Anti-adam9 antibody-drug conjugates and uses thereof

Abstract

The present application relates to an anti-ADAM9 antibody-drug conjugate and use thereof. The anti-ADAM9 antibody-drug conjugate provided by the present application has good binding and endocytosis ability to ADAM9 positive cells, excellent in-vitro physicochemical properties, and good killing effect in-vivo and in-vitro, and has good drug development prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody that specifically binds to ADAM9 or its antigen-binding fragment, as well as antibody-drug conjugates conjugated with a drug, and their tautomers, stereoisomers, or pharmaceutically acceptable salts. This invention also relates to a method for preparing an anti-ADAM9 antibody or its antigen-binding fragment, a nucleic acid molecule encoding the anti-ADAM9 antibody or its antigen-binding fragment, a vector containing the nucleic acid molecule, a host cell; and compositions containing the anti-ADAM9 antibody or its antigen-binding fragment, or antibody-drug conjugates, and their tautomers, stereoisomers, or pharmaceutically acceptable salts. This invention further relates to the use of the anti-ADAM9 antibody or its antigen-binding fragment, antibody-drug conjugates, and their tautomers, stereoisomers, or pharmaceutically acceptable salts, and compositions containing them. Background Technology

[0002] Studies have shown that ADAM9 is abnormally overexpressed in various malignant tumors, including lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, and kidney cancer. ADAM9 overexpression is closely associated with increased tumor invasiveness, increased risk of metastasis, immunosuppression in the tumor microenvironment, and poor patient prognosis. For example, in lung cancer, ADAM9 overexpression can shorten overall survival and participate in brain metastasis by promoting the adhesion of tumor cells to vascular endothelial cells. In prostate cancer, over 60% of recurrent tumors show elevated ADAM9 protein expression, which is associated with low recurrence-free survival. Given the differential expression of ADAM9 in tumor tissues and its limited expression in normal tissues, this target has become one of the popular targets for antibody-drug conjugates (ADCs).

[0003] Developing antibody-drug conjugates with anti-ADAM9 antibodies as the target carrier to achieve precise treatment of ADAM9-positive tumors has important clinical significance and application value. Summary of the Invention

[0004] The purpose of this invention is to provide an antibody or antigen-binding fragment thereof that has a strong binding affinity to ADAM9, and based on the antibody or fragment thereof, to provide anti-ADAM9 antibody-drug conjugates and their uses.

[0005] On the one hand, this invention provides an antibody or antigen-binding fragment thereof that has binding specificity to detegrin-like metalloproteinase 9 (ADAM9). The anti-ADAM9 antibody or antigen-binding fragment of this invention exhibits many excellent properties, including high affinity and specificity in binding to human ADAM9 protein, as well as good endocytic activity and inhibition of ADAM9 protease activity, showing promising drug development potential.

[0006] The present invention provides an anti-ADAM9 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VH CDR1, VH CDR2 and VH CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively, and the light chain variable region comprises VL CDR1, VL CDR2 and VL CDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively.

[0007] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and spaced framework regions (FRs), with the domains arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0008] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is of animal origin, such as mouse origin, or is a chimeric antibody; preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 6. In some specific embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6.

[0009] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the heavy chain variable region comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising a heavy chain FR region of human IgG1, IgG2 or IgG4; and the light chain variable region comprises a light chain FR region derived from human κ chain, λ chain or its variants, preferably comprising a light chain FR region derived from human κ chain.

[0010] In some embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 11-16, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences shown in SEQ ID NO: 11-16; the light chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 17-23, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the amino acid sequences shown in SEQ ID NO: 17-23.

[0011] In some specific embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein: (1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; (2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 18, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 18. (3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 19, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. (4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17. (5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 20, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; (6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 14, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 21, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; (7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 22; or (8) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 16, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 23, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 23.

[0012] In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 17. In other preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 18. In other preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19. In other preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 17. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22. In some preferred embodiments, the anti-ADAM9 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.

[0013] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region.

[0014] In some embodiments, the antibody or its antigen-binding fragment comprises a human or mouse constant region, preferably a human or mouse heavy chain constant region and / or a light chain constant region; preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, and / or a light chain constant region of human κ, λ chains or variants thereof.

[0015] In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 5. In one embodiment, the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0016] It should be understood that sequence variants of these constant region structural domains may also be used, for example, to contain one or more amino acid modifications, where the amino acid sites are identified by the EU indexing system of Kabat et al. (1991).

[0017] In some embodiments, the antibody is any form of monoclonal antibody (including full-length monoclonal antibody), polyclonal antibody, multispecific antibody (e.g., bispecific antibody), bifunctional antibody, murine antibody, fully or partially humanized antibody, or chimeric antibody. In one specific embodiment, the antibody is a monoclonal antibody. In a preferred embodiment, the antibody is a chimeric antibody. In another preferred embodiment, the antibody is a humanized antibody.

[0018] In some implementations, the antibody is a full-length antibody.

[0019] In some embodiments, the anti-ADAM9 antibody of the present invention is a complete antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody. In other embodiments, the anti-ADAM9 antibody of the present invention covers only its antigen-binding portion, such as: Fab, Fab', Fd, Fd', Fv, dAb, F(ab')2, scFv, diabodies, linear antibodies, or dsFv.

[0020] In some embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody, such as Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, BsFv, dsFv, (dsFv)2, or dAb.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain.

[0022] On the other hand, the present invention provides a biomaterial comprising: (i) A nucleic acid molecule encoding the anti-ADAM9 antibody or its antigen-binding fragment as described in this invention; (ii) a vector comprising the nucleic acid molecule described in (i); and / or (iii) A host cell comprising the nucleic acid molecule described in (i) and / or the vector described in (ii), or the host cell being transformed or transfected by the nucleic acid molecule described in (i) and / or the vector described in (ii).

[0023] In some embodiments, the nucleic acid molecule may be an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule is DNA, such as cDNA, genomic DNA, or recombinant DNA. In other embodiments, the nucleic acid molecule is RNA, such as mRNA or hnRNA. Those skilled in the art, based on the anti-ADAM9 antibody or its antigen-binding fragment disclosed in this application, can deduce the DNA or RNA sequence encoding the amino acid sequence and configure suitable expression elements thereon, enabling the DNA or RNA molecule to express the antibody or its antigen-binding fragment of the present invention.

[0024] In some implementations, the vector is an expression vector, such as a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector.

[0025] In some embodiments, the host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is another cell suitable for preparing antibodies or their antigen-binding fragments.

[0026] On the other hand, the present invention provides a method for preparing the anti-ADAM9 antibody or its antigen-binding fragment as described herein, comprising: (1) Chemical synthesis method: prepared by synthesizing the amino acid sequence of the anti-ADAM9 antibody or its antigen-binding fragment according to the present invention; (2) Bio-preparation method: culturing the host cells as described above, and optionally isolating and / or purifying the antibodies from the obtained culture.

[0027] On the other hand, the present invention provides an antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, wherein the antibody-drug conjugate comprises: (a) Anti-ADAM9 antibody or its antigen-binding fragment; and (b) Camptothecin-like toxin load; The anti-ADAM9 antibody or its antigen-binding fragment is conjugated to the camptothecin toxoid via a linker; The anti-ADAM9 antibody or its antigen-binding fragment is the anti-ADAM9 antibody or its antigen-binding fragment described in various aspects of this document.

[0028] The antibody-drug conjugates and their tautomers, stereoisomers or pharmaceutically acceptable salts provided by this invention have excellent thermodynamic stability, plasma stability and antitumor effects. In multiple CDX models, the antibody-drug conjugates and their tautomers, stereoisomers or pharmaceutically acceptable salts showed significant antitumor efficacy at 1 mpk, and the body weight fluctuation was stable at high doses. Therefore, they have good application prospects in the treatment of ADAM9 positive tumors.

[0029] In some embodiments, the camptothecin toxin payload comprises a structure as shown in formula (I):

[0030] Formula (I); in, Indicates the site connected to the connector.

[0031] In some embodiments, the connector has a structure as shown in formula (II) or formula (III):

[0032] Equation (II)

[0033] Formula (III); Among them, end a The b-terminus represents the site where the anti-ADAM9 antibody or its antigen-binding fragment is linked. This indicates the site that is connected to the camptothecin-like toxin load.

[0034] In some embodiments, the linker and the camptothecin toxin load form a structure as shown in formula (VI) or formula (VII):

[0035] Formula (VI)

[0036] Equation (VII); in, This indicates the site where the anti-ADAM9 antibody or its antigen-binding fragment is linked.

[0037] In some embodiments, the antibody-drug conjugate has a structure as shown in formula (IV) or formula (V):

[0038] Formula (IV)

[0039] Formula (V); Wherein, Ab is the anti-ADAM9 antibody or its antigen-binding fragment, and n is an integer selected from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) or a decimal (e.g., 1.2, 1.5, 1.8, 2.4, 2.6, 2.9, 3.5, 3.7, 4.1, 4.4, 4.8, 5.2, 5.6, 5.9, 6.1, 6.3, 6.7, 7.2, 7.6, 7.9); where, when n is an integer, it represents the molar ratio of camptothecin to the anti-ADAM9 antibody or its antigen-binding fragment (also known as DAR, i.e., drug-antibody conjugation ratio); when n is a decimal, it represents the average number of linkers-camptothecin toxin loads conjugated to each of the anti-ADAM9 antibody or its antigen-binding fragments; preferably, n is 8.

[0040] On the other hand, the present invention provides a composition comprising the anti-ADAM9 antibody or its antigen-binding fragment thereof, the antibody-drug conjugate or its tautomer, stereoisomer or pharmaceutically acceptable salt thereof, and / or the biological material thereof, and optionally a pharmaceutically acceptable carrier or excipient.

[0041] In some embodiments, the composition is a pharmaceutical composition.

[0042] On the other hand, the present invention provides the use of the anti-ADAM9 antibody or its antigen-binding fragment, the antibody-drug conjugate or its tautomer, stereoisomer or pharmaceutically acceptable salt, the biomaterial or the composition of the present invention in the preparation of a medicament for treating tumor diseases.

[0043] In some implementations, the tumor disease is selected from at least one of the group consisting of gastric cancer, lung cancer, colorectal cancer, prostate cancer, breast cancer, and pancreatic cancer.

[0044] In some embodiments, the anti-ADAM9 antibody of the present invention or its antigen-binding fragment, antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, biological materials (including nucleic acid molecules, carriers, host cells) and / or compositions of the present invention may also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for the treatment of the related oncological diseases mentioned herein.

[0045] On the other hand, the present invention provides a method for treating tumor diseases, comprising administering to a subject in need a prophylacticly effective amount or a therapeutically effective amount of the anti-ADAM9 antibody or its antigen-binding fragment thereof, the antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the biological material (including nucleic acid molecules, carriers, host cells) or the composition thereof. Preferably, the subject is a mammal; more preferably, the subject is a human.

[0046] On the other hand, the present invention provides the anti-ADAM9 antibody or its antigen-binding fragment thereof, the antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts thereof, the biological material (including nucleic acid molecules, carriers, host cells) or the composition thereof, which are used as drugs or for treatment.

[0047] In some implementations, the drug is a drug for treating tumor diseases.

[0048] On the other hand, the present invention provides a kit comprising the anti-ADAM9 antibody or its antigen-binding fragment as described in the present invention, the antibody-drug conjugate and its tautomers, stereoisomers or pharmaceutically acceptable salts, the biological material (including nucleic acid molecules, carriers, host cells) or the composition thereof.

[0049] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims. Attached Figure Description

[0050] Figure 1 Affinity detection results of humanized antibody and hADAM9-his protein.

[0051] Figure 2A and Figure 2B Results of humanized antibody binding to cells.

[0052] Figure 3 Results of endocytic activity of humanized antibodies on NCI-H1703 cells.

[0053] Figure 4 Results of the detection of the binding activity of humanized antibodies with ADAM9 family proteins ADAM8, ADAM10, ADAM15, and ADAM17.

[0054] Figure 5 Results of detection of the inhibitory effect of humanized antibody on the enzyme activity of ADAM9 protein.

[0055] Figure 6 Results of assay for the activity of humanized antibodies in inhibiting cell protein shedding.

[0056] Figure 7A and Figure 7B DAR value detection results of conjugate 12-2-MSP-CPT2C.

[0057] Figure 8 sec-HPLC detection results of conjugate 12-2-MSP-CPT2C.

[0058] Figure 9A and Figure 9B DAR value detection results of conjugate 12-2-Mc-CPT2C.

[0059] Figure 10 sec-HPLC detection results of conjugate 12-2-Mc-CPT2C.

[0060] Figure 11A and Figure 11B DAR value detection results of the conjugate IC-1-Mc-CPT2C.

[0061] Figure 12 : sec-HPLC detection results of the conjugate IC-1-Mc-CPT2C.

[0062] Figure 13 DAR value detection results of the conjugate PC-1-Exatecan.

[0063] Figure 14 : sec-HPLC detection results of the conjugate PC-1-Exatecan.

[0064] Figure 15 Results of plasma stability assay using the conjugate free payload method.

[0065] Figure 16 Results of plasma stability determination using the DAR value method for conjugates.

[0066] Figure 17 Results of binding experiments between the conjugate and SNU5 cells.

[0067] Figure 18A and Figure 18B Results of endocytosis experiments of conjugates in SNU5 cells.

[0068] Figure 19 Results of in vitro killing experiments of the conjugate on CLAU3 cells.

[0069] Figure 20Results of in vitro killing experiments of the conjugate on NCI-1703 cells.

[0070] Figure 21 Results of ADCC effect experiment using NCI-H1975 cells as target cells.

[0071] Figure 22 Results of ADCC effect experiment using SNU5 cells as target cells.

[0072] Figure 23 Results of drug efficacy test on the CDX model of NCI-H1975 (human lung adenocarcinoma cells).

[0073] Figure 24 Results of drug efficacy trials on the SNU-5 (human gastric cancer cells) CDX model.

[0074] Figure 25 Results of drug efficacy trial in the NUGC-4 (human gastric cancer cell) CDX model.

[0075] Figure 26 Results of drug efficacy trial of LS174T (human colorectal cancer cells) CDX model. Detailed Implementation

[0076] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims. 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. Unless otherwise stated, the invention will be practiced using conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of this art. For the purposes of this invention, the following terms are defined below.

[0077] Abbreviations and Definitions Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields.

[0078] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0079] The amino acid three-letter codes and single-letter codes used in this invention are as described in J.biol.chem, 243, p3558 (1968).

[0080] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0081] As used herein, the term "antibody" or "immunoglobulin" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (knO) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The heavy chain consists of a variable region (VH) and a constant region (CH). The constant region consists of three domains (CH1, CH2, and CH3). The light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of one domain, CL. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy / light chain pairs (VH and VL) form the antigen-binding sites.

[0082] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are primarily responsible for binding to the antigen. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as those defined in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16;9:2278), etc. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system.

[0083] Unless otherwise stated, the variable region and CDR sequence in the embodiments of the present invention are governed by the "Kabat" numbering rule. Although a numbering system (such as Kabat) is used to define amino acid residues in a specific implementation, the corresponding technical solutions of other numbering systems are considered equivalent.

[0084] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody, other than the CDR residues as defined above. The term "antibody" is not limited to any particular method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0085] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment containing a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab' F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragment, scFv, comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Exemplary examples include: (i) a Fab fragment having VL, CL, VH, and CH1 domains and a disulfide bond between the heavy and light chains; (ii) a Fab' fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment having VH and CH1 domains; (iv) an Fd' fragment having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment having VL and VH domains of one arm of the antibody; (vi) a dAb fragment consisting of a VH domain; and (vii) a hingeless antibody comprising at least VL, VH, and C1 domains. (viii) F(ab)2 fragment, which is a bivalent fragment containing two Fab' fragments linked by a disulfide bridge in the hinge region; (ix) Single-chain antibody molecule (scFv) refers to a molecule containing an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH; (x) "Diabodies" have two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) linked together in the same polypeptide chain; (xi) "Linear antibody" contains a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions; (xii) dsFv refers to a fragment formed by replacing one amino acid residue in VH and VL with a cysteine ​​residue and then linking the polypeptides together via SS bonds between those cysteine ​​residues.

[0086] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0087] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions.

[0088] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0089] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any particular method.

[0090] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0091] The monoclonal antibodies of this invention can be prepared using a variety of techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al., Nature 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0092] Antibodies can be purified using known techniques, such as affinity chromatography with protein A or protein G. Subsequently, or alternatively, the specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and purified by immunoaffinity chromatography to achieve immunospecific antibody purification. For purification of immunoglobulins, see, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0093] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is called the "donor," and the immunoglobulin providing the framework is called the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework or a sequence having approximately 85%, 90%, 95%, 99%, or higher identity compared to it. Humanized antibodies typically retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, and reactivity. Donor antibodies can be mouse, rat, rabbit, or non-human primate antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0094] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. Based on the given nucleic acid sequence, those skilled in the art can easily construct a suitable vector that enables the nucleic acid to be replicated or expressed.

[0095] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0096] The term "antigen" refers to a molecule or molecular moiety that can be selectively recognized by antigen-binding protein molecules (such as antibodies) or bound by a binding agent. An antigen may have one or more epitopes that can interact with different antigen-binding protein molecules (such as antibodies).

[0097] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 The equilibrium dissociation constant (K) of M or less D ( ) binds to antigens or epitopes. In some embodiments, the antibody binds to the K antigen. D This antibody binds to the K+ of non-specific antigens (such as BSA, casein). D 10% or less (e.g., 1%). K can be measured using known methods. DFor example, measurements can be taken using FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or its epitope may be cross-reactive to other related antigens, such as to corresponding antigens from other species (homologous) (e.g., humans or monkeys, such as the cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno) and the chimpanzee (Pan troglodytes) (chimpanzee, chimp)) or the common marmoset (Callithrix jacchus) (commonmarmoset, marmoset).

[0098] The term "non-binding" means that the antibody cannot bind to an antigen or its epitope within that antigen in the manner described above for specific binding. For example, when the antibody binds at approximately 1 × 10⁻⁶... -6 M or a larger equilibrium dissociation constant (K) D () Binds to antigens or their epitopes within antigens.

[0099] The terms “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical analogs of naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise stated, a particular peptide sequence also implicitly encompasses variants with conserved modifications.

[0100] The term "sequence identity" refers to the degree (percentage) to which two sequences share the same amino acids / nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0101] The antibodies or antigen-binding fragments thereof of the present invention may contain amino acid mutations and / or conserved modifications. The antibodies or antigen-binding fragments thereof of the present invention also include sequences having more than 95% identity with the specifically provided amino acid sequences. Furthermore, the antibodies or antigen-binding fragments thereof of the present invention also include variants thereof.

[0102] The sequence “variant” used in this article refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0103] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0104] Amino acid mutations can be amino acid substitutions, deletions, insertions, and / or additions. In some embodiments, an amino acid mutation is the substitution of one or more amino acids, such as a single amino acid substitution or a combination of multiple amino acid substitutions. Amino acid deletions and insertions include deletions and insertions at the amino and / or carboxyl ends of the polypeptide sequence, as well as deletions and insertions within the polypeptide sequence. The amino acid substitutions of the present invention optionally include conservative substitutions of amino acids.

[0105] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acid substitutions with chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0106] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0107] "Isolated nucleic acid molecules" are nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule. Isolated nucleic acid molecules differ from their naturally occurring form or environment. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in their natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in natural cells.

[0108] The term "antibody-drug conjugate" or "antibody-conjugated drug" (ADC) refers to the linking of a targeting ligand, such as an antibody (e.g., a monoclonal antibody), or an antibody fragment, to a biologically active molecule (e.g., via a linker).

[0109] Unless otherwise specified, the term "pharmaceutically acceptable salt" means a salt suitable for contact with mammalian, particularly human, tissues without excessive toxicity, irritation, allergic reactions, etc., and in a manner commensurate with a reasonable benefit / risk ratio, within the limits of reasonable medical judgment. Medically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final separation and purification of the compounds of this invention, or solely by reacting a free base or free acid with a suitable reagent. Exemplary salts include, but are not limited to: sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobromides, hydroiodates, nitrates, hydrogen sulfates, phosphates, acidic phosphates (-H₂PO₄) salts, phosphites, isonicotinate, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrate, ascorbic acid salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, ammonium salts, calcium salts, etc. Furthermore, pharmaceutically acceptable salts have more than one charged atom in their structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counter atoms. For example, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counter atoms.

[0110] Unless otherwise specified, the compounds described in this invention (including antibody-drug conjugates) also contain "isotope labels," which refer to compounds of this invention that can exist in an isotopically traced or enriched form, containing one or more atoms whose atomic weight or mass number differs from the atomic weight or mass number of the most abundant atoms found in nature. The isotope can be radioactive or non-radioactive. Commonly used isotopes for isotope labeling include hydrogen isotopes. 2 H and 3 H; Carbon isotopes: 13 C and 14 C; Chlorine isotopes: 35 Cl and 37 Cl; Fluorine isotopes: 18 F; Iodine isotopes: 123 I and 125 I; Nitrogen isotopes: 13 N and 15 N; oxygen isotopes: 15 O, 17 O and 18 O and sulfur isotopes 35 S. These isotope-labeled compounds can be used to study the distribution of pharmaceutical molecules in tissues. Especially 2 H and 13C, due to their ease of labeling and detection, are more widely used. Substitution of certain heavy isotopes, such as deuterium (2H), can enhance metabolic stability and prolong half-life, thereby achieving a therapeutic advantage through dose reduction. Isotope-labeled compounds are generally synthesized starting from a labeled starting material, using known synthetic techniques similar to those used to synthesize non-isotope-labeled compounds.

[0111] Unless otherwise specified, the compounds described in this invention (including antibody-drug conjugates) also comprise "solvents," where "solvent" refers to the physical association of the compound of this invention with one or more solvent molecules (whether organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. Solvent molecules in a solvate may be present in a regular and / or disordered arrangement. Solvents may contain stoichiometric or non-stoichiometric solvent molecules. "Solvent" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0112] Unless otherwise specified, the term "stereoisomer" refers to compounds having the same chemical structure but with different spatial arrangements of atoms or groups. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and inhibited isomers. Any mixture of stereoisomers obtained can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0113] The term "linker-drug" refers to the partial structure of an antibody-drug conjugate consisting of a linker and a bioactive compound.

[0114] The term "linker" refers to a chemical structural fragment, denoted by L, that is linked at one end to an antibody and at the other end to a cytotoxic drug. In some embodiments of the invention, the "linker" is formed by linking an antibody to a "linker-drug compound." In other embodiments, a portion of the "linker" is first linked to an antibody, and another portion is linked to a bioactive compound, and then the linker is formed by covalently linking the different portions of the linker. The linker of the present invention comprises a covalently linked unit linked to an antibody, an extension unit, peptide residues, and optionally a self-cleaving fragment.

[0115] In this invention, the linker-drug compound and the antibody are linked via conventional coupling methods in the art, including: lysine coupling, inter-light and heavy chain reducing disulfide bond coupling, and directional coupling. Preferably, this invention uses inter-light and heavy chain reducing disulfide bond coupling, i.e., the linkage is achieved through the reaction of one or more thiol groups (sulfur atoms of cysteine ​​residues) formed after reduction at one or more disulfide bond sites between the light and heavy chains (two sites between the heavy chain and two sites between the heavy and light chains).

[0116] The term "pharmaceutical composition" means a mixture containing one or more of the antigen-binding molecules or antibodies, antibody-drug conjugates and their tautomers, stereoisomers or pharmaceutically acceptable salts and / or compositions described herein, and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0117] The term "pharmaceuticalally acceptable carrier" refers to a component in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the test subject. Pharmaceutically acceptable carriers include, but are not limited to, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, and suspending agents.

[0118] The terms “subject” or “individual” include both humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless explicitly stated otherwise, the terms “patient” or “subject” are used interchangeably herein. As used herein, the term “cynomolgus monkey” refers to the cynomolgus monkey (Macacafascicularis). In some embodiments, the individual or subject is a human.

[0119] "Administration" or "giving," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or biological fluid.

[0120] "Treatment" and "treatment" (and their grammatical variations) refer to a clinical intervention intended to be applied to the individual being treated, and may be implemented for preventative purposes or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing / decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, the molecules disclosed herein are used to delay the onset of disease or slow its progression.

[0121] As used herein, the term "therapeutic effective dose" or "effective dose" refers to the amount of an anti-ADAM9 antibody or its antigen-binding fragment, administered alone or in combination with another therapeutic agent, that effectively prevents or alleviates the disease or condition to be treated. A therapeutic effective dose further refers to the amount of the antibody or its antigen-binding fragment sufficient to cause symptom relief, such relief being, for example, treatment, cure, prevention, or alleviation of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the symptom. The effective dose for a specific patient can vary depending on various factors, such as the disease to be treated, the patient's overall health condition, the route and dosage of administration, and the severity of side effects. An effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity. When administered to an individual as a single active ingredient, the therapeutic effective dose refers to that single ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0122] Example The present invention will be further illustrated by specific embodiments below, but the scope of the present invention is not limited thereto.

[0123] Unless otherwise specified, the reagents and instruments used in this invention are all conventional reagents and instruments that can be obtained commercially; the methods used are all conventional techniques, and those skilled in the art can undoubtedly complete the experiments and obtain the corresponding results based on the contents of the specification.

[0124] In this embodiment of the invention, the positive control antibody is an antibody against MacroGenics' ADC product IMGC-936, the sequence of which is shown below: PC-1(MGA021) Light Chain: DIVMTQSPDSLAVSLGERATISCKASQSVDYSGDSYMNWYQQKPGQPPKLLIYAASDLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSHEDPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 24) PC-1 (MGA021) heavy chain: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVGEIIPIFGHTNYNEKFKSRFTISLDNSKNTLYLQMGSLRAEDTAVYYCARGGYYYYPRQGFLDYW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPG (SEQ ID NO: 25) In this embodiment of the invention, the negative control antibody IC-1 was purchased from MCE, catalog number HY-P99001.

[0125] In this embodiment of the invention, the linker forms a structure with camptothecin as shown in formula (VI) (MSP-PEG-CPT2C) and formula (VII) (Mc-CPT2C).

[0126] Formula (VI)

[0127] Equation (VII); in, This indicates the site where the anti-ADAM9 antibody or its antigen-binding fragment is linked.

[0128] The compounds shown in formulas (VI) and (VII) are based on compounds I-4 and I-1 with specific structures disclosed in the applicant’s prior published patent application WO2025016467A1 (which is incorporated herein by reference in its entirety).

[0129] In this embodiment of the invention, the payload molecule BCN-VA-Exatecan used in the positive ADC was custom-synthesized by WuXi AppTec, and its structure is shown below:

[0130] BCN-VA-Exatecan.

[0131] Example 1: Preparation, screening, and activity detection of anti-ADAM9 antibodies (1) The protein antigen used for immunization was recombinant ADAM9 protein, namely human ADAM9-his protein (catalog number: AD9-H52H7), purchased from Beijing Biosys Biotechnology Co., Ltd., containing the antigenic active fragment Ala 206-Asp697. The experimental steps for verifying its activity were as follows: First, antigen coating was performed. Serially diluted huADAM9-his antigen solutions were prepared using PBS solution, and 96-well plates were coated overnight at 4°C. After overnight coating, the 96-well plates were washed three times with PBST solution and then blocked with antigen. The antigen was blocked with PBS (containing 3% BSA) and kept at room temperature for 1 hour. The ADAM9 antibody (Biointron, catalog number: 467DDD001) was quantitatively diluted with DPBS (containing 1% BSA) solution and coated at 37°C for 1 hour. Discard the reaction solution, wash three times with PBST solution, add 100 μl of secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) to each well, and incubate at 37°C for 1 hour. Finally, discard the reaction solution, wash three times with PBST solution; add TMB (Abcam, catalog number: ab171523) solution and stop solution successively, gently shake until a yellow color appears, and then measure the OD value at 450 nm.

[0132] The results of the activity verification of the human ADAM9-his antigen used in the immunization study showed that the huADAM9-his antigen has good antigen-antibody binding activity and can be used as a mouse immunogen in later experiments.

[0133] (2) Animal immunization was performed using huADAM9-his protein. Five female Balb / C mice, aged 6-8 weeks, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The initial immunization used Freund's complete adjuvant (Sigma, catalog number: F5881-6x10ML) at a dose of 50 μg / mouse. Subsequent immunizations used Freund's incomplete adjuvant (Sigma, catalog number: F5506-6x10ML) at a dose of 25 μg / mouse. The injection method was subcutaneous injection at multiple sites (sc). Serum titers were monitored during the immunization period. Mice with high antibody titers were selected and given a shock immunization of 50 μg huADAM9-his protein via intraperitoneal injection (ip). Fusion was performed 3 days later. The experimental animals and immunization information are shown in Table 1 below.

[0134] Table 1. Experimental animals and immunization information

[0135] Mice immunized using the above method were subjected to serum titer testing using huADAM9-his antigen. The experimental steps were as follows: A 1 μg / ml solution of huADAM9-his antigen (AcroBiosystem, catalog number: AD9-H52H7) was prepared using PBS and coated in 96-well plates overnight at 4°C. The overnight coated 96-well plates were washed three times with PBST solution. The antigen was blocked with PBS (containing 3% BSA) and incubated at room temperature for 1 hour. The serum solution was diluted with DPBS (containing 1% BSA). The negative control was serum from unimmunized mice. The positive control wells contained ADAM9 antibody (Biointron, catalog number: 467DDD001). The reaction was incubated at room temperature for 1 hour. Discard the reaction solution, wash three times with PBST solution, add secondary antibody (Goat anti-Ms IgG F(ab')2 Secondary Antibody, source: Invitrogen, catalog number: 31436), add secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) to the positive control well, and incubate at room temperature for 1 hour. Finally, discard the reaction solution, wash three times with PBST solution, add TMB (Abcam, catalog number: ab171523) solution and stop solution successively, gently shake until yellow, and then measure the OD value at 450 nm. Serum titer tests of multiple immunized mice showed that the immune response of immunized mouse No. 5 was better.

[0136] As described above, after titer testing of mice immunized with huADAM9-his protein, high-titer Balb / c mice were selected for pulse immunization to initiate the fusion experiment. Splenic cells and SP2 / 0 mouse myeloma cells were mixed at a 3:1 ratio, centrifuged, and co-precipitated. After removing the supernatant, the cells were resuspended in an appropriate amount of fusion buffer, and the mixed cell density was adjusted to 1-2 × 10⁻⁶ cells / mL. 7 Cells were electrofused at 100 cells / ml (BTX, ECM2001). After fusion, the cells were incubated in the electrode holder for 5 minutes, then added to 1×HAT selective DMEM medium (Gibco, catalog number: 11995-065) containing 20% ​​FBS at a concentration of 2.0×10⁶ cells / ml. 4 Cells / well were seeded into 96-well flat-bottom cell culture plates and cultured at 37°C in a 5% CO2 incubator for 7 days. The medium containing HAT (Sigma, catalog number: H0262-10VL) in the 96-well cell culture plates was replaced with DMEM selective medium containing 1×HT (Sigma, catalog number: H0137-10VL). After 10 days, the supernatant was collected, and the binding ability to CHO-K1 human ADAM9 overexpressing cell lines was detected by FACS. Positive clones were transferred to 24-well plates. The supernatant was screened and identified by FACS (CHO-K1 human ADAM9 overexpressing cell lines) and ELISA (huADAM9-his protein). Positive clones were selected for 1-2 rounds of subcloning, and hybridoma monoclonal cells stably expressing specific anti-ADAM9 antibodies were screened again. Based on this, the cell lines with the highest expression levels were selected, and cell samples and Trizol samples were cryopreserved. Total RNA was extracted from ADAM9-positive hybridoma monoclonal cells obtained through screening, and a transcriptome sequencing library was constructed for high-throughput sequencing. The sequencing data were analyzed using bioinformatics and compared with the IMGT database to obtain the full-length, functional antibody heavy chain and light chain variable region gene sequences. The VH and VL genes were synthesized and cloned into mammalian expression vectors containing the constant regions of human antibodies. The recombinant plasmids were transfected into CHO cells for antibody expression. The cell culture supernatant was collected and purified by Protein A affinity chromatography to obtain the anti-ADAM9 chimeric antibody.

[0137] (3) The interaction between the antibody and ADAM9 protein was determined using the ForteBio molecular interaction analyzer. The specific method is as follows: 0.05% Tween20 PBS was used as PBST Buffer for sample preparation. The antibody was diluted to 5 μg / ml antibody sample well. At the same time, Human ADAM9 Protein, His Tag (Acro, catalog number: AD9-H52H7-50μg) was diluted to 100nM with PBST. Then, 6 concentration gradients were obtained by 2-fold dilution and added to the antigen sample wells respectively. Anti-human IgG Fc Capture (AHC) Dip and Read Biosensors were used as detection probes for detection. pH 1.5 and 100mM glycine were used as regeneration solution.

[0138] Affinity assays for the chimeric antibody 12F5B12 showed that it could bind to human ADAM9 protein, with an affinity K0. D The value of (M) is 2.19E-09.

[0139] (4) The binding of the antibody to the ADAM9 overexpressing cell line or the naturally expressing ADAM9 NCI-H1703 cell line was detected by flow cytometry. The ADAM9 human / cyno overexpressing cell line or the naturally expressing ADAM9 NCI-H1703 cell line was seeded in a 96-well plate, and the test antibody prepared with 2% FBS (Gibco) PBS Buffer was added. The initial concentration was 30 μg / mL, and the plate was serially diluted 5 times to a total of 8 concentration points. The blank wells were filled with 2% FBS PBS as a negative control. The plate was incubated at 4℃ for 1 h, and then the secondary antibody was added. After incubation, the plate was tested by flow cytometry.

[0140] The results of cell binding assays showed that the chimeric antibody 12F5B12 could bind to human / cyno ADAM9 overexpressing cell lines or NCI-H1703 cells that naturally express ADAM9.

[0141] (5) Flow cytometry was used to detect the endocytic activity of the antibody. NCI-H1703 cells were seeded in 96-well plates, and 100 μL of diluted antibody with a final concentration of 15 μg / mL was added. After thorough mixing with the cells, the cells were incubated for 30 min. After centrifugation, the cells were washed twice with 200 μL of PBS containing 0.5% FBS. The cells were resuspended and cultured at 4℃ and 37℃ for 1, 2, and 4 h, respectively. Samples were taken at each time point, centrifuged at 4℃, and then incubated with secondary antibody (goat anti-human AF647, Thermo Fisher Scientific) for 30 min at 4℃ before being analyzed by flow cytometry. The results of the chimeric antibody endocytic activity experiment showed that the chimeric antibody 12F5B12 mediated endocytosis with good activity.

[0142] After sequencing, the sequence of antibody 12F5B12 is as follows: Heavy chain variable region (VH): EVQLVETGGGLVQPKGSLKVSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRDLDYYGSSYAFAYWGQGTLVTVSA (SEQ ID NO: 1) VH CDR1: NNVMN (SEQ ID NO: 2) VH CDR2: RIRSKSNNYEIYYADSVKD (SEQ ID NO: 3) VH CDR3: DLDYYGSSYAFAY (SEQ ID NO: 4) Heavy chain constant region (CH): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) Light chain variable region (VL): DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVFNAKTLAEGVPSRFSGGGSGTQFSLKINSLQPEDFGTYYCQHHYVIPLTFGAGTKLELK (SEQ ID NO: 6) VL CDR1: RASENIYSYLA (SEQ ID NO: 7) VL CDR2: NAKTLAE (SEQ ID NO: 8) VL CDR3: QHHYVIPLT (SEQ ID NO: 9) Light chain constant region (CL): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) (6) Based on the heavy chain variable region sequence of the human-mouse chimeric antibody ADAM9 antibody 12F5B12, the human antibody gene sequence library (NCBI Ig BLAST) was compared to find the corresponding sequence of the variable region of human germline antibody that is similar to the heavy chain variable region sequence of human-mouse chimeric antibody 12F5B12. Among them, there are 13 different amino acids in the heavy chain FR region.

[0143] Based on the framework sequence optimization, the humanized antibody heavy chain variable region sequence was designed as follows: VH1: EVQLVESGGGLVQPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVGRIRSKSNNYEIYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS (SEQ ID NO: 11) VH2: EVQLVESGGGLVQPGGSLRVSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS (SEQ ID NO: 12) VH3: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 13) VH4: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 14) VH5: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVARIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 15) VH6: EVQLVESGGGLVKPGGSLRLSCAASGFTFNNNVMNWVRQAPGKGLEWVGRIRSKSNNYEIYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRDLDYYGSSYAFAYWGQGTLVTVSS(SEQ ID NO: 16) By comparing the light chain variable region sequence of the human-mouse chimeric antibody ADAM9 antibody 12F5B12 with the human antibody gene sequence library (NCBI Ig BLAST), sequences corresponding to the variable regions of human germline antibodies with similar light chain variable region sequences to those of human-mouse chimeric antibody 12F5B12 were identified. Among these, 16 amino acids differed in the light chain FR region.

[0144] Based on the framework sequence optimization, the humanized antibody light chain variable region sequence was designed as follows: VL1: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVFNAKTLAEGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHYVIPLTFGQGTKLEIK (SEQ ID NO: 17) VL2: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPQLLVFNAKTLAEGVPSRFSGSGSGTQFTLTISSLQPEDFATYYCQHHYVIPLTFGQGTKLEIK (SEQ ID NO: 18) VL3: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPQLLVFNAKTLAEGVPSRFSGSGSGTQFSLTINSLQPEDFATYYCQHHYVIPLTFGQGTKLEIK (SEQ ID NO: 19) VL4: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLVFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 20) VL5: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLVFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 21) VL6: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 22) VL7: DIQMTQSPSSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKSPKLLVFNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYVIPLTFGGGTKVEIK(SEQ ID NO: 23) The combinations of the heavy chain variable region and light chain variable region of the humanized antibody of the present invention are shown in Table 2.

[0145] Table 2. Humanized anti-ADAM9 antibody combinations

[0146] (7) The binding of humanized antibody to ADAM9 protein was detected by ELISA. The hADAM9-his protein (Acro, catalog number: AD9-H52H7) solution prepared in PBS was added to a 96-well plate and coated overnight at 4°C. After washing with PBST and blocking with 3% BSA, 15 μg / mL antibody working solution was added and double-duplicate wells were set up for incubation at 37°C. After washing with PBST, HRP-labeled secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) was added and incubated at 37°C. After washing with PBST, TMB (Abcam, catalog number: ab171523) was added for color development and the reaction was terminated with stop solution. Finally, the absorbance value at 450 nm was detected by microplate reader.

[0147] Affinity assay results of humanized antibody and hADAM9-his protein are as follows: Figure 1 As shown, the results indicate that all humanized antibodies can bind to the hADAM9-His antigen, and the affinity of 12F5B12-4 for the hADAM9-His antigen is similar to that of the parent antibody 12F5B12.

[0148] (8) Flow cytometry was used to detect the binding of humanized antibodies to ADAM9-overexpressing cell lines or naturally ADAM9-expressing NCI-H1703 cells. ADAM9-overexpressing cell lines or naturally ADAM9-expressing NCI-H1703 cells were seeded in 96-well plates. The test antibody, prepared with 2% FBS in PBS, was added at an initial concentration of 30 μg / mL, with 8 concentration points obtained through a 5-fold serial dilution. Blank wells were treated with 2% FBS (Gibco) in PBS buffer as a negative control. The plates were incubated at 4°C for 1 h. Secondary antibodies were then added, and the plates were incubated before flow cytometry analysis.

[0149] Humanized antibody binding assay Figure 2A and Figure 2B As shown, the humanized antibodies 12F5B12-1, 12F5B12-1, 12F5B12-2, 12F5B12-3, and 12F5B12-4 can bind to human ADAM9 overexpressing cell lines or NCI-H1703 cells that naturally express ADAM9, and their binding ability is comparable to that of the parent 12F5B12. (9) Flow cytometry was used to detect the endocytic activity of the antibody in NCI-H1703 cells. NCI-H1703 cells were seeded in 96-well plates, and 100 μL of diluted antibody with a final concentration of 15 μg / mL was added. After thorough mixing with the cells, the cells were incubated for 30 min. After centrifugation, the cells were washed twice with 200 μL of PBS containing 0.5% FBS. The cells were then resuspended and cultured at 4℃ and 37℃ for 1, 2, and 4 h, respectively. Samples were taken at each time point, centrifuged at 4℃, and then incubated with secondary antibody at 4℃ for 30 min before being analyzed by flow cytometry.

[0150] The endocytic activity of humanized antibodies on NCI-H1703 cells at 37°C is as follows Figure 3 As shown, the ADAM9 humanized antibody exhibits significant endocytic activity at 37°C, which is comparable to that of the parent antibody 12F5B12.

[0151] (10) Using the ELISA method, the ADAM8 (Acro, catalog number: AD8-H5223), ADAM10 (R&D Systems, catalog number: 936-AD-020), ADAM15 (Sino Biological, catalog number: 10517-H08H), and ADAM17 (R&D Systems, catalog number: 930-ADB-010) family protein solutions prepared with PBS were added to a 96-well plate and coated overnight at 4°C. After washing with PBST and blocking with 3% BSA, 15 μg / mL antibody working solution was added and double replicates were set up. The plate was incubated at 37°C. The sample was then washed with PBST, incubated with HRP-labeled secondary antibody (HRP-Goat Anti-Human IgG, F(ab')2 Secondary Antibody, source: Jackson, catalog number: 109-035-006) at 37°C, washed again with PBST, and then incubated with TMB (Abcam, catalog number: ab171523) for color development. The reaction was terminated with stop solution, and the absorbance at 450 nm was measured using an ELISA reader.

[0152] Test results as follows Figure 4 As shown, both the maternal antibody and the humanized antibody bind to the human ADAM9 protein, but do not bind to the human ADAM8, ADAM10, ADAM15, or ADAM17 proteins.

[0153] (11) The inhibitory effect of the antibody on the enzymatic activity of ADAM9 protein was evaluated by fluorescence resonance energy transfer (FRET). A pH 9.0 buffer containing 25 mM Tris, 2.5 μM ZnCl2, and 0.005% (w / v) Brij-35 was prepared, and hADAM9 was diluted to 80 μg / mL and the antibody to 120 μg / mL with the buffer. The antigen and antibody solutions were mixed in equal volumes and incubated at room temperature for 15 min. Meanwhile, Mca-PLAQAV-Dpa-RSSSR-NH2 Fluorogenic Peptide Substrate (R&D Systems, catalog number: ES003) was diluted to 20 μM. Then, an equal volume of the above hADAM9-antibody mixture was mixed with the substrate solution to make the final concentration of the system 20 μg / mL hADAM9, 30 μg / mL antibody, and 10 μM substrate. A control group was set up with only substrate added and no hADAM9 added. After incubation at 37℃ for 30 min, 100 μL of reactants and controls were added to a black ELISA plate. The fluorescence intensity was detected using a fluorescence plate reader with an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Finally, a relevant curve was plotted based on the fluorescence values.

[0154] Test results as follows Figure 5 As shown, compared with hIgG1, both the humanized antibody 12F5B12 and the maternal antibody 12F5B1 showed a decrease in fluorescence value, indicating that the antibody has an enzyme activity inhibitory effect, and there was no significant difference in the value before and after humanization.

[0155] (12) Prepare CHOK1-hADAM9 cells and place them in 6-well plates. Transfect the experimental wells with PEI transfection reagent (Polysciences, catalog number: 23966-1) to hEphB4-AP-Puro plasmid, and transfect the control wells with Luc-GFP plasmid. Co-culture for 6 h. After 6 h, discard the supernatant and replace with 2 ml of complete culture medium. Add 1 ml of Opti-MEM serum-free culture medium (Gibco) and 5 μg / ml of test antibody protein or IgG1 control antibody protein (Novoprotein, catalog number: NC002). 48 h after transfection, collect the supernatant and place it in a centrifuge tube on ice. Mix the supernatant with an equal volume of 2 mg / mL 4-nitrophenyl phosphate disodium hexahydrate (Merck, catalog number: N2765-50TAB) and incubate at 37 °C for 1.5 h. Detect the absorbance at 405 nm using a microplate reader.

[0156] Test results as follows Figure 6As shown, compared to the transiently transfected Luc-GFP control plasmid, CHOK1-hADAM9 cells transfected with the hEPHB4-AP plasmid exhibited increased alkaline phosphatase activity in the supernatant. The maternal antibody 12F5B12 and the humanized antibody reduced the alkaline phosphatase activity in the supernatant, suggesting that they inhibited the cleavage of hEPHB4-AP by hADAM9 cells.

[0157] Example 2: Coupling of conjugate 12-2-MSP-CPT2C The stock solution of monoclonal antibody 12F5B12-2, with a concentration of 8.5 mg / mL, was prepared in 2000 μL and set aside for later use. 4.5 mg of TCEP-HCl (tris(2-carbonylethyl)phosphohydrochloride, purchased from Thermo Fisher Scientific) was weighed and added to 0.45 mL of PBS to prepare a 10 mg / mL TCEP solution. The molar equivalent was 10 eq. The corresponding volume of TCEP solution was added to the antibody solution, and the reaction was carried out at 37°C for 60 min. 2.5 mg of MSP-PEG-CPT2C (formula VI, 14.0 eq) was weighed and dissolved in 100 μL (5% volume) of DMSO. After complete dissolution, the solution was slowly added to the reaction system, followed by 16 μL of 1M Tris. The reaction was carried out at 37°C for 4 h, after which the reaction was stopped. The reaction solution was filtered through a 0.22 μm PVDF filter. The filtrate was replaced with 20 mM histidine using a G25 chromatography column (column volume 29.9 mL), and the eluent with a UV value greater than 150 mAU was collected to obtain the conjugate 12-2-MSP-CPT2C, the structure of which is shown in formula (IV).

[0158] like Figure 7A and Figure 7B As shown, the DAR value of 12-2-MSP-CPT2C determined using the reducing LCMS method was 7.99; Figure 8 As shown, the sec-HPLC purity of the conjugate 12-2-MSP-CPT2C is 96.8%.

[0159] Example 3: Coupling of conjugate 12-2-Mc-CPT2C The stock solution of monoclonal antibody 12F5B12-2, with a concentration of 8.5 mg / mL, was prepared in 2000 μL and set aside for later use. 4.5 mg of TCEP-HCl was weighed and added to 0.45 mL of PBS to prepare a 10 mg / mL TCEP solution. The molar equivalent was 10 eq. The corresponding volume of TCEP solution was added to the antibody solution, and the reaction was carried out at 37°C for 60 min. 2.5 mg of Mc-CPT2C (Formula VII) was weighed and dissolved in 100 μL (5% volume) of DMSO. After complete dissolution, the solution was slowly added to the reaction system at a molar equivalent of 14.0 eq. The reaction was carried out at 37°C for 2 h. The reaction was then terminated, and the reaction solution was filtered through a 0.22 μm PVDF filter. The filtrate was replaced with 20 mM histidine using a G25 chromatography column (column volume 29.9 mL), and the eluent with a UV value greater than 150 mAU was collected to obtain the conjugate 12-2-MSP-CPT2C, the structure of which is shown in formula (V).

[0160] like Figure 9A and Figure 9B As shown, the DAR value of 12-2-MSP-CPT2C determined using the reducing LCMS method was 7.92; Figure 10 As shown, the sec-HPLC purity of the conjugate 12-2-MSP-CPT2C is 95.2%.

[0161] Example 4: Coupling of the conjugate IC-1-CPT2C The monoclonal antibody IC-1, with a stock solution concentration of 10 mg / mL, was prepared in 2000 μL and set aside for later use. 4.5 mg of TCEP-HCl was weighed and added to 0.45 mL of PBS to prepare a 10 mg / mL TCEP solution (molar equivalent of 10 eq). The corresponding volume of TCEP solution was added to the antibody solution, and the reaction was carried out at 37°C for 60 min. 1.9 mg of CPT2C was weighed and dissolved in 100 μL (5% volume) of DMSO. After complete dissolution, approximately 14.0 eq of CPT2C was slowly added to the reaction system, and the reaction was carried out at 37°C for 2 h. The reaction was then stopped, and the reaction solution was filtered through a 0.22 μm PVDF filter. The filtrate was exchanged using a G25 column (29.9 mL column volume) to 20 mM histidine. The eluent with a UV value greater than 150 mAU was collected to obtain the conjugate IC-1-CPT2C.

[0162] like Figure 11A and Figure 11B As shown, the DAR value of IC-1-CPT2C determined using the reducing LCMS method was 7; Figure 12 As shown, the sec-HPLC purity of the conjugate IC-1-CPT2C is 97.9%.

[0163] Example 5: Conjugate PC-1-Exatecan The PC-1 antibody was replaced using an Amicon ultrafiltration tube to a 20 mM His, 150 mM NaCl buffer at pH 7.5, resulting in an antibody concentration of 15 mg / mL. An equal volume of β-(1,4)-GalNAc transferases (Ing. Biotech, catalog number: YJ-O-147-1) was replaced to a solution containing 20 mM His, 150 mM NaCl, and pH 7.5, resulting in a concentration of 1 mg / mL. 10 mg of antibody, 0.1 mg of Endo-SH enzyme (1% w / w) (MCE, CAS: 37278-88-9), 0.40 mg of β-(1,4)-GalNAc transferases (4% w / w), and 0.64 mg of UDP-N3-GalNAc (15 eq. mol / mol) (Biode Pharmaceuticals, CAS: 653600-61-4) were added to a total volume of 6% 100 mM MnCl2. After mixing, the mixture was reacted overnight at 30°C. The sample was centrifuged and the supernatant was collected. Protein A was purified, and the solution was changed to TBS at pH 7.5. Finally, 10 eq equivalents of BCN-VA-Exatecan molecules were added to make the final system contain 10% DMA. The mixture was reacted at 37°C for 12 h. After the reaction was completed, a G25 chromatography column (column volume 29.9 mL) was used to change the solution to 20 mM histidine. The eluent with a UV value greater than 150 mAU was collected to obtain the conjugate PC-1-Exatecan.

[0164] like Figure 13 As shown, the DAR value of PC-1-Exatecan was 7, determined using the reducing LCMS method; Figure 14 As shown, the sec-HPLC purity of the conjugate PC-1-Exatecan (also referred to as "PC-1" in this paper) was 98.2%.

[0165] Example 6: Plasma stability experiment Plasma stability was assessed using methods that detected the free payload and those that tracked changes in the DAR value, respectively. a. Detecting Free Payload: Sample processing in human plasma (Rubai Biotechnology PBPLSH010C): The ADC was incubated in the human plasma system at 37℃ with an incubation concentration of 0.5 mg / mL. 100 μL samples were collected at 0, 1, 3, 7, and 14 days and stored at -80℃. An acetonitrile / methanol mixture (containing 0.1% formic acid, 250 nM tolbutamide, and 250 nM labetalol) was added at a volume ratio of 1:3. The mixture was then incubated in a metal incubator at 25℃ and 1100 rpm for 20 min, followed by centrifugation at 4℃ and 3500 g for 20 min. The supernatant was collected and placed in a sample vial.

[0166] Preparation of standard curve: Prepare stock solution of free-drug compound K4 (preparation method see WO2025016467A1), with a stock solution concentration of 10 mg / mL (DMSO system). The standard curve samples were diluted according to the following concentration gradients: C1=2000 ng / L, C2=1000 ng / L, C3=500 ng / L, C4=200 ng / L, C5=100 ng / L, C6=50 ng / L, C7=20 ng / L, and C8=10 ng / L. 100 μL of each standard curve sample was added to a 1:1 mixture of acetonitrile and methanol (containing 0.1% formic acid, 250 nM tolbutamide, and 250 nM labetalol) at a volume ratio of 1:3. The mixture was then incubated in a metal incubator at 25°C and 1100 rpm for 20 min, followed by centrifugation at 4°C and 3500 g for 20 min. The supernatant was collected and collected in a sample vial for analysis using LC-MS.

[0167] DS-8201 was used as a control in the same manner as above, with DS-8201's free drug being DXD (MCE, catalog number: HY-13631D).

[0168] Figure 15 The results are for plasma stability testing using the Free payload method.

[0169] b. DAR level detection to assess plasma stability: Hu-ADAM9 biotinylation: 170 µL of ultrapure water was added to 2 mg of EZ-link NHS-PEG4-Biotin (Thermo catalog number A39259) to prepare a 20 mM NHS-PEG 4-Biotin stock solution.

[0170] Prepare two 100μL portions of Hu-ADAM9 protein (catalog number: AD9-H52H7, Beijing Baipusaisi Biotechnology Co., Ltd., 1.0mg / mL) + 20μL of 20mM biotin, and incubate at 25℃ and 1100rpm in a metal bath for 1 hour.

[0171] Biotin was removed using a desalting column (Thermo catalog number 89882) to obtain a final volume of 120 μL of 0.83 mg / mL Biotin-ADAM9, which was stored at -80°C. Plasma sample capture LCHC assay: (1) Take 25 μL of streptavidin magnetic beads (Promega product number V7820) into a 1.5 mL EP tube, place it on a magnetic rack, discard the solvent, and wash once with 20 μL of 200 mM NaCl PBS solution; (2) Add 30 μL Biotin-ADAM9 (0.83 mg / mL) and 260 μL 200 mM NaCl PBS solution to the centrifuge tube, shake in a metal bath at 25 °C and 1100 rpm for 2 hours, place on a magnetic rack to discard the supernatant, and wash once with 300 μL 200 mM NaCl PBS solution; (3) Add 30 μL of plasma sample and 290 μL of 200 mM NaCl PBS solution. Shake in a metal bath at 25 °C and 1100 rpm for 2 hours. Place on a magnetic rack and discard the supernatant. Wash twice with 200 μL and 300 μL of 200 mM NaCl PBS solution, then wash once with 200 μL of PBS and once with 200 μL of ultrapure water. (4) Add 50 μL of 0.1% TFA elution buffer, centrifuge at 25℃ and 1200 rpm for 0.5 hours, collect the solution into a new centrifuge tube, add 5 μL of 1M NH4HCO3 neutralization solution and 5 μL of 1M DTT reducing agent, centrifuge at 25℃ and 1100 rpm for 0.5 hours, centrifuge at 3200g for 5 min, and take the supernatant for LC-MS analysis.

[0172] DS-8201 was used as a control in the same manner as above.

[0173] Figure 16 The results are from a plasma stability experiment based on DAR value levels.

[0174] Example 7: Binding assay of ADC with SNU-5 cells (1) Digest SNU-5 cells, centrifuge and count them, and seed them into 96-well plates, with 2E5 cells per well. Take 20 μl of cell suspension and mix it with 20 μl of VisStain AOPI Staining Solution (Nexcelom Bioscience, catalog number CS2-0106-25mL). Take 20 μl of the liquid and count the cells (4.56E6 viable cells per mL, viability 89.9%). Centrifuge the cells to remove the supernatant.

[0175] (2) Prepare antibodies and negative control antibodies with PBS containing 1% FBS. The maximum concentration of the antibodies is 15 μg / ml. Dilute 5 times to 8 points (excluding 0 point). Add 100 μl to each tube and incubate at 4 degrees for 1 hour.

[0176] (3) Wash the plate three times with PBS containing 1% FBS.

[0177] (4) Add PE-labeled human IgG (Invitrogen catalog number 12-4998-82) to PBS with 1% FBS diluted 1:1500, add 100 μl to each well, and incubate at 4 degrees for 0.5 hours.

[0178] (5) Wash the plate twice with PBS containing 1% FBS. Resuspend the plate in 100 μl of PBS containing 1% FBS before analysis.

[0179] Figure 17 The results of the ADC binding experiment with SNU5 cells.

[0180] Example 8: Endocytosis of ADCs on SNU-5 cells (1) After trypsin digestion and centrifugation, SNU-5 cells were resuspended in FACS Buffer (PBS + 1% FBS) and the cell density was adjusted to 2E6 / ml. 100 μL was added to a 96-well plate (Corning #3799), 2E5 / well. 20 μl of cell suspension was aspirated and mixed with 20 μl of VisStain AOPI Staining Solution. 20 μl of the liquid was taken for cell counting (3.5E6 viable cells per ml, viability 92%).

[0181] (2) Centrifuge at 300×g for 5 min under pre-cooling conditions at 4℃, and discard the supernatant.

[0182] (3) Add 100 μL of diluted antibody (final concentration 15 μg / mL), mix thoroughly with the cells, and incubate at 4°C for 30 minutes.

[0183] (4) Centrifuge at 300×g for 5 min at 4℃ and discard the supernatant. Add 200 μL of FACS Buffer (PBS+0.5% FBS) to each well, centrifuge at 300×g for 5 min at 4℃, and wash twice.

[0184] (5) Add 100 μL of FACS Buffer to the sample wells for resuspension. Place the 4℃ sample in a 4℃ refrigerator and strictly control the temperature. Place the sample in a 5% CO2 incubator at 37℃ and incubate for 1, 2 and 4 hours respectively.

[0185] (6) Take out the sample at each time point, centrifuge at 300×g for 5 min at 4℃, discard the supernatant, add 100 μL of the working solution of LAPC anti-human IgG Fc (Jackson ImmunoResearch catalog number 109-605-098) and FACS Buffer (1:500) to each well, and incubate at 4℃ for 0.5 hours.

[0186] (7) Centrifuge at 300×g for 5 min at 4℃, discard the supernatant, and wash twice. Add 100 μL FACS Buffer to each well and read the data using a cell flow cytometer.

[0187] Figure 18A and Figure 18B The results are from the endocytosis experiment of ADC in SNU5 cells.

[0188] Example 9: Killing experiment of ADC on CLAU3 and NCI-H1703 cells CLAU3 and NCI-H1703 cells were cultured separately in T75 culture flasks. CLAU3 cells were digested with trypsin, centrifuged, the supernatant was removed, 3 mL of culture medium was added to resuspend the cells, and the cells were counted and adjusted to a cell density of 2.5E4 / mL. NCI-H1703 cells were digested with trypsin, centrifuged, the supernatant was removed, 3 mL of culture medium was added to resuspend the cells, and the cells were counted and adjusted to a cell density of 3E4 / mL.

[0189] Seed cells into 96-well cell culture plates. For CLAU3 cells, add 200 μl of cell dilution buffer to each well, seeding 5000 cells per well. Gently tap to mix. Incubate the cell culture plates at 37°C for 16 hours. For NCI-H1703 cells, add 200 μl of cell dilution buffer to each well, seeding 5000 cells per well. Gently tap to mix. Incubate the cell culture plates at 37°C for 16 hours.

[0190] The CLAU3 experimental group samples were diluted 5-fold with MEM complete medium from the maximum concentration of 200 μg / mL, with 10 spots, and set aside for use. The CLAU3 experimental group medium was removed, and 100 μl of MEM complete medium and 100 μl of sample solutions at different concentration gradients were added. After adjustment, the initial concentration of each sample group was 100 μg / mL, and the cell culture plates were incubated at 37℃ for 7 days.

[0191] In the NCI-1703 experimental group, the antibody was diluted 5-fold from its maximum concentration of 100 μg / ml using RPMI 1640 complete medium, and applied to 10 wells. After removing the culture medium from the culture plate, 200 μl of sample solutions with different concentration gradients were added to each well of the experimental group, and 200 μl of RPMI 1640 complete medium was added to the blank control. The cell culture plates were incubated at 37°C for 7 days.

[0192] After 7 days of culture, the cell culture plates were removed from the incubator. For the CLAU3 plates, the culture medium was first removed, and 20 μl of MEM complete culture medium and 20 μl of CTG were added to each well. The plates were gently shaken for 5 minutes and allowed to stand at room temperature for 10 minutes. 100 μl of the reaction solution was then transferred to a white transparent-bottomed microplate for data analysis. Similarly, for the NCI-1703 plates, the culture medium was aspirated, 20 μl of culture medium was added, and then 20 μl of CTG was added to each well. The plates were gently shaken for 5 minutes and allowed to stand at room temperature for 10 minutes. 30 μl of the reaction solution was then transferred to a white transparent-bottomed microplate for data analysis.

[0193] Calculation formula: Inhibition rate = 1 - (experimental group - blank) / (background value) × 100%, where blank is the value measured when only culture medium is added to the well plate; background value is the value measured when cells and culture medium are added to the well plate, but no drug is added.

[0194] Figure 19 , Figure 20 The results show the killing effects of ADC on CLAU3 and NCI-1703 cells, respectively.

[0195] Example 10: ADCC Effect Experiment 1. NCI-H1975 ADCC Experiment a. Inoculating target cells Adherent target cells: Seed the target cells in the logarithmic growth phase and in good condition the afternoon before the experiment, and count the cell density and viability. Remove an appropriate amount of cells from the culture flask, centrifuge at 1000 rpm for 5 min, discard the supernatant, and adjust the cell density to 2.5E5 cells / mL with complete culture medium. Seed 100 μL / well (25000 cells / well) into 96-well white plates. Add 100 μL / well of culture medium to the background (BG) wells and 200 μL / well of PBS to the edge wells. Incubate overnight at 37°C in a 5% CO2 incubator for cell adhesion.

[0196] b. Sample processing and incubation The sample was diluted to an initial concentration of 400 μg / mL using preheated detection buffer (phenol red-free 1640 medium), and then serially diluted 5-fold to a total of 10 concentration points.

[0197] Remove the well plate containing target cells prepared one day in advance, aspirate the culture supernatant, and wash once with 200 μL of buffer, being careful to aspirate all residual liquid from the wells without pipetting or aspirating. Add 25 μL / well of preheated 37°C ADCC detection buffer to the sample wells and negative control (NC) wells, and 75 μL / well of ADCC detection buffer to the BG wells.

[0198] Add 25 μL of each serially diluted sample to the wells of the plate inoculated with target cells, and add 25 μL of buffer to each NC well. Incubate the plate at 37°C with 5% CO2 for 45 min.

[0199] c. Effector cell treatment Effector cells in the logarithmic growth phase and in good condition were collected. Cell density and viability were counted, and the cells were centrifuged at 1000 rpm for 5 min, discarding the supernatant. Cells were washed once with assay buffer, centrifuged, and the supernatant was discarded. Passage: P22; Cell density: 5.4E6 cells / mL; Viability: 96%. Cells were resuspended and adjusted to a density of 3E6 cells / mL with buffer, with an effector-to-target ratio of 3:1. 25 μL / well (75,000 cells / well) was added to each well of a plate, except for the BG wells which were left unseeded. The plates were incubated at 37°C in a 5% CO2 incubator for 6 h.

[0200] d. On-machine testing Remove the assay plate and allow it to equilibrate to room temperature for 15 minutes. Add 75 μL of pre-equilibrated firefly luciferase assay reagent to each well, avoiding the formation of air bubbles. Incubate at room temperature for 5 minutes, then place the plate in a microplate reader to detect the light signal.

[0201] 2. SNU5 ADCC Experiment a. Target cell treatment Collect SNU5 target cells in the logarithmic growth phase and in good condition, count cell density and viability, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Adjust the target cell density to 1E6 cells / ml using detection buffer.

[0202] b. Effector cell treatment Collect effector cells in the logarithmic growth phase and in good condition. Count cell density and viability. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Wash cells once with assay buffer, centrifuge, and discard the supernatant. Resuspend and adjust cell density to 3E6 cells / ml with buffer, achieving an effector-to-target ratio of 3:1.

[0203] c. Incubation Seed 25 μl of target cells per well into a 96-well white plate and incubate at 37°C for 45 min. NC wells: 25 μl target cells + 25 μl detection buffer per well; BC wells: 75 μl detection buffer per well. Seed 25 μl of effector cells per well (75,000 cells / well). Gently tap the plate edge and incubate at 37°C with 5% CO2 for 6 h.

[0204] d. On-machine testing Remove the assay plate and allow it to equilibrate to room temperature for 15 minutes. Add 75 μL of pre-equilibrated firefly luciferase assay reagent to each well, avoiding the formation of air bubbles. Incubate at room temperature for 5 minutes, then place the plate in a microplate reader to detect the light signal.

[0205] Figure 21 , Figure 22 These are the experimental results of ADCC effect.

[0206] Example 11: Pharmacodynamic Experiment of an ADAM9-Positive CDX Model CDX model experiments were conducted using NCI-H1975 (human lung adenocarcinoma cells), SNU-5 (human gastric cancer cells), NUGC-4 (human gastric cancer cells), and LS174T (human colorectal cancer cells). Taking the NCI-1975 model as an example, the steps are as follows: (1) Resuscitate human lung cancer NCI-H1975 cells and expand them in vitro; (2) Purchase experimental animals and allow them to adapt for 3-7 days before experimental treatment; (3) Collect cells in the logarithmic growth phase and resuspend them in serum-free culture medium, adjusting the cell concentration to 5 × 10⁻⁶. 7 / mL; (4) Inject the cell suspension subcutaneously into the axilla of the right forelimb of the mouse using a 1 mL syringe. Inject 100 μL (5 × 10⁻⁶) per animal. 6 ); (5) Regularly observe the growth of the animals and the transplanted tumors; (6) Wait until the tumor volume grows to 100 mm 3 When the tumor size was around 6, the animals were randomly divided into groups using a block design, with 6 animals in each group. The drugs were administered according to the experimental protocol, and the administration method and cycle are detailed in Table 3-6. (7) The tumor volume of an individual animal reached 2000 mm. 3 Timely euthanasia; (8) At the end of the experiment, the tumor was weighed and photographed; and a gross dissection and visual observation of the organs were performed to check for any obvious abnormalities.

[0207] The experimental procedures for other cell line models were performed in accordance with the NCI-H1975 cell CDX model. The UHDE (DS-8201) used in the examples was purchased from AstraZeneca (National Drug Approval Number SJ20230005).

[0208] Table 3. Dosing regimen for the NCI-1975 model

[0209] Table 4. SNU-5 model dosing regimen

[0210] Table 5. Dosing regimen for NCGU-4 model

[0211] Table 6. Dosing regimen for the LS174T model

[0212] Figure 23 , Figure 24 , Figure 25 , Figure 26 The results are from the CDX model pharmacological experiment.

Claims

1. An antibody-drug conjugate, wherein, The antibody-drug conjugate comprises: (a) Anti-ADAM9 antibody; and (b) Camptothecin-like toxin load; The anti-ADAM9 antibody is conjugated to the camptothecin toxoid load via a linker; The anti-ADAM9 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises VH CDR1, VH CDR2, and VH CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. The light chain variable region comprises VL CDR1, VL CDR2, and VLCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. The linker and the camptothecin-like toxin load form a structure as shown in formula (VI) or formula (VII): Formula (VI) Equation (VII); in, This indicates the site that is linked to the anti-ADAM9 antibody.

2. The antibody-drug conjugate according to claim 1, wherein: (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 17; (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 18; (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 11; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 19; (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 12; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 17; (5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 20; (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 14; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 21; (7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 22; or (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 16; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

23.

3. The antibody-drug conjugate according to claim 1, wherein, The antibody is a monoclonal antibody, the heavy chain constant region of the monoclonal antibody is the heavy chain constant region of IgG1, IgG2, IgG3, and IgG4, and the light chain constant region of the monoclonal antibody is the light chain constant region of the κ and λ chains.

4. The antibody-drug conjugate according to claim 3, wherein, The heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 5; the light chain constant region contains the amino acid sequence shown in SEQ ID NO:

10.

5. The antibody-drug conjugate according to any one of claims 1-4, wherein, The antibody-drug conjugate has a structure as shown in formula (IV) or formula (V): Formula (IV) Formula (V); Wherein, Ab is the anti-ADAM9 antibody, and n is an integer or decimal selected from 1 to 8.

6. A composition comprising the antibody-drug conjugate of any one of claims 1-5, and a pharmaceutically acceptable carrier.

7. Use of the antibody-drug conjugate of any one of claims 1-5 and / or the composition of claim 6 in the preparation of a medicament for treating tumor diseases; wherein the tumor disease is selected from at least one of the group consisting of gastric cancer, lung cancer, colorectal cancer, prostate cancer, breast cancer, and pancreatic cancer.

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