Monoclonal antibody targeting ADAM9 protein

Abstract

The invention provides a monoclonal antibody of a targeted ADAM9 protein. Specifically, the invention provides an ADAM9-targeted antibody or an antigen binding fragment thereof, the antibody or the antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprise complementary determining regions CDR selected from the following groups: the heavy chain variable region comprises the following complementary determining regions CDR: VHCDR1-3 shown as SEQ ID NO: 6-8; and the light chain variable region comprises the following complementarity determining regions CDR: VLCDR1 to VLCDR3 as shown in SEQ ID NO: 2 to SEQ ID NO: 4. The antibody provided by the invention can specifically recognize and bind ADAM9 protein, can mediate internalization after the ADAM9 protein is expressed on the surface of a binding cell, and can bind human and monkey ADAM9 antigens and inhibit the growth and / or proliferation of tumors or tumor cells.

Description

Technical Field

[0001] This invention relates to the field of antibodies. Specifically, it relates to a monoclonal antibody targeting the ADAM9 protein. Background Technology

[0002] The detegrin and metalloproteinase (ADAM) family belongs to the zinc protease superfamily and consists of transmembrane proteins with multi-domain extracellular regions, a single transmembrane sequence, and a relatively short cytoplasmic domain. The extracellular region of an ADAM contains several distinct domains: a predomain, followed by a metalloproteinase, a detegrin, and a cysteine-rich domain. Extradomain shedding, typically mediated by members of the ADAM family, is a key mechanism regulating cell signaling. More than half of ADAMs contain a conserved zinc-binding motif in their metalloproteinase domain and can cleave (“shed”) the extradomain of cell surface substrates. Most ADAMs (except ADAM10 and ADAM17) also possess an epidermal growth factor (EGF)-like domain. ADAM proteins have been reported to have a wide range of biological functions, including development, reproduction, extracellular domain shedding, cell adhesion, cell-cell interactions, vascular endothelial cell function, inflammation, immunity, signal transduction, neurodegenerative diseases, and cancer biology. Therefore, ADAMs play important roles in various physiological settings.

[0003] ADAM9 (a distegrinin and a metalloprotease 9) is a tumor-associated antigen highly expressed in many types of solid tumors and involved in carcinogenesis and tumor progression. In prostate cancer, ADAM9 cleaves insulin β-chain, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-α, gelatin, β-casein, etc., and induces the shedding of epidermal growth factor (EGF), fibroblast growth factor receptor 2 (FGFR2)-IIIB, and heparin-bound EGF-like growth factor. Therefore, ADAM9 induces the degradation of specific substrates, releases active growth factors, and interacts with key regulatory factors; its expression is involved in various cancer processes such as cell growth and invasion. For example, in gene expression synthesis (GEO) studies, bladder cancer with peripheral carcinoma and invasive carcinoma showed significantly high ADAM9 mRNA expression; ADAM9 knockdown attenuated cell migration in pancreatic cancer-associated Panc-1 and ASPC-1 cells. Therefore, it is an ideal target for targeted cancer therapy.

[0004] Given the therapeutic potential of ADAM9, there is an urgent need in the field to develop an antibody that specifically binds to the ADAM9 protein. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody that specifically binds to the ADAM9 protein.

[0006] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting ADAM9 is provided, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region and light chain variable region comprising complementarity-determining regions (CDRs) as follows:

[0007] The heavy chain variable region includes the following complementary determinant region (CDR):

[0008] VHCDR1, as shown in SEQ ID NO:6,

[0009] VHCDR2 shown in SEQ ID NO:7, and

[0010] VHCDR3 as shown in SEQ ID NO:8;

[0011] Furthermore, the light chain variable region includes the following complementary determinant region (CDR):

[0012] VLCDR1 shown in SEQ ID NO:2,

[0013] VLCDR2 shown in SEQ ID NO:3, and

[0014] VLCDR3 as shown in SEQ ID NO:4.

[0015] In another preferred embodiment, the CDR sequences of SEQ ID NO:2-4 and SEQ ID NO:6-8 are shown below:

[0016] SEQ ID NO.2:QNVGTN;

[0017] SEQ ID NO.3:SAS;

[0018] SEQ ID NO.4:QQYNSYPLT;

[0019] SEQ ID NO.6:GFTFSDVW;

[0020] SEQ ID NO.7:IRSKANYHST;

[0021] SEQ ID NO.8:TRKWGN.

[0022] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it; and / or the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:1 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it.

[0023] In another preferred embodiment, the antibody has a heavy chain variable region as shown in SEQ ID NO:5 and a light chain variable region as shown in SEQ ID NO:1.

[0024] In another preferred embodiment, SEQ ID NO.1:

[0025] DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWHQQKPGQSPKPLIYSA

[0026] SYRYSGVPDRFTGSGSGTYFTLTISNVQSEDLAEYFCQQYNSYPLTFGAGTKL

[0027] ELK

[0028] In another preferred embodiment, SEQ ID NO. 5:

[0029] EVNLDESGGGLVQPGGSMKLSCAASGFTFSDVWMDWVRQSPEKGLEW VAEIRSKANYHSTYYAESVKGRFTISKDDSKGSVYLQMNSLRAEDTGIYYCT RKWGNWGQGTLVTVSA.

[0030] In another preferred embodiment, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:9 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it; and / or the amino acid sequence of the light chain is as shown in SEQ ID NO:10 or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with it.

[0031] In another preferred embodiment, the antibody has a heavy chain as shown in SEQ ID NO:9 and a light chain as shown in SEQ ID NO:10.

[0032] In another preferred embodiment, the antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; and the light chain variable region includes the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

[0033] In another preferred embodiment, the antibody targeting ADAM9 or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody (“scFv”), bisscFv, (scFv)2, micro antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, and disulfide-stabilized Fv protein (“dsFv”).

[0034] In another preferred embodiment, the antibody specifically binds to ADAM9 or a derivative thereof.

[0035] In another preferred embodiment, the antibody is specifically capable of binding to ADAM9 derived from humans, mice, and cynomolgus monkeys.

[0036] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0037] In another preferred embodiment, the light chain constant region is of human, mouse, or rabbit origin, preferably of human origin.

[0038] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0039] In another preferred embodiment, the heavy chain constant region is of human, mouse, or rabbit origin, preferably of human origin.

[0040] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0041] In another preferred embodiment, the antibody is a monoclonal antibody.

[0042] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0043] In a second aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0044] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0045] (ii) optional tag sequences to assist in expression and / or purification.

[0046] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag. In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0047] In another preferred embodiment, the recombinant protein is a fusion protein.

[0048] In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against ADAM9), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0049] In another preferred embodiment, the bispecific or multispecific antibody not only targets ADAM9 but also specifically binds to additional target antigens (such as other tumor antigens, such as other antigens of gastric cancer or other tumor antigens).

[0050] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0051] In another preferred embodiment, the recombinant protein further includes an additional fusion element (or fusion polypeptide fragment) fused together with said element (i).

[0052] In a third aspect of the invention, a chimeric antigen receptor (CAR) is provided, wherein the antigen-binding domain of the chimeric antigen receptor contains an antibody single-chain variable region sequence scFv targeting ADAM9, wherein the heavy chain variable region and the light chain variable region of the scFv include the following complementarity-determining regions (CDRs):

[0053] The heavy chain variable region includes the following complementary determinant region (CDR):

[0054] VHCDR1, as shown in SEQ ID NO:6,

[0055] VHCDR2 shown in SEQ ID NO:7, and

[0056] VHCDR3 as shown in SEQ ID NO:8;

[0057] Furthermore, the light chain variable region includes the following complementary determinant region (CDR):

[0058] VLCDR1 shown in SEQ ID NO:2,

[0059] VLCDR2 shown in SEQ ID NO:3, and

[0060] VLCDR3 as shown in SEQ ID NO:4.

[0061] In a fourth aspect of the invention, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, or a chimeric antigen receptor (CAR) as described in the third aspect of the invention.

[0062] In a fifth aspect of the invention, a carrier is provided, said carrier containing the polynucleotide described in the fourth aspect of the invention.

[0063] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0064] In another preferred embodiment, the vector is a retroviral vector.

[0065] In a sixth aspect of the invention, a host cell is provided, wherein the host cell contains an exogenous polynucleotide as described in the fourth aspect of the invention integrated into a vector or chromosome as described in the fifth aspect of the invention.

[0066] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0067] In another preferred embodiment, the cell is a mammalian cell.

[0068] In another preferred embodiment, the cells are NK cells or T cells.

[0069] In another preferred embodiment, the host cell is an engineered immune cell.

[0070] In a seventh aspect of the invention, a method for preparing CAR-NK cells or CAR-T cells, wherein the CAR-NK cells or CAR-T cells express the chimeric antigen receptor described in the third aspect of the invention, includes the following steps:

[0071] The polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention is introduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0072] In an eighth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a chimeric antigen receptor as described in the third aspect of the invention, a polynucleotide as described in the fourth aspect of the invention, a carrier as described in the fifth aspect of the invention, or a host cell as described in the sixth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0073] In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0074] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0075] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a host cell or a combination thereof as described in the sixth aspect of the invention, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentages are percentages by mass of the pharmaceutical composition.

[0076] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of cancer or tumors.

[0077] In another preferred embodiment, the tumor is selected from: gastric cancer, pancreatic cancer, bladder cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0078] In another preferred embodiment, the tumor is selected from: gastric cancer, bladder cancer, and pancreatic cancer.

[0079] In a ninth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0080] (a) An antibody portion, said antibody portion being selected from the group consisting of: antibodies or antigen-binding fragments thereof as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, or combinations thereof; and

[0081] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0082] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0083] In a tenth aspect of the invention, the use of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a chimeric antigen receptor as described in the third aspect of the invention, a polynucleotide as described in the fourth aspect of the invention, a vector as described in the fifth aspect of the invention, or a host cell as described in the sixth aspect of the invention, a pharmaceutical composition as described in the eighth aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention is provided.

[0084] (a) Preparation of detection reagents or kits; and / or

[0085] (b) Prepare medicines or formulations for the prevention and / or treatment of ADAM9-related diseases.

[0086] In another preferred embodiment, the ADAM9-related disease is cancer or tumor.

[0087] In another preferred embodiment, the tumor is selected from: gastric cancer, pancreatic cancer, bladder cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0088] In an eleventh aspect of the present invention, a method for in vitro detection of ADAM9 protein in samples (including diagnostic or non-diagnostic samples) is provided, the method comprising the steps of:

[0089] (1) Contact the sample with the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention;

[0090] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of ADAM9 protein in the sample.

[0091] In another preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0092] In another preferred embodiment, the method is an immunochemical staning (ICC) detection method, an immunohistochemistry (IHC) detection method, a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0093] In a twelfth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0094] (a) Culturing the host cells described in the sixth aspect of the present invention under suitable expression conditions;

[0095] (b) Isolate a recombinant polypeptide from the culture, wherein the recombinant polypeptide is an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention.

[0096] In a thirteenth aspect of the present invention, a detection plate is provided, the detection plate comprising: a substrate (support plate) and a test strip, the test strip containing an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, an immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof.

[0097] In a fourteenth aspect of the invention, a kit is provided, the kit comprising:

[0098] (1) A first container containing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and / or

[0099] (2) A second container containing a secondary antibody against the antibody of the present invention;

[0100] Alternatively, the kit may contain the detection plate described in aspect thirteen of the present invention.

[0101] In a fifteenth aspect of the invention, a method for treating a disease associated with abnormal expression or function of ADAM9 is provided, comprising administering to a subject requiring treatment an effective amount of an antibody or antigen-binding fragment thereof as described in a first aspect of the invention, a recombinant protein as described in a second aspect of the invention, a host cell as described in a twelfth aspect of the invention, or a pharmaceutical composition as described in an eighth aspect of the invention.

[0102] In another preferred embodiment, the disease associated with abnormal ADAM9 expression or function is a tumor or cancer.

[0103] In a sixteenth aspect of the invention, the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention, or the recombinant protein as described in the second aspect of the invention, or the host cell as described in the twelfth aspect of the invention, and / or the pharmaceutical composition as described in the eighth aspect of the invention, in the preparation of a medicament for treating diseases associated with abnormal ADAM9 expression or function is provided.

[0104] In another preferred embodiment, the ADAM9 expression abnormality refers to ADAM9 overexpression.

[0105] In another preferred embodiment, the overexpression refers to the ratio of the expression level (F1) of ADAM9 to the expression level (F0) under physiological conditions (i.e., F1 / F0) being ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0106] In another preferred embodiment, the drug is used for the prevention and / or treatment of tumors.

[0107] In another preferred embodiment, the drug is used to prevent and / or treat tumor occurrence, growth and / or metastasis.

[0108] In another preferred embodiment, the tumor includes solid tumors and hematologic malignancies.

[0109] In another preferred embodiment, the tumor is a tumor that highly expresses ADAM9.

[0110] In another preferred embodiment, the tumor is selected from: gastric cancer, pancreatic cancer, bladder cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, cholangiocarcinoma, lung cancer, and non-small cell lung cancer.

[0111] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0112] Figure 1 The image shows the binding of antibodies with different numbers to the surface of ADAM9-positive cells as detected by cell flow cytometry; the positive cells were NCI-H1975 cells, SNU5 cells, and HPAF-II cells.

[0113] Figure 2 The results show the killing effect of antibody 267 and secondary antibody conjugated to toxic substances (MMAE or DXD) on tumor cells (SNU-5 or BXPC-3 cells) that highly express ADAM9 protein; where 2nd-DXD represents secondary antibody conjugated to toxic substance DXD; 2nd-MMAE represents secondary antibody conjugated to toxic substance MMAE, and 267 represents ADAM9 antibody numbered 267, with IgG serving as a negative control.

[0114] Figure 3 The results show the recognition of human and cynomolgus ADAM9 antigens by chimeric antibody ch67, with the horizontal axis representing different concentrations of chimeric antibody ch267. Detailed Implementation

[0115] Through extensive and in-depth research, the inventors unexpectedly discovered a new class of monoclonal antibodies against ADAM9. The antibodies provided by this invention can specifically recognize and bind to the ADAM9 protein, and mediate internalization after binding to ADAM9 protein expressed on the cell surface. The antibodies of this invention exhibit excellent binding activity against ADAM9 from multiple species. Furthermore, the antibodies of this invention can also mediate the specific killing of ADAM9-overexpressing cancer cells by immune cells through ADCC. Based on these findings, this invention was completed.

[0116] Specifically, this invention provides an antibody that binds to the ADAM9 protein and its applications. The antibody or its antigen-binding fragment described in this invention possesses one or more of the following properties: 1) it binds to the ADAM9 protein with high affinity and specificity; 2) it specifically recognizes the ADAM9 protein expressed on the cell surface; 3) it can be internalized by cells, especially tumor cells; 4) it can be used to construct immunoconjugates (ADC molecules), and these immunoconjugates can also inhibit the growth and / or proliferation of tumors or tumor cells. This invention also provides a method for preparing the antibody and its applications.

[0117] On one hand, the present invention provides a plurality of antibodies or antigen-binding fragments thereof, comprising a variable region VL of the antibody light chain and a variable region VH of the antibody heavy chain, wherein the VL comprises any one of VLCDR1, VLCDR2 and VLCDR3, and the VH comprises any one of VHCDR1, VHCDR2 and VHCDR3, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:1, and the VH comprises the amino acid sequence shown in SEQ ID NO:5.

[0118] In some embodiments, the antibody or its antigen-binding fragment possesses one or more properties selected from the group consisting of:

[0119] 1) It can specifically recognize the ADAM9 protein expressed on the cell surface;

[0120] 2) It can inhibit the proliferation of tumors and / or tumor cells;

[0121] 3) Human-mouse chimeric antibodies can bind to human and monkey ADAM9 antigens.

[0122] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and immunoconjugates (ADC molecules).

[0123] the term

[0124] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0126] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0127] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, comprising the antibody against ADAM9 of the present invention and compositions thereof, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount that effectively relieves the symptoms of one or more diseases (therapeutic effective amount).

[0128] As used herein, the terms “optional” or “optionally” mean that the events or circumstances described below may occur but are not required to occur.

[0129] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0130] As used herein, the term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0131] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0132] As used herein, the terms “antibody of the present invention”, “ADAM9 antibody”, “#267 antibody”, and “CL267” are used interchangeably and all refer to the monoclonal antibody numbered 267.

[0133] As used in this article, the terms "ADAM9 chimeric antibody" and "ch267" are used interchangeably, both referring to the cloning of the antibody variable region sequence into a vector containing the human Fc fragment, transfection into 293F cells for antibody expression, and further purification by Protein A to obtain the chimeric antibody.

[0134] As used herein, the term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0135] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0136] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0137] As used herein, the term "antigen-binding domain" and the like encompasses any naturally occurring, enzymatically available, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains, can be used, for example, deriving antigen-binding fragments of antibodies from intact antibody molecules. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, such as to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0138] As used herein, non-limiting examples of antigen-binding fragments or antigen-binding domains include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., independent complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0139] As used herein, antigen-binding fragments or antigen-binding domains will generally contain at least one variable domain. Variable domains can have any size or amino acid composition and will typically contain at least one CDR adjacent to or conforming to one or more frame sequences. L V of domain association H In the antigen-binding fragment of the domain, the VH and VL domains can be arranged opposite each other in any suitable configuration. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Optionally, the antigen-binding domain can contain monomeric VH or VL domains.

[0140] In a given antibody's light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia based on the antibody's three-dimensional structure and the topology of the CDR loop; Kabat (Kabat, E., et al., USDapartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); the International Immuno GeneTics database (IMGT); the EU numbering system; and Chothia definitions based on loop structural positions.

[0141] It should be understood that the precise amino acid sequence boundaries of the CDR in this invention can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system.

[0142] A distegrinin and a metalloprotease 9(ADAM9)

[0143] ADAM9 is a tumor-associated antigen highly expressed in many types of solid tumors and involved in carcinogenesis and tumor progression. In prostate cancer, ADAM9 cleaves insulin β-chain, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-α, gelatin, β-casein, etc., and induces the shedding of epidermal growth factor (EGF), fibroblast growth factor receptor 2 (FGFR2)-IIIB, and heparin-bound EGF-like growth factor. Therefore, ADAM9 induces the degradation of specific substrates, releasing active growth factors and interacting with key regulatory factors; its expression is involved in various cancer processes such as cell growth and invasion. For example, in gene expression synthesis (GEO) studies, bladder cancer with peripheral carcinoma and invasive carcinoma showed significantly high ADAM9 mRNA expression; ADAM9 knockdown attenuated cell migration in pancreatic cancer-associated Panc-1 and ASPC-1 cells. Therefore, it is an ideal target for targeted cancer therapy.

[0144] Antibody

[0145] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0146] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0147] This invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0148] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0149] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0150] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0151] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0152] Table A

[0153] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0154] As used in this article, the terms “ADCC” or “antibody-dependent cell-mediated cytotoxicity” include cell-mediated reactions in which nonspecific cytotoxic cells expressing FcγR recognize antibodies bound to target cells, causing target cell lysis. In various contexts, enhanced ADCC effector function can refer to enhanced titer or enhanced efficacy. In the experimental context, “titer” refers to the concentration of the antibody (half-maximum effective concentration) at which a specific therapeutic efficacy EC50 is observed. In the experimental context, “efficacy” refers to the maximum possible effector function of an antibody at saturation levels.

[0155] Preferably, the antibody described herein is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0156] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0157] The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0158] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0159] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0160] The animal is preferably a mammal, such as a mouse.

[0161] The antibodies of this invention can be chimeric antibodies, humanized antibodies, CDR-grafted and / or modified antibodies that target ADAM9 (e.g., human ADAM9, mouse ADAM9 or cynomolgus monkey ADAM9).

[0162] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0163] In the above-described content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0164] Recombinant protein

[0165] The present invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VHCDR1), heavy chain CDR2 (VHCDR2), and heavy chain CDR3 (VHCDR3) of the antibody of the present invention, and / or one or more of the light chain CDR1 (VLCDR1), light chain CDR2 (VLCDR2), and light chain CDR3 (VLCDR3) of the antibody of the present invention.

[0166] Preferably, the recombinant protein further includes an antibody heavy chain constant region and / or an antibody light chain constant region. The antibody heavy chain constant region is conventional in the art, preferably a rat-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and more preferably a human-derived antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat-derived antibody light chain constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.

[0167] In another preferred embodiment, the recombinant protein comprises the antibody of the present invention.

[0168] The recombinant protein is a conventional protein in the art, preferably one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies.

[0169] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0170] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0171] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0172] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0173] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from an expression transformant or obtaining it through artificial synthesis of the protein sequence. The method for isolating the protein from the expression transformant is preferably as follows: cloning a polynucleotide molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0174] Polynucleotides

[0175] The present invention also provides a polynucleotide encoding the above-described antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0176] The preparation method of the polynucleotide is a conventional preparation method in the art. Preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full-sequence synthesis.

[0177] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the protein sequence, while maintaining antibody activity.

[0178] carrier

[0179] The present invention also provides a recombinant expression vector containing the nucleic acid.

[0180] The recombinant expression vector described herein can be obtained by conventional methods in the art, namely, by linking the nucleic acid molecule described in this invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. Preferably, the vector includes various plasmids, granules, bacteriophages, or viral vectors, etc.

[0181] The present invention also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0182] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any common host cells in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells (expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0183] Antibody preparation

[0184] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0185] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0186] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0187] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0188] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0189] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0190] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0191] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0192] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0193] Antibody-drug conjugates (ADCs)

[0194] As used in this article, the terms “antibody-drug conjugate”, “ADC”, and “antibody-drug conjugate” are used interchangeably.

[0195] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0196] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0197] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0198] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0199] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0200] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0201] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0202] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0203] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0204] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0205] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0206] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0207] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula:

[0208]

[0209] in:

[0210] Ab is an antibody.

[0211] LU stands for connector;

[0212] D is a drug;

[0213] Furthermore, the subscript p is a value selected from 1 to 8.

[0214] DNA topoisomerase I inhibitors (DxD)

[0215] DxD is a potent inhibitor of DNA topoisomerase I and has attracted considerable attention in antibody-drug conjugates (ADCs) as a binding agent for HER2-targeting ADCs (DS-8201a). DxD exhibits cytotoxicity against human cancer cell lines KPL-4, NCI-N87, SK-BR-3, and MDA-MB-468, with IC50 values ​​ranging from 1.43 nM to 4.07 nM. However, the control IgG-ADC (DxD as the effective load) showed no inhibition against these four cell lines (HER2-expressing). DS-8201a (DxD as the effective load) showed significant inhibition against HER2-positive KPL-4, NCI-N87, and SK-BR-3 cell lines, with IC50 values ​​of 26.8, 25.4, and 6.7 ng / mL, respectively, but no such inhibitory effect was observed against MDA-MB-468 (IC50, >10,000 ng / mL). The CAS number for DxD is 1599440-33-1, and its molecular formula is C2. 26 H 24 FN3O6, molecular weight: 493.48, DxD structural formula is as follows:

[0216]

[0217] Methylaurestatin E (MMAE)

[0218] Methylaurestatin E (MMAE) is an antimitotic agent that inhibits cell division by blocking microtubule polymerization, and is of considerable interest among antibody-drug conjugates (ADCs). MMAE is derived from a polypeptide called dolastatin found in the marine shell-less mollusc Dolabella auricularia. These polypeptides have shown effective in vitro and in vivo activity against various lymphomas, leukemias, and solid tumors in preclinical studies. CAS RN: 474645-27-7; Molecular formula: C 39 H 67 N5O7; Molecular weight: 717.97858; The structural formula of MMAE is shown below:

[0219]

[0220] VcMMAE(mc-vc-PAB-MMAE)

[0221] VcMMAE is part of an antibody-coupled device (ADC) and possesses anticancer activity. It consists of MMAE (a tubulin inhibitor) and valine-citrulline (VC). The structural formula of VcMMAE is shown below:

[0222]

[0223] application

[0224] The present invention also provides the use of the antibodies, recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0225] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal ADAM9 expression or function.

[0226] In this invention, the diseases associated with abnormal ADAM9 expression or function are conventional diseases associated with abnormal ADAM9 expression or function in the art. Preferably, the diseases associated with abnormal ADAM9 expression or function are cancer.

[0227] In this invention, the cancer is a conventional cancer in the art, preferably gastric cancer, pancreatic cancer, bladder cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, endometrial cancer, endometrioid adenocarcinoma, bile duct cancer, lung cancer, and non-small cell lung cancer.

[0228] Detection uses and kits

[0229] The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0230] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, resected tumor samples, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0231] The samples used in this invention include fixed or preserved cell or tissue samples.

[0232] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0233] Pharmaceutical Composition

[0234] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0235] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0236] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0237] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal expression or function of ADAM9.

[0238] The pharmaceutical composition of the present invention can be directly used to bind ADAM9 protein molecules, and therefore can be used for the prevention and treatment of diseases such as tumors.

[0239] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0240] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0241] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0242] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0243] This invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal ADAM9 expression or function. Preferably, the disease associated with abnormal ADAM9 expression or function is a tumor / cancer.

[0244] The main advantages of this invention include:

[0245] (1) The antibody or its antigen-binding fragment of the present invention can specifically recognize the ADAM9 protein expressed on the cell surface;

[0246] (2) The antibody or its antigen-binding fragment of the present invention can inhibit the proliferation of tumors and / or tumor cells;

[0247] (3) The antibody or its antigen-binding fragment of the present invention can be internalized by cells, especially tumor cells;

[0248] (4) The antibodies or antigen-binding fragments of the present invention can be used to construct immune conjugates (ADC molecules), and the immune conjugates can also inhibit the growth and / or proliferation of tumors or tumor cells;

[0249] (5) The human-mouse chimeric antibody of the present invention can bind to human and monkey ADAM9 antigens.

[0250] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0251] Example 1: Preparation of mouse monoclonal antibody hybridoma cells containing ADAM9 protein

[0252] 1.1 Preparation of antigen

[0253] A polypeptide fragment (Val 760-Asn 771) from the intracellular region of ADAM9 was used as the immunogen. This sequence is present in both human and monkey ADAM9 proteins, and the antigen was purchased from Kaijing Biotechnology.

[0254] 1.2 Immunization of mice

[0255] The antigen will be used to immunize 12 Balb / c mice (8-12 weeks old), and their serum titers will be monitored to determine the optimal number of immunizations. Optimized adjuvants and immunization methods generate high-affinity antibodies (IgG subtype) against most antigenic peptides. After the primary immunization, there will be 3 to 4 booster immunizations. After each booster, mouse serum titers will be measured (recombinant protein coated as anti-antigen). Mice with acceptable titers will be given one dose and used for fusion, while mice with unacceptable titers will receive one to two more boosters until the highest titer is achieved, at which point fusion will take place.

[0256] 1.3 Serum detection and screening

[0257] Blood was collected from the orbital sinus of immunized mice, and serum titers were detected by ELISA (recombinant protein was used as the antigen coating). The serum titer must be greater than 10K; otherwise, booster immunization should be performed.

[0258] 1.4 Fusion and Screening

[0259] The entire spleen and half of the lymph nodes were harvested and fused with myeloma SP2 / 0 cell lines. The fusion process used was an optimized PEG fusion method. The fused cells were seeded onto four 384-well plates (10 cells per well). 2 Up to 10 4Cells were cultured. Supernatants from all wells were collected, and positive cell lines were screened by detecting the reaction of the cell supernatants to the ADAM9 recombinant protein using ELISA. Positive wells containing cells were transferred to 96-well plates for further culture. After several days of growth, supernatants from all wells were collected, and the reactivity of the cell supernatants to the ADAM9 recombinant protein was detected by ELISA. The binding affinity of different dilutions of cell supernatants to the ADAM9 recombinant protein in the positive wells was further analyzed to rank the cell lines by affinity. The 36 parental clones with the highest affinity were selected for subcloning.

[0260] 1.5 Subcloning and Screening

[0261] Subcloning was performed using limiting dilution and ELISA to obtain monoclonal hybridoma cells. Cells were seeded into 96-well plates and cultured until approximately 1 / 6 of the bottom was covered. ELISA was used to detect the reaction of the supernatant in each well to the ADAM9 recombinant protein. Two wells with high OD values ​​and good cell condition were selected for the next round of subcloning. The above steps were repeated until the cell line positivity rate in the wells reached 100%. At this point, monoclonal cell lines were obtained. After the final round of subcloning, all positive cells (hybridoma cells) were immediately expanded into larger cultures; some were cryopreserved for later use, and the rest were used for ascites fluid preparation.

[0262] 1.6 Ascites preparation and antibody purification

[0263] Five monoclonal cell lines were ultimately obtained and injected into F1 mice via abdominal injection for antibody production. The resulting ascites fluid was purified using Protein A / G and used for subsequent assays.

[0264] Example 2: Antibody specifically recognizes ADAM9 antigen expressed on cell surface

[0265] Using NCI-H1975 cells (human lung adenocarcinoma cells), SNU5 cells (human gastric cancer cells), and HPAF-II (human pancreatic cancer cells) as positive cells, flow cytometry was used to analyze the binding of ADAM9 on the cell surface to five ADAM9 monoclonal antibodies. Cells in logarithmic growth phase were collected, suspended in blocking buffer (1×PBS containing 10% goat serum), and blocked at 4°C for 30 min. Then, 50 μl of cells were added to 96-well plates, and the cell density was adjusted to 2 × 10⁶ cells / well. 5Cells / well were incubated with an equal volume of five 20 μg / ml ADAM9 monoclonal antibodies diluted with blocking buffer at 4°C for 1 h. After incubation, cells were washed twice with 1×PBS (1500 rpm, 5 min) and the supernatant was discarded. Secondary antibody (goat anti-mouse IgG, ImmunoResearch) was diluted with blocking buffer at a ratio of 1:1000, and 100 μL of the solution was resuspended in the cells. Cells were incubated at 4°C in the dark for 45 min. After incubation, cells were washed twice with 1×PBS (1500 rpm, 5 min). Cells were resuspended in 100 μL of 1×PBS. Flow cytometry (BD Calibur) was used to analyze the binding affinity of the antibodies to cell surface antigens.

[0266] The results are as follows Figure 1 As shown, antibody number 267 can specifically recognize NCI-H1975 cells (human lung adenocarcinoma cells), SNU5 cells (human gastric cancer cells), and HPAF-II (human pancreatic cancer cells); antibody number 276 can specifically recognize HPAF-II (human pancreatic cancer cells).

[0267] Example 3: In vitro tumor-inhibiting activity of ADAM9 antibody

[0268] After linking goat anti-mouse secondary antibodies to the toxic substance MMAE or DXD, cell killing assays were used to verify whether the ADAM9 antibody could recognize and bind to tumor cell surface antigens in vitro, thereby inhibiting tumor activity.

[0269] 3.1 Preparation of toxic substances linked to goat anti-mouse secondary antibodies

[0270] First, the disulfide bonds in the structure of the goat anti-mouse secondary antibody (Goat anti-mouse IgG, purchased from Abdominal Pharmaceuticals (Shanghai) Co., Ltd.) were opened using the reducing agent TCEP (Thermo#20490). Then, the small molecule drug MMAE (VcMMAE, HY-15575 in this example) or DXD was added to the reduced goat anti-mouse secondary antibody at a ratio of goat anti-mouse secondary antibody:drug = 1:14 for DAR8 conjugation. The unconjugated free small molecules were removed by a salt removal column and activated carbon to obtain the secondary antibody conjugated drug (2nd-MMAE or 2nd-DXD). Among them, IgG represents the negative control antibody conjugated drug.

[0271] 3.1.1 Buffer Adjustment

[0272] The pH of the ch267 antibody was adjusted by adding prepared PBS (50mM phosphate + 2mM EDTA, pH=7.0).

[0273] 3.1.2 Reduction of disulfide bonds

[0274] Add antibody, TCEP, 5 / 10xPB and dimethyl methacrylate (DMDM) to a 1.5 mL centrifuge tube, mix well, and incubate at 37 °C for 2 h for reduction.

[0275] 3.1.3 Conjugation of small molecule drugs

[0276] After reduction in a 37℃ constant temperature incubator, place it on ice to cool for 10 minutes.

[0277] First, add the DMA organic reagent (at room temperature) on ice, then add 10-15x of the small molecule drug and mix thoroughly.

[0278] The coupling was performed by placing the sample on a shaker at 22℃ (room temperature) for 1 hour.

[0279] 3.1.4 Desalination

[0280] First, equilibrate the desalting column using 1xFB. Desalinate the experimental sample twice using the equilibrated desalting column. If the sample size is small in the second run, less than the desalting range of the column, a small amount of FB can be added.

[0281] 3.1.5 Activated carbon adsorption of free small molecule drugs

[0282] Dissolve 300 mg of activated carbon in 1 mL of water / FB and add it to the sheep anti-mouse secondary antibody-linked toxic substance system at a volume ratio of 10% (if adding powder directly, add 30 mg of activated carbon per 1 mL of sample).

[0283] Rotate at 22℃ or room temperature for 1–2 hours;

[0284] Centrifuge and aspirate the supernatant (subsequent experiments will be conducted in a clean bench).

[0285] 3.1.6 Filter Installation

[0286] The sample was filtered into a liquid chromatography vial containing a liner using a 0.22 μM filter on a clean bench.

[0287] HPLC analysis was performed on the samples. The DAR value of the goat anti-mouse secondary antibody-linked toxic substance was determined using a HIC column, and the degree of polymerization and free small molecular weight of the goat anti-mouse secondary antibody-linked toxic substance were determined using a SEC column.

[0288] 3.1.7 Concentration Determination

[0289] The concentration of toxic substances linked to goat anti-mouse secondary antibodies was determined using the BCA method.

[0290] 3.2 In vitro tumor suppression test

[0291] SNU5 cells (human gastric cancer cells) and BXPC-3 cells (human orthotopic pancreatic adenocarcinoma cells) were cultured in 96-well cell culture plates, 100 μl per well. 50 μl of supernatant was discarded, and 50 μl of ADAM9 monoclonal antibody #267 diluted to 11 concentrations (300,000 pM, 100,000 pM, 30,000 pM, 10,000 pM, 3,000 pM, 1,000 pM, 300 pM, 100 pM, 30 pM, 10 pM, 3 pM) and negative control IgG were added sequentially. Then, 50 μl of each solution was added to a final concentration of 4 μg / ml. Add 2nd-MMAE or 2nd-DXD, gently pat to mix, and continue culturing in a 37℃ incubator for 4 days, observing cell killing activity daily during this period; after 4 days, remove the cell culture plate, discard the culture medium, add 100μl of 10-fold diluted CCK8 to each well, incubate at 37℃ for 0.5-2 hours, and then measure the absorbance at 450nm.

[0292] Table 1

[0293]

[0294] Note: In the table, 267 antibody refers to ADAM9 monoclonal antibody #267; IgG antibody refers to negative control antibody; SNU-5vs2nd-DXD refers to the cell viability test results after adding different concentrations of 267 antibody or IgG antibody to SNU-5 cells, followed by the addition of the secondary antibody-drug conjugate 2nd-DXD; SNU-5vs2nd-MMAE refers to the cell viability test results after adding different concentrations of 267 antibody or IgG antibody to SNU-5 cells, followed by the addition of the secondary antibody-drug conjugate 2nd-MMAE; similarly, BXPC-3vs2nd-DXD and BXPC-3vs2nd-MMAE are as described above.

[0295] The results are shown in Table 1 and Figure 2 As shown, ADAM 9 antibody (267) exhibits the ability to inhibit tumor activity in vitro by binding to tumor cell surface antigens. Specifically, in the results of SNU-5 cell killing with the secondary antibody conjugate 2nd-MMAE, antibody 267 showed approximately 20% cell killing at a concentration of 3 nM (3000 pM) and approximately 85% cell killing at a concentration of 300 nM (300000 pM). In the results of BXPC-3 cell killing with the secondary antibody conjugate 2nd-MMAE, antibody 267 showed approximately 20% cell killing at a concentration of 3 nM (3000 pM) and approximately 65% ​​cell killing at a concentration of 300 nM (300000 pM). Furthermore, in the results of SNU-5 cell killing with the secondary antibody conjugate 2nd-MMAE, 267 at the same concentration showed no significant killing effect on SNU-5 cells.

[0296] The above results suggest that the antibodies and ADCs of the present invention can be endocytosed by binding to the ADAM 9 receptor on the cell surface, thereby exerting the killing effect of ADC drugs in a targeted and efficient manner.

[0297] Example 4: Antibody Variable Region Sequencing

[0298] Hybridoma cells containing antibody number 267 were selected for expanded culture. After Trizol lysis, cellular RNA was extracted and reverse transcribed to obtain cDNA. The variable region of the antibody was amplified using specific degenerate primers and cloned into a T-plasmid. Sequencing was performed using SP6 primers.

[0299] The amino acid sequence of the antibody light chain variable region VL is as shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region VH is as shown in SEQ ID NO:5.

[0300] Example 5: Preparation of chimeric antibodies

[0301] Preparation of ADAM9 chimeric antibody

[0302] Using the variable region sequence obtained from sequencing in Example 4 above, recombinant plasmids were constructed by ligating them with human heavy / light chain Fc fragments. After obtaining the heavy / light chain plasmids, DNA / PEI transfection complexes were prepared in equal quantities. The complexes were transfected into 293F cells for culture. Five days after transfection, the cell supernatant was collected and purified using Mabselect SuRe to obtain the ADAM9 chimeric antibody ch267.

[0303] Example 6: Chimeric antibodies recognize human and monkey ADAM9 antigens respectively.

[0304] ELISA was used to analyze whether the antibody recognized the human and cynomolgus ADAM9 antigen. First, 1 μg / mL of human and cynomolgus ADAM9 protein were coated onto 96-well plates. A gradient concentration of ADAM9 chimeric antibody ch267 (500 ng / ml, 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, 15.625 ng / ml, 7.8125 ng / ml, 3.90625 ng / ml, 1.953125 ng / ml, 0.9765625 ng / ml, 0.48828125 ng / ml, 0.244140625 ng / ml) was loaded. After washing and incubation with secondary antibody (goat anti-mouse HRP secondary antibody, Jackson), the plates were washed again, and chromogenic and stop solutions were added. The absorbance at 450 nm was measured using a microplate reader.

[0305] Table 2

[0306]

[0307]

[0308] The results are shown in Table 2 and Figure 3 As shown, the ADAM9 chimeric antibody ch267 can not only recognize human ADAM9 antigens, but also specifically recognize monkey ADAM9 antigens; it has excellent human-monkey cross-reactivity; and it is helpful to use monkeys as a model for performance verification when developing antibody drugs.

[0309] The sequences involved in this invention are shown in Table 3 below:

[0310] Table 3

[0311]

[0312]

[0313] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof targeting ADAM9, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region include the following complementarity-determining regions (CDRs): The heavy chain variable region includes the following complementary determinant region (CDR): VHCDR1, as shown in SEQ ID NO:6, VHCDR2 shown in SEQ ID NO:7, and VHCDR3 as shown in SEQ ID NO:8; Furthermore, the light chain variable region includes the following complementary determinant region (CDR): VLCDR1 shown in SEQ ID NO:2, VLCDR2 shown in SEQ ID NO:3, and VLCDR3 as shown in SEQ ID NO:

4.

2. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the antibody or antigen-binding fragment thereof as described in claim 1; (ii) optional tag sequences to assist in expression and / or purification.

3. A chimeric antigen receptor CAR, characterized in that, The antigen-binding domain of the chimeric antigen receptor contains an antibody single-chain variable region sequence scFv targeting ADAM9, wherein the heavy chain variable region and light chain variable region of the scFv include the following complementarity-determining regions (CDRs): The heavy chain variable region includes the following complementary determinant region (CDR): VHCDR1, as shown in SEQ ID NO:6, VHCDR2 shown in SEQ ID NO:7, and VHCDR3 as shown in SEQ ID NO:8; Furthermore, the light chain variable region includes the following complementary determinant region (CDR): VLCDR1 shown in SEQ ID NO:2, VLCDR2 shown in SEQ ID NO:3, and VLCDR3 as shown in SEQ ID NO:

4.

4. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 2, or the chimeric antigen receptor CAR as described in claim 3.

5. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, The host cell contains the vector of claim 5 or the chromosome in which the exogenous polynucleotide of claim 4 is integrated.

7. A method for preparing CAR-NK cells or CAR-T cells, characterized in that, The CAR-NK cells or CAR-T cells expressing the chimeric antigen receptor of claim 3 include the following steps: The polynucleotide of claim 4 or the vector of claim 5 is introduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as claimed in claim 1, the recombinant protein as claimed in claim 2, the chimeric antigen receptor as claimed in claim 3, the polynucleotide as claimed in claim 4, the carrier as claimed in claim 5, or the host cell as claimed in claim 6, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. An immunoconjugate comprising: (a) Antibody portion, wherein the antibody portion is selected from the group consisting of: The antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 2, or a combination thereof; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

10. Use of an antibody or antigen-binding fragment thereof as claimed in claim 1, a recombinant protein as claimed in claim 2, a chimeric antigen receptor as claimed in claim 3, a polynucleotide as claimed in claim 4, a vector as claimed in claim 5, a host cell as claimed in claim 6, a pharmaceutical composition as claimed in claim 8, or an immunoconjugate as claimed in claim 9, characterized in that, (a) Preparation of detection reagents or kits; and / or (b) Prepare medicines or formulations for the prevention and / or treatment of ADAM9-related diseases.

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