A kit comprising 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, or the pharmaceutical composition according to claim 10.

12. Use 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 preparation of an agent for preventing, treating, detecting, or diagnosing a disease associated with CD228; preferably, the CD228-associated disease comprises one or more of melanoma, lung cancer, gastric cancer, colon cancer, mesothelioma, pancreatic cancer, and breast cancer.

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