Anti-CD228 antibodies and their drug conjugates

Abstract

An antibody or antigen-binding fragment thereof that binds to CD228, and an antibody-drug conjugate containing said antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment has a strong affinity for CD228 protein and the ability to mediate ADCC, and said antibody-drug conjugate exhibits inhibitory effects against various tumors. Nucleic acid encoding said antibody or antigen-binding fragment thereof; cells containing said nucleic acid; pharmaceutical compositions containing said antibody or antigen-binding fragment thereof, said nucleic acid, said cells, or said antibody-drug conjugate; and a kit. The use 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 CD228-related diseases.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine or biopharmaceutical technology, and in particular to an anti-CD228 antibody and its drug conjugate. Background Technology

[0002] CD228 (also known as melanin transferrin, MTF, melanoma-associated antigen p97, MFI2, or MAP97) is a 90-97 kDa salivary glycoprotein member of the transferrin family. CD228 is typically found to be tethered 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 CD228 expression can lead to accelerated melanoma tumor growth. 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, such as 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 melanoma and 79% of pancreatic cancer, as well as in 83% of mesothelioma, 100% of colon cancer, 57% of breast cancer, and 69% of squamous cell carcinoma, indicating significant clinical demand.

[0005] Antibody-drug conjugates (ADCs) link antibodies to small-molecule chemotherapeutic drugs via linkers. They possess the high targeting specificity of antibodies while fully leveraging the cytotoxicity of chemotherapeutic drugs, achieving highly efficient tumor cell killing. Currently, several ADCs have been successfully marketed, and the number of products in development is gradually increasing, indicating that the technology is mature. CD228 is highly expressed in many tumor tissues but lowly expressed or not expressed in normal tissues. Based on this differential expression, CD228 may be an ideal target for ADCs.

[0006] Therefore, providing a novel CD228 antibody-drug conjugate as an effective anticancer drug has broad application value in the pharmaceutical field. Summary of the Invention

[0007] This invention provides an anti-CD228 antibody or its antigen-binding fragment, capable of binding to the CD228 protein; the invention also provides a nucleic acid encoding the antibody or its antigen-binding fragment; a cell containing the nucleic acid; a pharmaceutical composition containing the antibody or its antigen-binding fragment, the nucleic acid, and the cell; a kit containing the antibody or its antigen-binding fragment, the nucleic acid, and the pharmaceutical composition; the use of the antibody or its antigen-binding fragment, the nucleic acid, and the pharmaceutical composition in the prevention, treatment, detection, or diagnosis of CD228-related diseases; the use of the CD228 antibody or its antigen-binding fragment in the preparation of antibody-drug conjugates (ADCs); and an anti-CD228 antibody-drug conjugate.

[0008] One aspect of the present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising three light chain complementarity-determining regions and / or three heavy chain complementarity-determining regions.

[0009] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions including LCDR1 shown in SEQ ID NO:13, LCDR2 shown in SEQ ID NO:14, and LCDR3 shown in SEQ ID NO:15, and / or the antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions including HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:18;

[0010] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions including LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:21, and / or the antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions including HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:22;

[0011] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions including LCDR1 shown in SEQ ID NO:23, LCDR2 shown in SEQ ID NO:20, and LCDR3 shown in SEQ ID NO:21, and / or the antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions including HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17, and HCDR3 shown in SEQ ID NO:22;

[0012] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions including LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:26, and / or the antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions including HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28;

[0013] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions comprising LCDR1 (SEQ ID NO:29), LCDR2 (SEQ ID NO:25), and LCDR3 (SEQ ID NO:30), and / or the antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions comprising HCDR1 (SEQ ID NO:16), HCDR2 (SEQ ID NO:17), and HCDR3 (SEQ ID NO:28); or

[0014] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions comprising LCDR1 as shown in SEQ ID NO:31, LCDR2 as shown in SEQ ID NO:14, and LCDR3 as shown in SEQ ID NO:21, and / or the antibody or its antigen-binding fragment has three heavy chain complementarity-determining regions comprising HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO:32; or

[0015] The antibody or its antigen-binding fragment has three light chain complementarity-determining regions (CLDs) comprising LCDR1 (SEQ ID NO:19), LCDR2 (SEQ ID NO:44), and LCDR3 (SEQ ID NO:45), and / or the antibody or its antigen-binding fragment has three heavy chain CLDs comprising HCDR1 (SEQ ID NO:16), HCDR2 (SEQ ID NO:17), and HCDR3 (SEQ ID NO:49); or, the antibody or its antigen-binding fragment has three light chain CLDs comprising LCDR1 (SEQ ID NO:46), LCDR2 (SEQ ID NO:47), and LCDR3 (SEQ ID NO:48), and / or the antibody or its antigen-binding fragment has three heavy chain CLDs comprising HCDR1 (SEQ ID NO:16), HCDR2 (SEQ ID NO:17), and HCDR3 (SEQ ID NO:28).

[0016] In one specific 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, when binding to CD228, binds to at least one of the following residues shown in SEQ ID NO:41: E312A, L313A, R282A, R275A.

[0017] In one specific embodiment of the present invention, the present invention provides an anti-CD228 antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising:

[0018] The antibody or its antigen-binding fragment includes the light chain variable region shown in SEQ ID NO:1 and / or the heavy chain variable region shown in SEQ ID NO:2;

[0019] The antibody or its antigen-binding fragment includes the light chain variable region shown in SEQ ID NO:3 and / or the heavy chain variable region shown in SEQ ID NO:4;

[0020] The antibody or its antigen-binding fragment includes the light chain variable region shown in SEQ ID NO:5 and / or the heavy chain variable region shown in SEQ ID NO:6;

[0021] The antibody or its antigen-binding fragment includes the light chain variable region shown in SEQ ID NO:7 and / or the heavy chain variable region shown in SEQ ID NO:8;

[0022] The antibody or its antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:9 and / or the heavy chain variable region shown in SEQ ID NO:10; the antibody or its antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:11 and / or the heavy chain variable region shown in SEQ ID NO:12; the antibody or its antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:38 and / or the heavy chain variable region shown in SEQ ID NO:37; or

[0023] The antibody or its antigen-binding fragment contains the light chain variable region shown in SEQ ID NO:40 and / or the heavy chain variable region shown in SEQ ID NO:39.

[0024] In one specific embodiment of the present invention, the sequence of the heavy chain constant region of the antibody or its antigen-binding fragment is SEQ ID NO:33.

[0025] Furthermore, the sequence of the light chain constant region of the antibody or its antigen-binding fragment is SEQ ID NO:34.

[0026] In the present invention, the antibody or its antigen-binding fragment includes monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, scFv or dsFv fragment, etc.

[0027] A second aspect of the present invention provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0028] A third aspect of the present invention provides a vector comprising the nucleic acid encoding the antibody or an antigen-binding fragment thereof. The vector can be used to express the antibody or the antigen-binding fragment thereof. Preferably, the vector is a viral vector; preferably, the viral vector includes, but is not limited to, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or retroviral vectors; preferably, the vector is a non-viral vector; preferably, the vector is a mammalian cell expression vector; preferably, the expression vector is a bacterial expression vector; preferably, the expression vector is a fungal expression vector.

[0029] A fourth aspect of the present invention provides a cell comprising the nucleic acid or the carrier, the cell being capable of expressing the antibody or its antigen-binding fragment. Preferably, the cell is a bacterial cell; preferably, the bacterial cell is Escherichia coli or the like; preferably, the cell is a fungal cell; preferably, the fungal cell is a yeast cell; preferably, the yeast cell is Pichia pastoris or the like; preferably, the cell is a mammalian cell; preferably, the mammalian cell is Chinese hamster ovary cell (CHO), human embryonic kidney cell (293), B cell, T cell, DC cell, or NK cell, etc.

[0030] 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 conjugation portion conjugated to the antibody portion, wherein the conjugation portion is selected from one or more of detectable markers, drugs, toxins, cytokines, radionuclides and enzymes.

[0031] In another preferred embodiment, the antibody-drug conjugate (ADC) is shown in Formula 1 below:

[0032]

[0033] In Formula 1: Ab is the anti-CD228 antibody of the present invention or its antigen-binding fragment, LU is the linker (also known as the linker), and D is the 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] The drug is selected from chemotherapy drugs, radiotherapy drugs, hormone therapy drugs, or immunotherapy drugs. Optionally, the drug is selected from the group consisting of: taxanes, maytansinoids, camptothecin, tubulin, orlistatin, calicheamicin, anthracyclines, docetaxel, cathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, ribonuclease, or radioisotope.

[0035] Furthermore, the connector LU is composed of the general formula R'-L1-L2-L3;

[0036] In this general formula, L3 is:

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

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

[0039] In this general formula, L2 is: Where A consists of phenylalanine residues, glycine residues, alanine residues, glutamic acid residues, aspartic acid residues, cysteine ​​residues, histidine residues, lysine residues, proline residues, or valine, guanidine residues, β-glycine residues, and β-alanine residues; 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 end of R' is connected to L1, and the d end is connected to A;

[0042] In a preferred embodiment, LU-D in Formula 1 of the antibody-drug conjugate (ADC) is VcMMAE, where 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 Formula 1 of the antibody-drug conjugate (ADC) is BNLD11, where LU is MC-β-Ala-(glucuronide)PAB and D is MMAE, and the structure of BNLD11 is as follows:

[0044] The exact mass of BNLD11 is 1322.690; BNLD11 is synthesized using conventional methods in the prior art; in a preferred example, BNLD11 is obtained by... Figure 30 The synthesis route shown is used to obtain the product.

[0045] In a preferred embodiment, the three light chain complementarity-determining regions of the anti-CD228 antibody or its antigen-binding fragment (Ab) in the ADC formula 1 include LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:26, and the three heavy chain complementarity-determining regions of the antibody or its antigen-binding fragment include 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 its antigen-binding fragment includes 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 its antigen-binding fragment 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, Ab is an anti-CD228 antibody or its antigen-binding fragment, the three light chain complementarity-determining regions of the anti-CD228 antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:26, and the three heavy chain complementarity-determining regions of the antibody or its antigen-binding fragment include 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 its antigen-binding fragment includes a light chain variable region shown in SEQ ID NO:7 and a heavy chain variable region shown in SEQ ID NO:8; even more preferably, the sequence of the heavy chain constant region of the antibody or its antigen-binding fragment is SEQ ID NO:33 and / or the sequence of the light chain constant region is SEQ ID NO:34.

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

[0048] Ab is an anti-CD228 antibody or its antigen-binding fragment, wherein the three light chain complementarity-determining regions of the anti-CD228 antibody or its antigen-binding fragment include LCDR1 shown in SEQ ID NO:24, LCDR2 shown in SEQ ID NO:25, and LCDR3 shown in SEQ ID NO:26, and the three heavy chain complementarity-determining regions of the antibody or its antigen-binding fragment include 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 its antigen-binding fragment includes a light chain variable region shown in SEQ ID NO:7 and a heavy chain variable region shown in SEQ ID NO:8.

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

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

[0051] The eighth aspect of the invention provides the use of the antibody or its antigen-binding fragment, nucleic acid, carrier, 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 its antigen-binding fragment or nucleic acid in the preparation of diagnostic and detection kits.

[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, reagent 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 said antibody or its antigen-binding fragment, nucleic acid, carrier, cell, pharmaceutical composition or antibody-drug conjugate for the treatment or prevention of disease.

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

[0057] The fourteenth aspect of the invention provides the use of the antibody or its antigen-binding fragment, the nucleic acid, the pharmaceutical composition, or the antibody-drug conjugate in the preparation of formulations for the prevention, treatment, detection, or diagnosis of CD228-related diseases.

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

[0059] The fifteenth aspect of the invention also provides the use of the CD228 antibody of the invention or its antigen-binding fragment in the preparation of antibody-drug conjugates (ADCs).

[0060] The anti-CD228 antibody and its antibody-drug conjugate provided by this invention have one or more of the following advantages:

[0061] 1. The anti-CD228 antibody or its antigen-binding fragment 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 this invention has good killing ability against human melanoma SK-MEL-5 cells.

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

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

[0065] 5. The anti-CD228 antibody-drug conjugate provided by this invention has few side effects and toxicity, and is highly safe.

[0066] 6. The anti-CD228 antibody of the present invention has better internalization and endocytosis effects compared with known antibodies on the market, such as Seagen's CD228-targeting antibody hL49. Attached Figure Description

[0067] Figure 1 This represents the expression level of CD228 on the TMA chip in Example 1.

[0068] Figure 2 The titer is the serum titer of CD228-immunized mice in Example 2.

[0069] Figure 3 This refers to the binding of each anti-CD228 antibody to the human CD228 protein in Example 4.

[0070] Figure 4 The binding activity of each anti-CD228 antibody in Example 5 to human melanoma SK-MEL-5 cells expressing CD228 protein is shown.

[0071] Figure 5A The results of the internalization experiments of each anti-CD228 antibody in human melanoma SK-MEL-5 cells in Example 6 are as follows. Figure 5B The results of the internalization experiments of each anti-CD228 antibody in Example 6 based on the human lung cancer cell line A549-CD228 are shown.

[0072] Figure 6 The results of ADCC experiments on human melanoma SK-MEL-5 cells for each anti-CD228 antibody in Example 7 are shown.

[0073] Figure 7 This is a schematic diagram showing the alignment results of the full-length hCD228 sequence with the soluble antigen sMFI2 sequence in Example 8.

[0074] Figure 8The image shows the HPLC chromatogram of CA149-BNLD11 in Example 9.

[0075] Figure 9A The results of the CA13-VcMMAE killing experiment based on human melanoma SK-MEL-5 cells in Example 10 are as follows. Figure 9B The results of the CA67-VcMMAE killing experiment based on human melanoma SK-MEL-5 cells in Example 10 are as follows. Figure 9C The results of the CA149-VcMMAE killing experiment based on human melanoma SK-MEL-5 cells in Example 10 are as follows. Figure 9D The results of the killing experiment of BA352-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10 are as follows. Figure 9E The results of the CA518-VcMMAE killing experiment based on human melanoma SK-MEL-5 cells in Example 10 are as follows. Figure 9F The results of the killing experiment of CA185-VcMMAE based on human melanoma SK-MEL-5 cells in Example 10 are shown.

[0076] Figure 10 The efficacy data (3 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model are shown.

[0077] Figure 11 The efficacy data (5 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model are shown.

[0078] Figure 12 The efficacy data of each anti-CD228 ADC (3 mg / kg) in Example 11 in the NCI-H226 animal model are presented.

[0079] Figure 13 The efficacy data of each anti-CD228 ADC (5 mg / kg) in Example 11 in the NCI-H226 animal model are presented.

[0080] Figure 14 The pharmacokinetic curves of each anti-CD228 ADC mouse in Example 12 are shown.

[0081] Figure 15A To evaluate the inhibitory activity of CA149-BNLD11 on the proliferation of MC38-CD228 cells in Example 13, Figure 15B To evaluate the inhibitory activity of CA149-BNLD11 on the proliferation of A375-CD228 cells in Example 13, Figure 15C To evaluate the inhibitory activity of CA149-BNLD11 on the proliferation of SK-MEL-5 cells in Example 13, Figure 15DTo evaluate the inhibitory activity of CA149-BNLD11 on the proliferation of A549-CD228 cells in Example 13, Figure 15E This study evaluates the inhibitory activity of CA149-BNLD11 on the proliferation of A375-CD228 cells in Example 13.

[0082] Figure 16 The image shows the tumor growth inhibition curve of the antibody-drug conjugate in the Calu-1 lung cancer model in Example 14.

[0083] Figure 17 This is a bar chart showing the tumor weight of the antibody-drug conjugate group in the Calu-1 lung cancer model in Example 14.

[0084] Figure 18 The image shows the tumor growth inhibition curve of the antibody-drug conjugate group in Example 15 on the human melanoma cell SK-MEL-5 nude mouse xenograft model.

[0085] Figure 19 This is a bar chart showing the tumor weight of the antibody-drug conjugate group in the SK-MEL-5 nude mouse xenograft model of human melanoma cells, as shown in Example 15.

[0086] Figure 20 The image shows the tumor growth inhibition curve of the antibody-drug conjugate group in Example 16 on a nude mouse xenograft model of human gastric cancer cells NUGC4 Balb / c nude.

[0087] Figure 21 This is a bar chart showing the tumor weight of the antibody-drug conjugate group in a nude mouse xenograft model of human gastric cancer cells NUGC4 Balb / c nude, as described in Example 16.

[0088] Figure 22 The tumor volume growth curve of the human lung squamous cell carcinoma NCI-H226 Balb / c nude mouse xenograft in Example 17 is shown.

[0089] Figure 23 The tumor volume growth curve of the human melanoma cell SK-MEL-5 Balb / c nude nude mouse xenograft in Example 18 is shown.

[0090] Figure 24 The image shows the tumor weight growth curve of the human melanoma cell SK-MEL-5 Balb / c nude nude mouse xenograft in Example 18.

[0091] Figure 25 The metabolic curve of the antibody-drug conjugate CA149-BNLD11 in Example 19 in mice.

[0092] Figure 26This study investigates the toxicity of the antibody-drug conjugate CA149-BNLD11 in male rats, as described in Example 20.

[0093] Figure 27 This study investigates the toxicity of the antibody-drug conjugate CA149-BNLD11 in female mice, as described in Example 20.

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

[0095] Figure 29 Toxicokinetic assays were performed 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 This is a schematic diagram of the BNLD11 synthesis circuit. Detailed Implementation

[0097] The present invention will be further illustrated below with reference to specific embodiments. The described embodiments are only some, not all, of the embodiments of the present invention. It should be understood that the following embodiments are provided to give a complete disclosure and description to those skilled in the art on how to utilize the methods and compositions of the present invention, and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0098] Example 1: CD228 Expression Experiment

[0099] PDX sample chips for gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, bile duct cancer, pancreatic cancer, cervical cancer, sarcoma, esophageal cancer, and gastric cancer were all purchased from Crown Bioscience, Inc. Tissue slides from 12 mesothelioma patients were purchased from Shanghai Lidi Biotechnology Co., Ltd. (4 cases) and Shanghai Xinchao Biotechnology Co., Ltd. (8 cases). Crown Bioscience, Inc. was commissioned to perform CD228 immunohistochemical staining on all PDX sample tissue chips and the 8 mesothelioma tissue slides purchased from Shanghai Xinchao Biotechnology Co., Ltd. Shanghai Lidi Biotechnology Co., Ltd. was commissioned to perform CD228 immunohistochemical staining on the 4 mesothelioma tissue slides. The CD228 immunohistochemical antibody was purchased from Novus Biologicals (catalog number: NBP1-85777), with a primary antibody dilution concentration of 1:200.

[0100] Immunohistochemical experiments were performed using an automated IHC and ISH system (Bond RX automatic IHC & ISH system, Leica). The immunohistochemical 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 independently three times, and the final H-score was the average of the three scores. All IHC score results are shown below. Figure 1 As shown. From Figure 1 The expression levels of CD228 on the TMA chip and the expression rates of CD228 shown in Table 1 indicate that CD228 is expressed at a relatively high proportion in gallbladder cancer, colon cancer, breast cancer, melanoma, lung cancer, cholangiocarcinoma, pancreatic cancer, and cervical cancer.

[0101] Table 1. Statistical analysis of CD228 expression levels in various tumors

[0102]

[0103] Example 2. Production of anti-CD228 monoclonal antibody

[0104] 1.1 Protein Production

[0105] The amino acid sequences of the three proteins (see Table 2) were sent to Jiangsu GenScript Biotech Co., Ltd., and the genes were synthesized. These genes were then transfected into CHO cells and cultured in a shaker at 37℃, 8% CO2, and 125 rpm. After transient expression for 10 days, the supernatant was collected. The expression supernatant was purified using a Ni (GenScript, L00250) column, and then further purified using an SP column (GE, 17-1087-01) to obtain human CD228, mouse CD228, and monkey CD228 proteins.

[0106] Table 2. Amino acid sequences of the three proteins

[0107]

[0108] 1.2 Mouse Immunization Methods

[0109] The mice used in the immunization experiments were fully human antibody transgenic mice (10 mice in total) independently developed by Shandong Boan Biotechnology Co., Ltd. Immunization was performed using CD228 antigen protein (0.23 mg / mL, Boan, 20200924, SEQ ID NO:41) independently produced by Shandong Boan Biotechnology Co., Ltd.

[0110] Immunization was performed via subcutaneous injection in the abdomen and groin at multiple sites, with an antigen protein dose of 20 μg per mouse. The initial immunization used Freund's complete adjuvant emulsified antigen, while the second to fourth immunizations used Freund's incomplete adjuvant emulsified antigen. The first batch of mice received three immunizations and one booster immunization; the second batch received four immunizations and one booster immunization. Each immunization was spaced 14 days apart. Starting with the second immunization, peripheral blood serum was collected on day 7 after each immunization to detect antibody titers, and mice with substandard titers were excluded. The serum titer results after immunization are shown below. Figure 2 As shown, 2500X, 12500X, and 62500X represent dilution ratios. Three days after booster immunization, mice were euthanized, and spleens were harvested to create single-cell banks.

[0111] 1.3 Establishment of the phage library

[0112] Mice were euthanized, and their spleens were dissected and removed. The spleens were then crushed using a syringe stopper and filtered through a screen. The filtered spleen cells were frozen, and RNA was extracted to obtain cDNA. The phage library was constructed using standard methods. The library size data are shown in Table 3.

[0113] Table 3. Phage library capacity for each immunized mouse group

[0114]

[0115] 1.4 Screening using two methods

[0116] 1.4.1 Plate screening: Plates were coated with CD228-His protein (homemade). The next day, phage libraries were added and incubated for 2 hours. After washing 4-10 times, the specifically bound phages were eluted with elution buffer.

[0117] 1.44.2 Magnetic bead screening: CD228-His protein was biotinylated according to the kit instructions, then bound to Thermo magnetic beads, blocked with BSA, and incubated with a phage library for 2 hours. After washing 4-10 times, the specifically bound phages were eluted with elution buffer. The antibody clones obtained and their sources are shown in Table 4.

[0118] Table 4. Sources of anti-CD228 antibodies obtained through screening

[0119]

[0120] Example 3. Molecular construction and production of complete antibodies

[0121] 133 positive IgG1 clones were constructed and sequenced. The amino acid sequences of the variable regions of 8 lead antibodies are shown in Table 5 below: (CDR regions are underlined, and the analysis system is the IMGT system). The variable region sequences of each antibody in this application are shown in Table 5, and the heavy and light chain constant region sequences are shown in Table 6.

[0122] Table 5. Variable region amino acid sequences of 8 antibodies

[0123]

[0124]

[0125] The antibody variable region gene was amplified using conventional molecular biology techniques PCR (2×Phanta Max Master Mix, manufacturer: Vazyme, catalog number: P515-P1-AA, batch number: 7E512E1). The antibody heavy chain variable region gene was then ligated into a vector pCDNA3.4 (Life Technology) containing the antibody heavy chain constant region sequence via homologous recombination. The antibody light chain variable region gene was also ligated into a vector pCDNA3.4 containing the antibody light chain constant region sequence.

[0126] Table 6. Amino acid sequences of the heavy and light chain constant regions of antibodies.

[0127]

[0128]

[0129] After sequencing, the positive clones were used to extract plasmids and co-transfect them into HEK293 cells. The cells were cultured in a shaker at 37°C, 8% CO2, and 125 rpm. After transient expression for 7 days, the supernatant was purified by Protein A affinity chromatography to obtain antibodies. The antibody concentration was determined by UV280 and the theoretical extinction coefficient.

[0130] The control antibody HL49 sequence was synthesized based on the sequence in patent US20200246479A1, and the amino acid sequence is shown in Table 7 below.

[0131] Table 7. Amino acid sequence of control antibody hL49

[0132]

[0133] Example 4. Characterization of anti-CD228 monoclonal antibody molecules

[0134] 4.1 Binding of anti-hCD228 antibody to human CD228 protein

[0135] Human CD228 protein (self-made by Boan, 20201014, SEQ ID NO:41) 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, and incubated overnight at 4℃; after washing, the plates were blocked with skim milk powder. After washing, 100 μL of antibody diluted with PBST (phosphate buffer, Solarbio P1010, +0.05% Tween 20) was added to each well (the diluted antibody is a complete antibody serially diluted with PBST, the complete antibody contains the Fc and Fab regions, and is an antibody with constant regions, starting at a concentration of 0.1 μg / mL, three-fold dilution in 8 gradients); after washing, 100 μL of goat anti-human IgG (H+L) / HRP (1:5000 dilution, KPL, 474-1006) was added to each well, and the plates were incubated at 37℃ for 1 h. After washing the plate, add 100 μL of TMB (Beijing Meikewande, 1001) to each well for color development. After 10 min, add 50 μL of 2M H2SO4 to each well to stop the color development. Read the OD450 using a microplate reader. In this experiment and the following experiments, Seagen's CD228-targeting antibody hL49 was used as a control antibody. Figure 3 Table 8 shows the binding curves of each antibody to human CD228 protein. Figure 3 The calculated EC50 values ​​of the antibodies show that the six antibodies exhibited similar binding activity at the ELISA level, and were better than the control antibody HL49.

[0136] Table 8. EC50 values ​​of antibodies

[0137]

[0138] 4.2 Detection of the affinity of the antibody for CD228 human, monkey, and mouse proteins

[0139] Table 9 shows the sources of CD228 protein used in the detection.

[0140]

[0141] The binding kinetics of 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 using the 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 2-fold with HBS-EP+ buffer to five concentrations, starting at 50 nM. Biacore was used for CD228 protein binding kinetic analysis, and the affinity activity KD value was calculated.

[0142] Table 10. Biacore detection of the binding of anti-CD228 antibody to human, monkey, and mouse proteins.

[0143]

[0144] As shown in Table 10, the antibodies exhibit similar affinity to human CD228 and monkey CD228 proteins, but do not bind to mouse CD228.

[0145] 4.3 Epitope Analysis of Anti-hCD228 Antibody

[0146] His microarrays captured CD228 protein at a concentration of 10 μg / mL with a threshold of 0.5 nm. First, a first CD228 antibody (30 μg / mL) was bound, and then the competitive binding of a second CD228 antibody (30 μg / mL) was analyzed. Octet 8K was used to analyze the response of antibody 2, determining whether antibody 1 and antibody 2 competed for binding. Table 11 shows the Octet antibody epitope response values.

[0147] Table 11 Octet antibody epitope response values

[0148]

[0149] The final competition analysis was performed using the following calculation 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 other antibodies compete with each other. Epitopes are similar (values ​​above 75% indicate epitope correlation).

[0150] Table 12 Final Competition Analysis Results

[0151]

[0152] Example 5. Flow cytometry detection of cellular-level binding activity of anti-hCD228 antibody

[0153] 50 μL of human melanoma SK-MEL-5 cells (ATCC, HTB-70) were added to each well of a 96-well round-bottom plate, at a cell count of 7E4 / well. Antibodies were serially diluted with FACS buffer (PBS, Boster Biologics, catalog number PYG0021), and added to each well at a dose of 50 μL / well. The plates were incubated at 4°C for 1 h. After centrifugation at 400 g for 4 min, the supernatant was discarded, and the cells were washed once with FACS buffer. 100 μL / well of fluorescent secondary antibody (Jackson, 109545-008) was added, and the plates were incubated at 4°C in the dark for 30 min. After centrifugation at 400 g for 4 min, the supernatant was discarded, and the cells were washed once with FACS buffer. The cells were then resuspended in 100 μL / well of FACS buffer and analyzed using a flow cytometer (NovoCyte 2060). Results are shown below. Figure 4 , Figure 4The binding of various anti-CD228 antibodies to human melanoma SK-MEL-5 cells (cells expressing CD228 protein) was shown. It can be seen that all six antibodies have high binding activity to SK-MEL-5 cells, and are superior to hL49. Figure 4 The isotype control antibody is an unrelated antibody targeting other targets that has the same constant region but a different variable region as the anti-CD228 antibody in the experimental group.

[0154] Example 6. Internalization experiment of anti-hCD228 monoclonal antibody molecules

[0155] 1. SK-MEL-5 cells

[0156] Add 50 μL / well of human melanoma SK-MEL-5 cells diluted with PBS (Boster Biologics, catalog number PYG0021) to each well of a 96-well round-bottom plate, resulting in a cell count of 5E4 cells / well. Dilute the antibody with buffer to a final concentration of 20 μg / mL. Add 50 μL / well of the 20 μg / mL antibody to each well containing 50 μL / well of cells. Incubate for 30 min, then centrifuge at 400g for 4 min and discard the supernatant. Wash twice with pre-chilled buffer, then add 100 μL / well of buffer. Incubate at 37°C and 4°C, stopping at different time points. Centrifuge at 400g for 4 min and discard the supernatant. Add 100 μL / well of pre-chilled fluorescent secondary antibody (Jackson, 109-545-008) at 4°C and incubate at 4°C in the dark for 30 min. Wash once with pre-chilled FACS buffer, then resuspend in 100 μL / well FACS buffer and analyze using a flow cytometer (Eisen, NovoCyte 2060). Results are shown below. Figure 5A , Figure 5A The results of internalization experiments of various anti-CD228 antibodies based on human melanoma SK-MEL-5 cells are shown. Figure 5A It can be seen that CA13, CA149 and BA352 have higher internalization rates compared with control hL49.

[0157] 2. A549-CD228 cells

[0158] Human lung cancer cell line A549-CD228 (KC-2150) stably expressing the exogenous CD228 gene in logarithmic growth phase was digested, and the digestion was terminated with serum-containing medium and diluted. The cells were then added to 96-well round-bottom plates (NEST, catalog number 701111), 50 μL / well, 1E5 / well. The antibody was diluted with serum-containing medium, and the antibody was mixed with 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 min, the labeled antibody mixture was added to the cells-containing wells, 50 μL / well. Cells were incubated at 37°C for 0 h, 2 h, 6 h, and 24 h, respectively. After washing once with PBS, the cells were resuspended in 100 μL / well of PBS, and the MFI values ​​were read using a flow cytometer (NovoCyte 2060). The results showed that CA149 antibody internalization increased with time; Figure 5B It can be seen that CA149 has superior internalization activity compared with the control antibody hL49.

[0159] Example 7. Antibody-dependent cell-mediated cytotoxicity (luciferase reporter effector cells)

[0160] Prepare ADCC working solution (RPMI 1640 medium containing 1% FBS); collect Bioassay Effector Cells (Promega, G7011) and adjust the cell density to 2.4 × 10⁻⁶ cells using ADCC working solution. 6 Collect Target Cells SK-MEL-5 (ATCC, HTB-70) and adjust the cell density to 8 × 10⁻⁶ cells using ADCC working solution. 5 Dilute the test samples with ADCC working solution, starting at a concentration of 5 μg / mL, and then perform 4-fold dilutions to obtain 8 concentrations. Add 25 μL each of effector cells, target cells, and test samples to a reaction plate (Costar, 3917), for a total reaction volume of 75 μL. Incubate the reaction system at 37°C for 6 h. Add 75 μL of Bio-Glo Luciferase System (Promega, G7940) to each well, and after 15 min of reaction, read the chemiluminescence value using a microplate reader (BioTek, Synergy Neo2). Results are shown below. Figure 6 , Figure 6 The results of ADCC experiments using various anti-CD228 antibodies on human melanoma SK-MEL-5 cells are shown. Figure 6 It can be seen that the signal gradually increases with the increase of antibody sample concentration, indicating that it has an ADCC effect on SK-MEL-5 cells.

[0161] Example 8: Study on the binding epitope of CA149 antibody to CD228

[0162] We prepared full-length antigen CD228 and antibody Fab complexes (CA149-Fab) separately, and commissioned the cryo-electron microscopy center of Tsinghua Future (Hangzhou) Technology Co., Ltd. to perform antigen-antibody structural analysis. By analyzing the amino acid types and side-chain interactions at the epitopes in the three-dimensional model, the researchers found that CA149 antibody Fab binds to the antigen through 7 hydrogen bonds and 1 salt bridge. Specific interaction sites are shown in Table 13, where superscript * indicates amino acids on the light chain of CA149 Fab, and italicized text without superscript indicates amino acids on the heavy chain of CA149 Fab.

[0163] Table 13 Interacting Sites

[0164]

[0165] Meanwhile, based on epitope results, we validated specific site mutations in the CD228 antigen (sequence shown in SEQ ID NO:41) independently produced by Shandong Boan Biotechnology Co., Ltd. We constructed hCD228 antigens with single-point mutations (R275A), (R282A), (E312A), and (L313A), as well as hCD228 antigens with double-point mutations (E312A, L313A), (R282A, E312A), and (R275A, R282A), and performed affinity analysis. Affinity results showed that the mutated antigens exhibited reduced or absent binding activity with CA149 antibodies, indicating that the four sites E312A, L313A, R282A, and R275A are key sites for antibody-antigen binding. The affinity test results are recorded in Table 14.

[0166] Table 14 Affinity Test

[0167]

[0168] In addition, since another alternative shearing product, soluble MFI2, also exists in the human body, namely sMFI2, and there are literature (J Neurochem.2002Nov;83(4):924-33.doi:10.1046 / j.1471-4159.2002.01201.x.; JCereb 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, in order to reduce the possible off-target risk, CD228-targeting antibodies should not bind to sMFI2. According to epitope results and the sequence alignment results of full-length hCD228 and soluble antigen sMFI2 (e.g. Figure 7 From this, we can see that the four key binding sites mentioned above are not on the sMFI2 antigen, meaning that the CA149 antibody will not bind to sMFI2.

[0169] Example 9. Conjugation experiment of anti-hCD228 antibody with drug

[0170] Prepare antibody-drug conjugates with the following molecular formulas.

[0171]

[0172] Where: Ab is any of the above anti-CD228 antibodies or their antigen-binding fragments, LU is the linker (also known as the linker), D is the drug, and the subscript p is the average DAR value of the antibody-drug conjugate.

[0173] 9.1 Preparation of anti-hCD228 antibody-vcMMAE antibody-drug conjugate

[0174] Phosphate buffer (pH 7.5, containing 11 mM DTPA) containing antibody (5-10 mg / mL) was treated with 2 equivalents of TCEP and incubated at 25°C for approximately 2 h. 5 equivalents of DMSO solution of vcMMAE were added to the reduced antibody-PBS solution and incubated at 25°C for approximately 1 h. Then, 10 equivalents of n-acetylcysteine ​​(NAC) were added to the mixture and incubated at 25°C for 5 min to quench all unreacted linker-drug conjugates. Ultrafiltration was performed to replace the buffer and remove free small molecules. The samples were then analyzed by HIC-HPLC. The analytical results are shown in Table 15. It can be seen that the average DAR value of the antibody-drug conjugates used in this application is between 4.01 and 4.42.

[0175] Table 15 Results of DC HIC-HPLC

[0176]

[0177] 9.2 Preparation of CA149-BNLD11 antibody-drug conjugate

[0178] Taking the anti-CD228 antibody CA149 as an example, an ADC composition with a homogeneous drug loading (DAR) of approximately 4 was prepared. Phosphate buffer (pH 7.5, containing 11 mM DTPA) containing antibody (5-10 mg / mL) was treated with 2 equivalents of TCEP and incubated at 25°C for approximately 2 hours. 5 equivalents of BNLD-11 in DMSO solution were added to the reduced antibody in PBS solution and incubated at 25°C for approximately 1 hour. Then, 10 equivalents of n-acetylcysteine ​​(NAC) were added to the mixture and incubated at 25°C for 5 minutes to quench all unreacted linker-drug conjugates. HICI-HPLC analysis was used to quantify the drug loading of the antibody-drug conjugate. Analytical results are shown below. Figure 8 As shown in Table 16, the average DAR value of the antibody-drug conjugate CA149-BNLD11 prepared in this embodiment is approximately 4.

[0179] Table 16 shows the DAR values ​​after integral analysis.

[0180]

[0181] Example 10. In vitro cell killing experiment of anti-hCD228 ADC

[0182] In a 96-well plate (Corning, catalog number 3917), add 50 μL / well of SK-MEL-5 cells, resulting in a cell count of 1E4 / well, and use 10% FBS / EMEM culture medium. Dilute each anti-CD228-ADC with the above culture medium, starting at a concentration of 1 μg / mL, and serially dilute 4-fold to obtain 6 concentrations. Add 50 μL / well of the diluted ADC to each 96-well plate and incubate at 37°C and 5% CO2 for 4 days. Then, equilibrate to room temperature using a CellTiter-Glo kit (Promega, G7571, protected from light during use), mix the buffer and substrate in the kit, and incubate for 1 hour. Add 100 μL of cell viability assay reagent to each well of the 96-well plate, vortex horizontally at 300 rpm for 2 minutes, and incubate for 10 minutes. Detect using a microplate reader (BioTek, SYNERGY neo, USA).

[0183] The results are as follows Figures 9A-9F As shown, Figures 9A-9FThe results of the killing experiments of various anti-CD228 ADCs based on human melanoma SK-MEL-5 cells are shown. It can be seen that two anti-hCD228 ADCs, CA13-vcMMAE and CA67-vcMMAE, showed similar in vitro cell killing activity to hL49-vcMMAE; four anti-hCD228 ADCs, CA149-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and CA185-vcMMAE, showed in vitro cell killing activity similar to the control group hL49-vcMMAE. Figures 9A-9F The isotype control antibody is an irrelevant antibody targeting other targets that has the same constant region as the six antibodies but a different variable region.

[0184] Example 11. In vivo efficacy experiment of anti-CD228 ADC on mouse xenografts

[0185] 11.1 Efficacy data in a human melanoma cell line SK-MEL-5 mouse xenograft model

[0186] Human melanoma cells SK-MEL-5 were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in EMEM medium containing 10% FBS. NOD / SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The concentration of SK-MEL-5 cells was adjusted to 2.5 × 10⁻⁶ cells using EMEM medium containing 50% Matrigel. 7 The tumor was injected subcutaneously on the right side of NOD / SCID mice at a dose of 0.1 mL per mouse. The tumor volume was approximately 76 mm². 3 Based on tumor volume and body weight, mice were divided into 8 experimental groups of 5 mice each. Drug administration began on the day of grouping, with a dosage of 3 mg / kg. Figure 10 The efficacy data (3 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model are shown, i.e., the change in tumor volume in mice after administration. It can be seen that the tumor volume decreased after administration of all 6 antibodies. Figure 10The graph is 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; the tumor weight inhibition rates were also 49.0%. The inhibition rates (IR%) were 59.8%, 65.9%, 61.4%, 55.4%, 68.2%, 65.7%, and 56.1%, respectively. It is evident that in the SK-MEL-5 mouse xenograft model of human melanoma cells, under a drug administration condition of 3 mg / kg, the six anti-CD228-vcMMAEs prepared in this project were superior to the control group hL49-vcMMAE in both tumor volume growth inhibition rate (TGI%) and tumor weight inhibition rate (IR%).

[0187] Table 17. Tumor volume changes after administration of 3 mg / kg

[0188]

[0189] Figure 11 The efficacy data (5 mg / kg) of each anti-CD228 ADC in the SK-MEL-5 animal model are shown. NCG mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The SK-MEL-5 cell concentration was adjusted to 3 × 10⁶ cells / kg using EMEM medium containing 50% Matrigel. 7 The tumor was injected subcutaneously on the right side of NCG mice at a dose of 0.1 mL per mouse. The tumor volume was approximately 83 mm². 3 Mice were divided into eight experimental groups of six mice each, based on tumor volume and body weight. Drug administration began on the day of grouping, at a dose of 5 mg / kg. The screened antibody showed better efficacy compared to the control hL49. Figure 11The graph is based on the volume change data in Table 18. As shown in Table 18, the tumor volume growth inhibition rates (TGI%) of the CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE groups at the experimental endpoint were 82%, 84%, 81%, 80%, 81%, and 74%, respectively; 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 SK-MEL-5 mouse xenograft model of human melanoma cells, under the condition of 5 mg / kg administration, the anti-CD228-vcMMAE prepared in this project was superior to the control group hL49-vcMMAE in terms of both the tumor volume growth inhibition rate (TGI%) and the tumor weight inhibition rate (IR%).

[0190] Table 18. Tumor volume changes after administration of 5 mg / kg

[0191]

[0192] 11.2 Efficacy data in human NCI-H226 lung cancer cells / mouse xenograft model

[0193] Human lung cancer cells NCI-H226 were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator on RPMI 1640 medium containing 10% FBS. CB-17 / SCID mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0194] The concentration of NCI-H226 cells was adjusted to 5.0 × 10⁻⁶ using RPMI 1640 medium containing 50% Matrigel. 7 The tumor was injected subcutaneously on the right side of CB-17 / SCID mice at a dose of 0.1 mL per mouse. The tumor volume was increased when the average tumor volume reached approximately 156 mm². 3 Based on tumor volume and body weight, mice were divided into 8 experimental groups of 5 mice each. Drug administration began on the day of grouping, with a dosage of 3 mg / kg. Figure 12 The efficacy data (3 mg / kg) of each anti-CD228 ADC in the NCI-H226 animal model are shown. It can be seen that after administration of all six antibodies, the tumor volume decreased, showing in vivo efficacy similar to HL49. Figure 12The graph is based on the volume change data in Table 20. As shown in Table 19, the tumor volume growth inhibition rates (TGI) of the CA13-vcMMAE, CA67-vcMMAE, CA149-vcMMAE, CA185-vcMMAE, CA352-vcMMAE, CA518-vcMMAE, and hL49-vcMMAE groups at the experimental endpoint 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.

[0195] Table 19. Tumor volume changes after administration of 3 mg / kg

[0196]

[0197] Figure 13 The efficacy data (5 mg / kg) of each anti-CD228 ADC in the NCI-H226 animal model are shown. NCG mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The NCI-H226 cell concentration was adjusted to 5 × 10⁶ cells using RPMI 1640 medium containing 50% Matrigel. 7 The tumor was injected subcutaneously on the right side of NCG mice at a dose of 0.1 mL per mouse. The tumor volume was approximately 10⁸ mm². 3 Mice were divided into eight experimental groups of six mice each, based on tumor volume and body weight. Drug administration began on the day of grouping at a dose of 5 mg / kg. The screened antibody showed better efficacy compared to the control HL49. Figure 13 The graph is based on the volume change data in Table 20. As shown in Table 16, the tumor volume growth inhibition rates (TGI) of the CA13-vcMMAE, CA149-vcMMAE, CA518-vcMMAE, CA523-vcMMAE, CA579-vcMMAE, and hL49-vcMMAE groups at the experimental endpoint were 94.4%, 92.3%, 94.6%, 98.3%, 103.9%, and 75.6%, respectively; the tumor weight inhibition rates were 73.1%, 69.2%, 72.1%, 75.5%, 77.4%, and 56.7%, respectively.

[0198] Table 20. Tumor volume changes after administration of 5 mg / kg

[0199]

[0200] Example 12. Pharmacokinetics of Anti-CD228 ADC in Mice

[0201] Three Balb / c mice were selected for each ADC and administered the drug subcutaneously at a dose of 10 mg / kg. Serum samples were collected at 0 h before administration, and at 1 h, 4 h, 10 h after administration, and at 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.

[0202] Figure 14 The pharmacokinetic curves of CA13-vcMMAE, CA67-vcMMAE, and CA149-vcMMAE are recorded. Specific results can be found in [link to relevant documentation]. Figure 14 It can be seen that CA149-vcMMAE has better pharmacokinetic levels than CA13-vcMMAE and CA67-vcMMAE constructed from CA13 and CA67 antibodies.

[0203] Example 13. Cell proliferation inhibitory activity of antibody-drug conjugate CA149-BNLD11

[0204] 13.1 Inhibitory activity of CA149-BNLD11 on the proliferation of MC38-CD228 and A375-CD228 cells

[0205] Logarithmically growing MC38-CD228 (Kangyuan Bochuang, KC-2023) and A375-CD228 (Kangyuan Bochuang, KC-2110) cells were digested, diluted and resuspended in 10% FBS / 1640 medium, and added to 96-well plates (SARSTED, catalog number 94.6120.096), 50 μL / well, 1E4 / well. The antibody-drug conjugate CA149-BNLD11 prepared in part 8.2 of Example 8 was diluted with serum-containing medium at initial concentrations of 60 and 12 μg / mL, followed by 5-fold serial dilutions. The antibody with antibody ID CA521 from CN202180003751.7 was used as the control group (Isotype) along with nCov-CA521-vcMMAE prepared using the same method as in Example 8. The diluted CA149-BNLD11 was added at 50 μL / well to the above-mentioned 96-well flat-bottom cell culture plates and incubated at 37°C and 5% CO2 for 96 h. The kit (Novazia, DD1101-01) must be protected from light during use. After equilibration to room temperature, invert and mix thoroughly. Add 100 μL / well to a 96-well plate, vortex horizontally at 300 rpm for 2 min, and then incubate for 10 min. Cell viability is then measured using a microplate reader (BioTek, SYNERGY neo, USA).

[0206] The results showed that CA149-BNLD11 had excellent inhibitory activity against the proliferation of MC38-CD228 and A375-CD228 cells, as shown in Table 21. 50The concentrations were 101.5 ng / mL and 61.8 ng / mL, respectively. Figures 15A-15B Its vertical axis represents the viable cell percentage (%), which is the ratio of the number of viable cells in the drug-treated group to the number of viable cells in the control group. Figures 15A-15B It can be seen that CA149-BNLD11 has a significant inhibitory activity on the proliferation of CD228-expressing cells compared to the control group (Isotype).

[0207] Table 21. Data on the proliferation inhibition activity of CA149-BNLD11

[0208]

[0209] 13.2 Inhibitory activity of CA149-BNLD11 on the proliferation of SK-MEL-5, A549-CD228, and A375-CD228 cells

[0210] Logarithmic growth phase SK-MEL-5 (ATCC, HTB-70), A549-CD228 (Kangyuan Bochuang, KC-2150), and A375-CD228 (Kangyuan Bochuang, KC-2110) cells were digested, digestion was terminated with serum-containing medium, and the cells were diluted and added to 96-well flat-bottom plates (SARSTED, catalog number 94.6120.096), 50 μL / well, 1E4 / well. CA149-BNLD11 was diluted with serum-containing medium at initial concentrations of 1.2, 60, and 6 μg / mL, followed by 5-fold, 6-fold, and 5-fold serial dilutions. The diluted ADC was added to the above 96-well flat-bottom cell culture plates at 50 μL / well and incubated at 37℃, 5% CO2 for 96 h or 120 h. The kit (Novazia, DD1101-01, must be protected from light during use) was equilibrated to room temperature, inverted and mixed, and added to 96-well plates at 100 μL / well. The plates were then vortexed horizontally at 300 rpm for 2 min, followed by incubation for 10 min. Cell viability was detected using a microplate reader (BioTek, SYNERGY neo, USA). Results showed that CA149-BNLD11 exhibited excellent inhibitory activity against the proliferation of 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). Figure 15C-15E It can be seen that CA149-BNLD11 has a significant inhibitory activity on the proliferation of CD228-expressing cells compared to the control group (Isotype).

[0211] Table 22 Data on the inhibitory activity of CA149-BNLD11 on the proliferation of CD228-expressing cells.

[0212]

[0213] Example 14. Evaluation of the efficacy of antibody-drug conjugate CA149-BNLD11 against human lung cancer cell CaLu-1 xenografts in nude mice.

[0214] CaLu-1 human lung cancer cells were purchased from ATCC and cultured at 37°C in a 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 × 10⁻⁶ cells using serum-free McCoy's 5A medium containing 50% Matrigel. 7 The tumor volume was 0.1 mL / mouse, injected subcutaneously into the right side of Balb / c nude mice. When the average tumor volume reached 135 mm³, the mice were divided into 5 experimental groups of 5 mice each, and the drug was administered on the same day as grouping. This experiment used a single-dose administration, and the mice were observed for 28 days after administration.

[0215] The results are recorded in Figure 16-17 middle, Figure 16 The image shows the tumor growth inhibition curve of the antibody-drug conjugate group in the Calu-1 lung cancer model. Figure 17 This is a bar chart showing tumor weight in the Calu-1 lung cancer model using antibody-drug conjugates.

[0216] like Figure 16 As shown, compared with the solvent control group (PBS phosphate buffer), all antibody-drug conjugate groups significantly inhibited tumor volume growth, with statistically significant differences (P < 0.05); the tumor-inhibiting effects of 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, 0.0016, and 0.0006, respectively); C No significant differences were found among the three groups: A149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd (P > 0.05 for all groups); 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.

[0217] like Figure 17As shown, at the experimental endpoint, the average tumor weight of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups was significantly lower than that of the solvent control group (PBS phosphate buffer), with statistically significant differences (all P < 0.05); there was no statistically significant difference in tumor weight between the nCov-CA521-vcMMAE group and the control group (P = 0.3250); no significant differences were found among the three groups (all P > 0.05); the tumor weight inhibition rates of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 96.3%, 87.3%, 87.6%, and 44.3%, respectively.

[0218] During the experiment, the experimental animals maintained good activity and appetite during the drug administration period, and their body 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).

[0219] Example 15. Evaluation of the efficacy of antibody-drug conjugate CA149-BNLD11 against human melanoma cell line SK-MEL-5 xenografts in nude mice.

[0220] SK-MEL-5 human melanoma cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in EMEM medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The concentration of SK-MEL-5 cells was adjusted to 5.0 × 10⁻⁶ cells using serum-free EMEM medium containing 50% Matrigel. 7 The tumor volume was 0.1 mL / mouse, injected subcutaneously into the right side of Balb / c nude mice. When the average tumor volume reached 98 mm³, the mice were divided into four experimental groups of five mice each, and the drug was administered on the same day as grouping. This experiment used a single-dose administration, and the mice were observed for 28 days after administration.

[0221] The results are recorded in Figure 18-19 middle, Figure 18 The image shows the tumor growth inhibition curve of the antibody-drug conjugate group in a nude mouse xenograft model of human melanoma cells SK-MEL-5. Figure 19 This is a bar chart showing the tumor weight of the antibody-drug conjugate in a nude mouse xenograft model of human melanoma cells (SK-MEL-5). Figure 18The tumor growth curves shown indicate that at the experimental endpoint, compared with the solvent control group (PBS phosphate buffer), all treatment groups significantly inhibited tumor volume growth, with statistically significant differences (P < 0.0001). The tumor-inhibiting effects of CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly better than those of the nCov-CA521-vcMMAE group (P < 0.0001), and the CA149-BNLD11 group showed a 2 / 3 increase in tumor volume. 5. Complete tumor clearance in mice; no statistically significant differences were observed among the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups (P > 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.

[0222] like Figure 19 As shown, at the experimental endpoint, the average tumor weight of all treatment groups was significantly lower than that of the solvent control group (PBS phosphate buffer), with statistically significant differences (P < 0.05). There were no significant differences among the four groups: CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE (P > 0.05). The tumor weight inhibition rates of the CA149-BNLD11, CA149-vcMMAE, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 88.3%, 90.7%, 90.5%, and 58.7%, respectively.

[0223] During the experiment, the experimental animals maintained good activity and feeding status during the drug administration period. Except for the solvent control group (PBS phosphate buffer), the body weight of all other groups increased to some extent, indicating that the animals tolerated the test product well. There were no significant differences between the groups (P>0.05).

[0224] Example 16. Evaluation of the efficacy of antibody-drug conjugate CA149-BNLD11 against human gastric cancer cells NUGC4 Balb / c nude xenografts in nude mice.

[0225] NUGC4 human gastric cancer cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. Cells were cultured in a 37℃, 5% CO2 incubator using RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The concentration of NUGC4 cells was adjusted to 1.8 × 10⁻⁶ cells using serum-free RMPI-1640 medium containing 50% Matrigel. 7 The tumor volume was 0.1 mL / mouse, subcutaneously injected into the right side of Balb / c nude mice. When the average tumor volume reached 108 mm3, the mice were divided into 4 experimental groups of 5 mice each, and the drug was administered on the same day as grouping. This experiment used a single-dose administration, and the mice were observed for 23 days after administration.

[0226] The results are recorded in Figure 20-21 middle, Figure 20 The image shows the tumor growth inhibition curve of the antibody-drug conjugate group in a nude mouse xenograft model of human gastric cancer cells NUGC4 Balb / cnude. Figure 21 This is a bar chart showing the tumor weight of the antibody-drug conjugate group in a nude mouse xenograft model of human gastric cancer cells NUGC4 Balb / c nude.

[0227] like Figure 20 The tumor growth curves show that at the experimental endpoint, compared with the solvent control group (PBS phosphate buffer), the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups all significantly inhibited tumor volume growth, with statistically significant differences (P < 0.0001). The tumor-inhibiting effects of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups were significantly better than those of the nCov-CA521-vcMMAE group (P < 0.0001). The tumor-suppressing effect of the 149-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.

[0228] like Figure 21As shown: At the experimental endpoint, the average tumor weight of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups was significantly lower than that of the solvent control group (PBS phosphate buffer), with statistically significant differences (all P < 0.0001); there was no statistically significant difference in tumor weight between the nCov-CA521-vcMMAE group and the control group (P = 0.8144); the tumor weight of the CA149-BNLD11, CA149-vcMMAE, and CA149-GGFG-Dxd groups was significantly less than that of the nCov-CA521-vcMMAE group (all P < 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.

[0229] During the experiment, the experimental animals maintained good activity and appetite during the drug administration period, and their body 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).

[0230] Example 17: Evaluation of the efficacy of antibody-drug conjugate CA149-BNLD11 against human lung squamous cell carcinoma NCI-H226 Balb / c nude mouse xenografts.

[0231] NCI-H226 human lung squamous cell carcinoma cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator on RMPI-1640 medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The NCI-H226 cell concentration was adjusted to 4.0 × 10⁶ cells using serum-free RMPI-1640 medium containing 50% Matrigel. 7 The tumor was injected subcutaneously on the right side of Balb / c nude mice at a dose of 0.1 mL per mouse. The tumor volume was increased to 145 mm². 3 Mice were divided into four experimental groups (n=6 per group) based on tumor volume, and drug administration began on the day of grouping. The dosage was 3.3 mg / kg, administered as a single dose. Results are expressed as mean ± standard error (Mean ± SEM). Data analysis and processing were performed using Graphpad 8.0 software. Tumor volume and body weight were compared between groups at each time point using two-way ANOVA; tumor weight was analyzed using one-way ANOVA for statistical difference analysis; and t-tests were used to compare between two groups. P < 0.05 was considered statistically significant.

[0232] The trial ended on day 24 of the group administration. Figure 22 The documented human lung squamous cell carcinoma NCI-H226 Balb / c The tumor volume growth curves of nude mouse xenografts are shown in the figure. Compared with the solvent control group (PBS phosphate buffer), the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups significantly inhibited tumor volume growth, with statistically significant differences (P < 0.05). The nCov-CA521-vcMMAE group had no tumor-inhibiting effect (P > 0.05). There were no statistically significant differences among the CA149-vcMMAE, CA149-BNLD11, and CA149-GGFG-Dxd groups (P > 0.05). The tumor volume growth inhibition rates (TGI%) of the CA149-vcMMAE, CA149-BNLD11, CA149-GGFG-Dxd, and nCov-CA521-vcMMAE groups were 86.6%, 81.1%, 93.5%, and 19.1%, respectively.

[0233] Example 18: Evaluation of the efficacy of multiple doses of antibody-drug conjugate CA149-BNLD11 on human melanoma cell line SK-MEL-5 Balb / cnude nude mouse xenografts.

[0234] SK-MEL-5 human melanoma cells were purchased from ATCC and cultured in a 37°C, 5% CO2 incubator in EMEM medium containing 10% FBS. Balb / c nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The concentration of SK-MEL-5 cells was adjusted to 3.0 × 10⁶ cells / mL using serum-free EMEM medium containing 50% Matrigel. 7 The tumor was injected subcutaneously on the right side of Balb / c nude mice at a dose of 0.1 mL per mouse. The tumor volume was increased to 10³ mm². 3 Mice were divided into four experimental groups (n=6 per group) based on tumor volume. 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 as a single dose. Results are expressed as mean ± standard error (Mean ± SEM). Data analysis and processing were performed using Graphpad 8.0 software. Tumor volume and body weight were compared between groups at each time point using two-way ANOVA; tumor weight was analyzed using one-way ANOVA. A t-test was used to compare between groups; P < 0.05 indicated statistical significance.

[0235] The trial ended on day 21 of the dosing regimen. Figure 23The tumor volume growth curves of SK-MEL-5 Balb / c nude nude mouse xenografts of human melanoma cells are shown in the figures. Compared with the solvent 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 solvent control group (PBS phosphate buffer) (P = 0.4030). The tumor volume growth inhibition rates (TGI%) of CA149-BNLD11 in the 1.0 mg / kg, 2.5 mg / kg, and 5.0 mg / kg groups were 25.1%, 72.1%, and 91.1%, respectively. This indicates that the antitumor activity of CA149-BNLD11 is dose-dependent.

[0236] At the end of the experiment, if Figure 24 The tumor weight growth curves of SK-MEL-5 Balb / c nude human melanoma cell xenografts in nude mice are shown: compared with the solvent control group (PBS phosphate buffer), the tumor weight of the 2.5 mg / kg and 5.0 mg / kg groups was significantly reduced, with statistically significant differences (P < 0.05); there was no significant difference between the 1.0 mg / kg group and the solvent 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.

[0237] During the experiment, the experimental animals maintained good activity and appetite during the drug administration period, and all groups showed a certain degree of weight gain, indicating good tolerance to the test product. There were no significant differences between the groups (P>0.05).

[0238] Example 19: Metabolic study of antibody-drug conjugate CA149-BNLD11 in mice.

[0239] Three ICR mice were selected and administered CA149-BNLD11 and CA149-vcMMAE via tail vein injection at a dose of 10 mg / kg. Serum samples were collected before administration and at 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 detected using the ELISA method. The specific test results are shown in the table below.

[0240] Table 23 Key metabolic parameters of CA149-BNLD11 and CA149-vcMMAE in ICR mice

[0241]

[0242] From Table 23 and Figure 25The demonstrated in vivo metabolic studies in mice show that CA149-BNLD11 has a longer half-life than CA149-vcMMAE. The total antibody exposure of CA149-BNLD11 is 1.4 times that of CA149-vcMMAE, and the total ADC exposure of CA149-BNLD11 is 2.3 times that of CA149-vcMMAE. Furthermore, the metabolic curves ( Figure 25 It can also be seen that the shedding rate of BNLD11 in mice is much lower than that of mc-vcMMAE. In conclusion, CA149-BNLD11 is metabolized more stably in mice.

[0243] Example 20: Toxicity study of antibody-drug conjugate CA149-BNLD11 in mice.

[0244] Balb / c mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. Mice were divided into 6 experimental groups (n=3 per group) according to sex and weight. Body weight and food weight were measured on the day of grouping, and drug administration began two days later. The drug administration regimen is shown in Table 24.

[0245] Table 24 In vivo drug administration regimens for mice

[0246]

[0247] The trial ended on day 14 of drug administration. Results were expressed as mean and standard error (Mean ± SEM). Data analysis and processing were performed using Graphpad 8.0 software. Body weight and food intake were compared using the T-test; P < 0.05 indicated statistical significance.

[0248] like Figure 26-27The study demonstrated that, compared to the solvent control group (PBS phosphate buffer), both 50 mg / kg of CA149-vcMMAE and CA149-BNLD11 significantly reduced mouse body weight. The maximum weight loss occurred on day 4 post-administration. The maximum weight loss in females was 17.44% and 10.04%, respectively; in males, it was 17.97% and 5.24%, respectively. Both male and female mice in the CA149-vcMMAE group exhibited varying degrees of ruffled fur and lethargy on days 4 and 6 post-administration, recovering to normal by day 8. Mice in the CA149-BNLD11 group showed no abnormal reactions and remained normal. On days 4 and 6 after administration, CA149-vcMMAE was significantly more toxic than CA149-BNLD11 (female: P < 0.05; male: P < 0.05). Compared with the solvent control group (PBS phosphate buffer), both 50 mg / kg CA149-vcMMAE and CA149-BNLD11 significantly reduced food intake in mice; there was no significant difference between the two groups of CA149-vcMMAE and CA149-BNLD11.

[0249] Example 21: Pretoxicological experiments and accompanying toxicokinetics of antibody-drug conjugate CA149-BNLD11 in cynomolgus monkeys.

[0250] Four cynomolgus macaques, half male and half female, were selected. At the start of drug 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 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) had one male and one female, the medium-dose CA149-BNLD11 group (6 mg / kg) had one male, and the high-dose CA149-BNLD11 group (10 mg / kg) had one female. The administration volume for each group was 5 mg / mL, corresponding to concentrations of 0.4, 1.2, and 2 mg / mL, respectively. The low-dose and high-dose groups were administered twice, while the medium-dose group was administered once, via intravenous infusion over 30 min per animal.

[0251] In addition to clinical observation, food intake and weight monitoring, hematological and blood biochemistry tests, a toxicokinetic study was also conducted after drug administration. Blood collection points included the medium-dose (6 mg / kg) and high-dose (10 mg / kg) groups before the first administration and at 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.

[0252] During the experiment, no animals in any group experienced near-death experiences or died, and no abnormalities were observed in any dosage group. The overall body weight of animals in each group fluctuated little, and no abnormalities related to the administration of the test substance were observed. Food intake in each group showed no regular changes, and no abnormalities related to the administration of the test substance were observed. Figure 28 As shown: Compared with pre-administration levels, CA149-BNLD11 at doses of 6 mg / kg or higher caused a decrease in WBC, #NEUT, and %NEUT in cynomolgus monkeys 5–14 days after administration, with a recovery trend after 21 days. Apart from this, other hematological indicators in all groups of animals at each measurement time point were generally within the normal range, with no dose-response or time-response correlation changes. No abnormalities related to the administration of the test substance were observed. Compared with pre-administration levels, CA149-BNLD11 at a dose of 10 mg / kg caused an increase in serum AST in cynomolgus monkeys 5 days after administration, which recovered after 8 days. Apart from this, other blood biochemical indicators in all groups of animals at each measurement time point were generally within the normal range, with no abnormalities related to the administration of the test substance.

[0253] like Figure 29 As shown, the accompanying toxicokinetic assay results indicated that the toxin shedding rate in the 10 mg / kg dose group was significantly lower than that in the 2 mg / kg dose group in cynomolgus monkeys. 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 its antigen-binding fragment thereof, said antibody or its antigen-binding fragment comprising three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 and three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, wherein The amino acid sequences of the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively, and the amino acid sequences of the three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO:16, SEQ ID NO:27, and SEQ ID NO:28, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes the light chain variable region shown in SEQ ID NO:7 and the heavy chain variable region shown in SEQ ID NO:

8.

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

34.

4. A nucleic acid encoding the anti-CD228 antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. A host cell comprising the nucleic acid of claim 4.

6. An antibody-drug conjugate (ADC), characterized in that, The ADC structure is shown in Equation 1 below: Wherein: Ab is the anti-CD228 antibody or its antigen-binding fragment as shown in any one of claims 1-3; LU stands for connector; D is a drug; p corresponds to the average DAR value of the antibody-drug conjugate; p is a value of 1-4 or 4-8. The LU-D structure is VcMMAE, where Vc is valine-citrulline and MMAE is monomethyl auristatin E, or The LU-D structure is as follows:

7. The antibody-drug conjugate according to claim 6, characterized in that, p is 4.

8. A pharmaceutical composition comprising the anti-CD228 antibody or its antigen-binding fragment as described in any one of claims 1-3, the nucleic acid as described in claim 4, the host cell as described in claim 5, or the antibody-drug conjugate as described in any one of claims 6-7.

9. A kit comprising the anti-CD228 antibody or its antigen-binding fragment as described in any one of claims 1-3, the nucleic acid as described in claim 4, the antibody-drug conjugate as described in any one of claims 6-7, or the pharmaceutical composition as described in claim 8.

10. The use of the anti-CD228 antibody or antigen-binding fragment thereof according to any one of claims 1-3, the nucleic acid according to claim 4, the antibody-drug conjugate according to any one of claims 6-7, the pharmaceutical composition according to claim 8, or the kit according to claim 9 in the preparation of reagents for the prevention, treatment, or diagnosis of CD228-related diseases; wherein the CD228-related diseases are one or more of melanoma, lung cancer, and gastric cancer.

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