Humanized antibody targeting ADAM9, antibody-drug conjugate thereof, and its use

Abstract

This invention discloses a humanized antibody targeting ADAM9, an antibody-drug conjugate thereof, and its use. The humanized antibody targeting ADAM9 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. The antibody-drug conjugate comprises the humanized antibody, a linker unit L, and a cytotoxic drug. The humanized antibody has good affinity, high hydrophilicity, solubility, and stability, and exhibits a high internalization rate in ADAM9-expressing cells. The antibody-drug conjugate has a good growth inhibitory effect on ADAM9-positive expressing cells and exhibits a significant antitumor effect in the body.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to a humanized antibody targeting ADAM9, an antibody-drug conjugate thereof, and its use.

Background Art

[0002] ADAM (A Disintegrin And Metalloproteinase) is a protein family involved in various physiological and pathological processes. At least 40 gene members of this family have been identified, and at least 21 of them are considered to be functional in humans. ADAM family members have a relatively conserved structure consisting of eight domains, including a metalloprotease domain and an integrin-binding (disintegrin) domain. The ADAM metalloprotease domain functions as a sheddase and has been reported to regulate a series of biological processes by cleaving transmembrane proteins, and the transmembrane proteins can function as soluble ligands and control cell signaling.

[0003] ADAM9 is a member of the ADAM molecular family. It is synthesized as an inactive form, and the inactive form is cleaved by proteolysis to generate an active enzyme. Processing at the upstream site is particularly important for the activation of the proenzyme. ADAM9 is expressed in fibroblasts, activated vascular smooth muscle cells, monocytes, and activated macrophages.

[0004] The metalloprotease activity of ADAM9 is involved in the degradation of substrate components, thereby enabling the migration of tumor cells. The disintegrin domain of ADAM9 shows high homology with many snake venom disintegrins, enables the interaction between ADAM9 and integrins, and positively or negatively regulates cell adhesion phenomena. It has been shown that the ADAM9 disintegrin domain interacts with α6β1, α6β4, αvβ5, and α9β1 integrins.

[0005] ADAM9 expression has been found to be associated with disease (particularly cancer). ADAM9 has been shown to cleave and release numerous molecules that play important roles in tumorigenesis and angiogenesis, including TEK, KDR, EPHB4, CD40, VCAM1, and CDH5. ADAM9 is expressed in a wide variety of tumor cells, including those of breast cancer, colorectal cancer, gastric cancer, glioma, liver cancer, non-small cell lung cancer, melanoma, myeloma, pancreatic cancer, and prostate cancer.

[0006] In particular, increased ADAM9 expression has been found to be positively correlated with tumor malignancy and metastatic potential. Furthermore, ADAM9 and its secreted soluble isoforms are considered to be extremely important for the spread of cancer cells. Therefore, many studies have identified ADAM9 as a potential target for anticancer therapy. Patent WO2018119196A1 discloses a mouse-derived anti-human ADAM9 monoclonal antibody "MAB-A" (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 7 of the same patent, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 11 of the same patent), in which the VH and VL domains of MAB-A have been humanized, and the CDR has been optimized to improve affinity and / or eliminate potential unfavorable amino acid conditions. However, this patent had a problem: the humanized antibody hMAB-A(2I.2), which had entered the clinical research stage of MAB-A (the amino acid sequence of its heavy chain is shown in SEQ ID NO: 52 of the same patent, and the amino acid sequence of its light chain is shown in SEQ ID NO: 68 of the same patent), was highly hydrophobic.

[0007] Therefore, there is still a need for the development of humanized anti-ADAM9 antibodies that are less hydrophobic, have higher solubility, and offer superior coupling yield and drug potential as ADC (Antibody-Drug Conjugate) drugs. [Overview of the Initiative]

[0008] This application claims priority to Chinese Patent Application No. 2023103024365, filed on March 24, 2023, and to Chinese Patent Application No. 202410263320X, filed on March 7, 2024. This application incorporates the full text of the above Chinese Patent Application.

[0009] To address the technical problems of prior art, such as the strong hydrophobicity, low solubility, and low coupling yield and drug discovery potential of humanized anti-ADAM9 antibodies, the present invention provides a humanized antibody targeting ADAM9, its antibody-drug conjugate, and its use by re-humanizing the mouse monoclonal antibody MAB-A disclosed in patent WO2018119196A1. This improves the solubility of the humanized version antibody, reduces its hydrophobicity, and enhances its coupling yield and drug discovery potential as an ADC drug.

[0010] The present invention solves the above technical problems through the following technical solutions. The first aspect of the present invention provides an antibody or antigen-binding fragment thereof that targets ADAM9, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3. The HCDR1 comprises the amino acid sequence indicated by LYWMX1, the HCDR2 comprises the amino acid sequence indicated by X2IIPIFGHTX3YX4EKFX5X6, the HCDR3 comprises the amino acid sequence indicated by SEQ ID NO: 11, the LCDR1 comprises the amino acid sequence indicated by SEQ ID NO: 12, the LCDR2 comprises the amino acid sequence indicated by SEQ ID NO: 16, and the LCDR3 comprises the amino acid sequence indicated by SEQ ID NO: 19. However, X1 is N, D, H, or E; X2 is R or D; X3 is D or K; X4 is N or E; X5 is K or R; and X6 is D or N.

[0011] In some embodiments of the present invention, X1 in HCDR1 is N or H. In some embodiments of the present invention, in HCDR2, X2 is R, X3 is K, X4 is N, X5 is K, and X6 is D or N.

[0012] In some specific embodiments of the present invention, If X1 is N, then X2 is R, X3 is K, X4 is N, X5 is K, and X6 is D. If X1 is H, then X2 is R, X3 is K, X4 is N, X5 is K, and X6 is N.

[0013] In some embodiments of the present invention, the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 8, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0014] In another embodiment of the present invention, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0015] In another embodiment of the present invention, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 5, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0016] In another embodiment of the present invention, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 6, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0017] In another embodiment of the present invention, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0018] In another embodiment of the present invention, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 9, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0019] In another embodiment of the present invention, HCDR1 includes the amino acid sequence shown in SEQ ID NO: 4, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 10, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19.

[0020] In some specific embodiments of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0021] In another specific embodiment of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0022] In another specific embodiment of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0023] In another specific embodiment of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0024] In another specific embodiment of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0025] In another specific embodiment of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 9, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0026] In another specific embodiment of the present invention, the amino acid sequence of HCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19.

[0027] In the present invention, the heavy chain variable region and / or light chain variable region further include a framework region, the framework region being a human framework region.

[0028] In some embodiments of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 31, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 23.

[0029] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 42, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 43.

[0030] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 22, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 23.

[0031] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 24, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 25.

[0032] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 26, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 27.

[0033] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 28, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 30.

[0034] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 34, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 35.

[0035] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 36, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 30.

[0036] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 37, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 38.

[0037] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 36, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 27.

[0038] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 39, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 27.

[0039] In another embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 44, and / or the light chain variable region comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 45.

[0040] In the present invention, the variable region of the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence.

[0041] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.

[0042] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 42, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43.

[0043] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.

[0044] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25.

[0045] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27.

[0046] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30.

[0047] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 34, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35.

[0048] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30.

[0049] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38.

[0050] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27.

[0051] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27.

[0052] In another specific embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 44, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 45.

[0053] In the present invention, ADAM9 is preferably human or monkey ADAM9, and in some specific embodiments of the present invention, ADAM9 is human ADAM9.

[0054] In some embodiments of the present invention, the antibody or its antigen-binding fragment is: (1) a full-length antibody, Fab, Fab', F(ab')2, Fv, sdAb or scFv, and / or (2) a monoclonal antibody, a bispecific antibody or a multispecific antibody.

[0055] In the present invention, if the antibody or its antigen-binding fragment is a full-length antibody, it includes the heavy chain constant region of the heavy chain of a human antibody, preferably the heavy chain constant region of human antibody IgG1, and / or it includes the light chain constant region of the light chain of a human antibody, preferably the light chain constant region of the human antibody κ chain.

[0056] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is represented by SEQ ID NO: 48, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 48, and / or the amino acid sequence of the light chain constant region of the human antibody κ chain is represented by SEQ ID NO: 49, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 49.

[0057] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is shown in SEQ ID NO: 48, and the amino acid sequence of the light chain constant region of the human antibody κ chain is shown in SEQ ID NO: 49.

[0058] In some embodiments of the present invention, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 50, and / or the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 51.

[0059] In some embodiments of the present invention, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 52, and / or the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 53.

[0060] In the present invention, the amino acid sequences having at least 90%, at least 95%, or at least 99% sequence identity maintain at least the same antigen-binding function as the original sequence.

[0061] In some specific embodiments of the present invention, the amino acid sequence of the antibody or its antigen-binding fragment heavy chain is shown in SEQ ID NO: 50, and / or the amino acid sequence of the light chain is shown in SEQ ID NO: 51.

[0062] In some specific embodiments of the present invention, the amino acid sequence of the antibody or its antigen-binding fragment heavy chain is shown in SEQ ID NO: 52, and / or the amino acid sequence of the light chain is shown in SEQ ID NO: 53.

[0063] A second aspect of the present invention provides an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0064] A third aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid described in the second aspect. In some specific embodiments of the present invention, the recombinant expression vector's backbone plasmid is PTT5.

[0065] A fourth aspect of the present invention provides a transformant comprising the recombinant expression vector described in the third aspect. In some embodiments of the present invention, the host cell of the transformed organism is a eukaryotic cell. In some specific embodiments of the present invention, the eukaryotic cell is a CHO cell.

[0066] The fifth aspect of the present invention provides a method for producing an antibody targeting ADAM9 or an antigen-binding fragment thereof, comprising culturing the transformant according to the fourth aspect and isolating the antibody targeting ADAM9 or an antigen-binding fragment thereof from the culture.

[0067] The sixth aspect of the present invention provides a method for detecting ADAM9, comprising contacting the antibody according to the first aspect or an antigen-binding fragment thereof with a test sample. In some embodiments of the present invention, the detection is for non-diagnostic purposes and / or therapeutic purposes.

[0068] The seventh aspect of the present invention provides an antibody-drug conjugate comprising the above-mentioned antibody or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug. In some embodiments of the present invention, the cytotoxic drug is a structure represented by formula (A-1), a stereoisomer thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of the salt.

Chemical formula

[0069] In some embodiments of the present invention, L 1 is, -(C(R 1a )(R 1b )) m -CH2-, and each R 1a Each R is independently hydrogen, halogen, or C1-C6 alkyl, and each R 1b These are independently hydrogen, halogen, or C1-C6 alkyl.

[0070] In some embodiments of the present invention, L 1 teeth, [ka] That is the case.

[0071] In some embodiments of the present invention, L 1 is a C3-C6 saturated cycloalkyl group, and the C3-C6 saturated cycloalkyl group may optionally contain one or more R groups. 2a Replaced by each R 2a These are independently hydrogen, halogen, or C1-C6 alkyl.

[0072] In some embodiments of the present invention, [ka] That is the case.

[0073] In some embodiments of the present invention, the cytotoxic drug has one of the following structures. [ka]

[0074] In some embodiments of the present invention, -L a -teeth, [ka] Preferably [ka] Here, the a-terminus is ligated to Ab, and the b-terminus is L b It is connected to.

[0075] In some embodiments of the present invention, -L b - has one of the following structures: [ka] Preferably [ka] and, more [ka] And here, the c-terminus is L a It is linked to the d-terminus, and the d-terminus is L c It is connected to.

[0076] In some embodiments of the present invention, -L c -teeth [ka] That is the case.

[0077] In some embodiments of the present invention, the linker unit L is [ka] Preferably [ka] That is the case.

[0078] In some embodiments of the present invention, the structure of the antibody-drug conjugate is represented by formula (A-2). [ka] p represents the average number of connections or the number of connections, and p is one integer or decimal number between 1 and 10. Ab is the antibody or its antigen-binding fragment as described in the first section. M is as described in Section 7, L is the linker unit.

[0079] In some embodiments of the present invention, p is an integer or decimal between 3 and 9. In some embodiments of the present invention, p is an integer or decimal between 7 and 8, for example, 7.8 or 7.9.

[0080] In some embodiments of the present invention, the antibody-drug conjugate is selected from the following structural formulas. [ka] [ka] however, p is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 3 to 9, and Ab is the antibody or its antigen-binding fragment.

[0081] In some embodiments of the present invention, the antibody-drug conjugate is one of the following conjugates. [ka] p is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 3 to 9, more preferably an integer or decimal number from 6 to 8, and more preferably p is 7.8. [ka] p is an integer or decimal number between 1 and 10, preferably between 3 and 9, and more preferably between 6 and 8. For example, p is 7.9. [ka] p is an integer or decimal number between 1 and 10, preferably between 3 and 9, and more preferably between 6 and 8. For example, p is 7.8. Ab16 is an antibody or antigen-binding fragment that targets ADAM9. The amino acid sequence of the heavy chain of Ab16 is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain is shown in SEQ ID NO: 51. Ab15 is an antibody or antigen-binding fragment thereof that targets ADAM9. The amino acid sequence of the heavy chain of Ab15 is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain is shown in SEQ ID NO: 53.

[0082] In some embodiments of the present invention, the antibody-drug conjugate is the following conjugate. [ka] p represents the number of connections, and p is one integer from 1 to 10, preferably one integer from 3 to 9, more preferably one integer from 4 to 8, for example, p is 4, 5, 6, 7, or 8. Ab16 is an antibody or antigen-binding fragment thereof that targets ADAM9. The amino acid sequence of the heavy chain of Ab16 is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain is shown in SEQ ID NO: 51.

[0083] The eighth aspect of the present invention provides a pharmaceutical composition comprising an antibody or its antigen-binding fragment as described in the first aspect and / or an antibody-drug conjugate as described in the seventh aspect, and a pharmaceutically acceptable carrier.

[0084] The ninth aspect of the present invention provides the use of the antibody or its antigen-binding fragment described in the first aspect, the antibody-drug conjugate described in the seventh aspect, and / or the pharmaceutical composition described in the eighth aspect in the manufacture of a pharmaceutical for the diagnosis, prevention, and / or treatment of cancers that highly express ADAM9. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0085] A tenth aspect of the present invention provides a method for diagnosing, preventing, and / or treating cancers that highly express ADAM9, comprising administering to a patient in need a therapeutically effective amount of the antibody described in the first aspect or its antigen-binding fragment, the antibody-drug conjugate described in the seventh aspect, and / or the pharmaceutical composition described in the eighth aspect. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0086] The eleventh aspect of the present invention provides an antibody or its antigen-binding fragment as described in the first aspect, an antibody-drug conjugate as described in the seventh aspect, and / or a pharmaceutical composition as described in the eighth aspect, for use in the diagnosis, prevention, and / or treatment of cancers that highly express ADAM9. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0087] A twelfth aspect of the present invention provides a combination therapy comprising administering to a patient in need an antibody or antigen-binding fragment thereof described in the first aspect, an antibody-drug conjugate described in the seventh aspect and / or a pharmaceutical composition described in the eighth aspect, and a second therapeutic agent, respectively. In some embodiments of the present invention, the second therapeutic agent includes other anti-ADAM9 antibodies or their antigen-binding fragments, or antibody-drug conjugates or pharmaceutical compositions comprising the other anti-ADAM9 antibodies or their antigen-binding fragments, and / or pharmaceuticals for treating cancers that highly express other ADAM9. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0088] A thirteenth aspect of the present invention provides a method for diagnosing, preventing, and / or treating cancer, comprising the step of administering to a patient in need a therapeutically effective amount of the antibody described in the first aspect or its antigen-binding fragment, the antibody-drug conjugate described in the seventh aspect, and / or the pharmaceutical composition described in the eighth aspect. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0089] A fourteenth aspect of the present invention provides an antibody or its antigen-binding fragment as described in the first aspect, an antibody-drug conjugate as described in the seventh aspect, and / or a pharmaceutical composition as described in the eighth aspect, for diagnosing, preventing and / or treating cancer. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0090] A fifteenth aspect of the present invention provides a combination therapy comprising the step of administering to a patient in need an antibody or antigen-binding fragment thereof described in the first aspect, an antibody-drug conjugate described in the seventh aspect and / or a pharmaceutical composition described in the eighth aspect, and a second therapeutic agent, respectively. In some embodiments of the present invention, the second therapeutic agent includes other anti-ADAM9 antibodies or their antigen-binding fragments, or antibody-drug conjugates or pharmaceutical compositions comprising the other anti-ADAM9 antibodies or their antigen-binding fragments, and / or pharmaceuticals for treating other cancers. In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer. In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0091] A sixteenth aspect of the present invention provides a method for producing the antibody-drug conjugate described in the seventh aspect, which includes reacting the antibody described in the first aspect or its antigen-binding fragment with a compound represented by formula II to obtain the antibody-drug conjugate. [ka] L' forms the linker unit L described in the seventh aspect with the antibody or its antigen-binding fragment. The cytotoxic drugs mentioned above are as described in Section 7.

[0092] In some embodiments of the present invention, the antibody-drug conjugate satisfies one or more of the following conditions. (1) The compound represented by formula II is [ka] And, (2) The antibody or its antigen-binding fragment is Ab16 or Ab15, The amino acid sequence of the heavy chain of Ab16 is preferably shown in SEQ ID NO: 50, and the amino acid sequence of the light chain is preferably shown in SEQ ID NO: 51. The amino acid sequence of the heavy chain of Ab15 is preferably shown in SEQ ID NO: 52, and the amino acid sequence of the light chain is preferably shown in SEQ ID NO: 53.

[0093] A 17th aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the 1st aspect, the antibody-drug conjugate described in the 7th aspect, and / or the pharmaceutical composition described in the 8th aspect in the manufacture of a pharmaceutical for diagnosing, preventing and / or treating cancer.

[0094] In some embodiments of the present invention, the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer.

[0095] In some specific embodiments of the present invention, the cancer is a non-small cell lung cancer such as lung adenocarcinoma.

[0096] As long as it does not violate common sense in the art, the above preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. The reagents and raw materials used in this invention are commercially available.

[0097] The positive progressive effects of this invention are: The ADAM9-targeting antibody of the present invention has one or more of the following advantages: 1. Compared to the humanized anti-ADAM9 antibody hMAB-A(2I.2) in prior art, the antibody of the present invention has higher hydrophilicity, and the antibody molecule has better stability and drug discovery potential. 2. Compared to the humanized anti-ADAM9 antibody hMAB-A(2I.2) in the prior art, the antibody of the present invention exhibits significantly improved expression levels, a lower proportion of high molecular weight polymers, and superior drug discovery potential. 3. The antibodies of the present invention have good affinity for human and monkey ADAM9 antigens. 4. The antibody of the present invention has a very high internalization rate in cells expressing ADAM9.

[0098] The antibody-drug conjugate of the present invention has one or more of the following advantages: 1. The antibody-drug conjugate of the present invention exhibits excellent inhibitory activity against the proliferation of ADAM9-positive human colorectal cancer cells LS174T and Colo205, and human lung adenocarcinoma cells Calu-3. 2. The antibody-drug conjugate of the present invention exhibits significant antitumor activity against mice bearing human colon cancer cells DLD-1, mice bearing colon cancer cells LS174T, and mice bearing human lung cancer cells Calu-3. [Brief explanation of the drawing]

[0099] [Figure 1] These are the results of a tumor inhibition study of ADAM9-ADC in human colon cancer cell DLD-1 tumor-bearing mice. [Figure 2] These are the results of a tumor inhibition study of ADAM9-ADC in human colorectal cancer cell LS174T tumor-bearing mice. [Figure 3] These are the results of a tumor inhibition study of ADAM9-ADC in human lung cancer cell Calu-3-bearing mice. [Figure 4a] This is the result of detecting FACS binding in LS174T cells. [Figure 4b] These are the results of an experiment inhibiting the proliferation of LS174T-ADC. [Figure 5a] This is the result of detecting FACS binding in HepG2 cells. [Figure 5b] These are the results of an experiment inhibiting the proliferation of HepG2-ADC. [Modes for carrying out the invention]

[0100] definition In this invention, the letters in the amino acid sequence represent single-letter abbreviations for amino acids known in the art, as described, for example, in J. Biol. Chem, 243, p3558 (1968): Alanine: Ala-A, Arginine: Arg-R, Aspartic acid: Asp-D, Cysteine: Cys-C, Glutamine: Gln-Q, Glutamic acid: Glu-E, Histidine: His-H, Glycine: Gly-G, Asparagine: Asn-N, Tyrosine: Tyr-Y, Proline: Pro-P, Serine: Ser-S, Methionine: Met-M, Lysine: Lys-K, Valine: Val-V, Isoleucine: Ile-I, Phenylalanine: Phe-F, Leucine: Leu-L, Tryptophan: Trp-W, Threonine: Thr-T.

[0101] In the present invention, the term "and / or" should be understood to mean any one of the items, or any two or more of the items in any combination.

[0102] In this invention, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items within a list, “or” or “and / or” should be interpreted as inclusive, i.e., including at least one of the quantities or elements in the list, and more, and optionally additional items not listed. “Consists of” refers to only one number or one element of the list that is listed, only when a term such as “only” or “exactly one” explicitly states the opposite, or when used in a claim.

[0103] In the present invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linking unit. In this application, "antibody-drug conjugate" refers to an antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug fragment via a stable linking unit.

[0104] In this invention, the term "cytotoxic drug" generally refers to a toxic drug having a chemical molecule that potently disrupts the normal growth of tumor cells within those cells. Cytotoxic drugs can kill tumor cells at sufficient concentrations. The "cytotoxic drug" may include small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioisotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 It may also contain radioactive isotopes of Lu, toxic drugs, chemotherapeutic agents, antibiotics or nucleases, or derivatives thereof, for example, toxic drugs including but not limited to camptothecin derivatives, such as the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0105] In the present invention, the term “antibody” generally refers to an immunoglobulin that reacts with a specific protein or peptide or a fragment thereof. Antibodies may include, but are not limited to, any class of antibodies, including IgG, IgA, IgM, IgD, and IgE, and any subclass (such as IgG1, IgG2, IgG3, and IgG4). An antibody may be selected from, for example, IgG1, IgG2, IgG3, or IgG4, and may have a heavy chain constant region. An antibody may further have a light chain selected from, for example, kappa (κ) or lambda (λ). Antibodies in this application may originate from any species. The term “antibody” includes complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and other modified immunoglobulin molecules, insofar as these antibodies exhibit the desired biological activity.

[0106] In this invention, the term "antigen-binding fragment" generally refers to a part of an antibody molecule containing amino acids that are responsible for specific binding between the antibody and the antigen. The part of the antigen that is specifically recognized and bound by the antibody is called an "epitope." As described above, the antigen-binding domain can typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH), although not necessarily both. For example, an Fd fragment has two VH regions, which usually retain part of the antigen-binding function of the complete antigen-binding domain. Examples of antibody antigen-binding fragments include (1) Fab fragments, which are monovalent fragments having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab′)2 fragments, which are bivalent fragments of two Fab fragments linked by a disulfide bond in the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in a single antibody arm; (5) dAb fragments having a VH domain (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), the whole of which is incorporated into the specification by reference); (6) isolated complementarity-determining regions (CDRs); and (7) single-stranded Fv (scFv) derived from the scFV library.The two domains of the Fv fragment, VL and VH, are encoded by independent genes, but can be linked using a recombination method via a synthetic linker, which allows the VL and VH regions to pair up and form a monovalent molecule, thus enabling production as a single protein chain (single-chain Fv (referred to as scFv)) (see Huston et al., "Protein Engineering of AntibodyBinding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-ChainFv Analogue Produced in Escherichia coli," Proc.Natl.Acad.Sci.USA 85:5879-5883 (1988)), (8) "VHH" refers to the variable antigen-binding domain of heavy chain antibodies of camelids (camels, dromedaries, llamas, alpacas, etc.) (Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH is also called nanobody (Nb).

[0107] In this invention, the term "variable region" or "variable domain" generally refers to the domain of the antibody heavy or light chain involved in antibody-antigen binding. In this application, the term "variable" generally refers to a significant change in a specific portion of the sequence of the antibody's variable domain, resulting in the binding and specificity of each particular antibody to a specific antigen. Mutations are not evenly distributed throughout the antibody's variable domain. They are concentrated in three segments of the light and heavy chain variable regions, called complementarity-determining regions (CDRs) or highly variable regions (HVRs): LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively. The more highly conserved portion of the variable domain is called the framework region (FR). The natural heavy and light chain variable domains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which employ a β-sheet structure and are connected by three CDR structural loop regions. The CDRs of each chain are in close proximity to each other via the FR region, and together with the CDRs of other chains, they form the antigen-binding site of the antibody.

[0108] In this invention, all listed CDR amino acid sequences are shown according to Kabat's definition rules. However, as is well known to those skilled in the art, antibody CDRs can be defined in several ways, such as Chothia's definition based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al. "Standard conformations for the canonical structures of immunoglobulins" Journal of Molecular Biology, 273, 927-948 (1997)), Kabat's definition based on the variability of the antibody sequence (Kabat et al. "Sequences of Proteins of Immunological Interest" 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using numerous crystal structures. Those skilled in the art will understand, unless otherwise specified, that the terms “CDR” and “complementarity-determining region” for a given antibody or region thereof (e.g., variable region) encompass the complementarity-determining region as defined by any of the known schemes described in the present invention. While the claims of the present invention are based on sequences shown under the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of the present invention. Accordingly, when limiting an antibody using a specific CDR sequence as defined in the present invention, the range of that antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundary differs from the specific CDR boundary defined in the present invention as a result of applying a different scheme (e.g., a different assignment system rule or combination).

[0109] Sequence identity between sequences is calculated as follows: To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison (for example, gaps can be introduced between the first and second amino acid sequences for optimal alignment, or non-homologous sequences can be excluded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence to be aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. Next, amino acid residues at the corresponding amino acid positions are compared. If the position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, then the molecules are identical at that position. Mathematical algorithms can be used for sequence comparison between two sequences and for calculating the percentage of identity. In a preferred embodiment, the Needlema and Wunsch algorithm ((1970) J. Mol. Biol. 48:444-453) in the GAP program integrated into the GCG software package (available at http: / / www.gcg.com) is used to determine the identity percentage between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, with gap weights 16, 14, 12, 10, 8, 6, or 4, and length weights 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and parameter set to be used unless otherwise described) is a gap penalty of 12, a gap stretching penalty of 4, and a frameshift gap penalty of 5, using the Blossum 62 scoring matrix. Furthermore, the percentage of identity between two amino acid sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0), utilizing the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4.Additionally or alternatively, the protein sequence described in the present invention can be used as a "query sequence" to perform a search on a public database to identify, for example, other family member sequences or related sequences.

[0110] In this invention, the term "complete antibody" is used interchangeably to refer to a glycoprotein in which at least two heavy chains (HC) and two light chains (LC) are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified into α, δ, ε, γ, and μ. Mammalian light chains are classified into λ or κ. Immunoglobulins containing α, δ, ε, γ, or μ heavy chains are classified into immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The Y chain is formed by the joining of the second and third constant regions (and a fourth constant region in the case of IgE and IgM) of two heavy chains, with disulfide bonds (interchain) formed at the hinge region. Heavy chains γ, α, and δ have a constant region consisting of three tandem (aligned in a row) Ig domains and a hinge region to increase flexibility, while heavy chains μ and ε have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of Y consists of a single heavy chain containing a variable region that binds to a single light chain and a first constant region. The variable regions of the light chain and heavy chain are responsible for antigen binding.

[0111] In the present invention, "Fab" consists of one light chain and one heavy chain comprising the CH1 and variable regions. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. "Fab'" consists of one light chain and a portion of one heavy chain containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of the two Fab' molecules to form an F(ab')2 molecule. "F(ab')2" consists of two light chains and two heavy chains constituting a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab′)2 fragment consists of two Fab′ fragments linked by a disulfide bond between two heavy chains. The term "Fv" refers to an antibody fragment that consists of the VL and VH domains of one arm of the antibody but lacks a constant region.

[0112] In the present invention, the scFv (single-chain antibody fragment) is a typical single-chain antibody in the art, comprising a heavy-chain variable region, a light-chain variable region, and a short peptide of 5 to 20 amino acids. Here, the VL and VH domains are paired by a linker that allows them to be expressed as a single polypeptide chain, forming a monovalent molecule [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules generally have the structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable prior art linker consists of a repeat or variant thereof of the G4S amino acid sequence. For example, a linker having the amino acid sequence (G4S)4 or (G4S)3 can be used, but variants thereof can also be used.

[0113] In this invention, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies; that is, the individual antibodies constituting the group are identical except for any potentially present trace amounts of spontaneous mutations. Monoclonal antibodies are highly specific because they target a single antigen epitope. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of antibodies targeting (or specific to) various epitopes. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by a specific method.

[0114] The term "multispecific antibody" is used in its broadest sense and includes antibodies that possess multiple epitope specificities. These multispecific antibodies include, but are not limited to, antibodies containing a heavy chain variable region (VH) and a light chain variable region (VL) (where the VH-VL unit has multi-epitope specificity), antibodies having two or more VL and VH regions (each VH-VL unit binds to a different target or a different epitope on the same target), antibodies having two or more single variable regions (each single variable region binds to a different target or a different epitope on the same target), full-length antibodies, antibody fragments, bispecific antibodies (diabodies), trispecific antibodies (triabodies), and antibody fragments linked by covalent or non-covalent bonds.

[0115] In the present invention, the term "human antibody" refers to antibody forms that include sequences of human antibodies and non-human antibodies (e.g., mouse, rat). Generally, a human antibody contains substantially all of at least one, usually two, variable domains, where all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all framework (FR) regions are framework regions of human immunoglobulin sequences. A human antibody may optionally contain at least a portion of the constant region (Fc) of a human immunoglobulin.

[0116] In this invention, "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity between molecule X and its partner Y is generally expressed by the dissociation rate constant and the binding rate constant (k, respectively). dis and k on The equilibrium dissociation constant (K) is the ratio of ). D It is expressed as ). Affinity can be measured by common methods known in the art. In some embodiments of the present invention, surface plasmon resonance (SPR) technique is used to measure affinity, such as the affinity between the antibody and antigen of the present invention. In some preferred embodiments of the present invention, a specific method for measuring affinity is the BIAcore method described herein.

[0117] In the present invention, the term "halogen" usually refers to fluorine, chlorine, bromine, and iodine, and may be, for example, fluorine or chlorine. In the present invention, the term "alkyl" usually refers to a residue obtained by removing a hydrogen atom from an alkane. Alkyls may be substituted or unsubstituted, and may be substituted or unsubstituted. The term "alkyl" generally refers to a saturated linear or branched aliphatic hydrocarbon group having a residue derived by removing a hydrogen atom from the same carbon atom or two different carbon atoms of the parent alkane, and may be a linear or branched group containing 1 to 20 carbon atoms, for example, a linear alkyl group containing 1 to 12 carbon atoms, or for example, a chain alkyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkyls include, but are not limited to, methyl, ethyl, propyl, propyl, and butyl.

[0118] In the present invention, the term "cycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituents, where the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, and cyclooctane, while polycyclic cycloalkyls include spiro, condensed, and crosslinked cycloalkyls.

[0119] In the present invention, the term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, which comprises 3 to 20 ring atoms, of which one or more ring atoms are selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it comprises 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; more preferably, it comprises 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; and most preferably, it comprises 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranil, piperidinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyls include spirocyclic, fused, and bridging heterocyclyls. The heterocyclyl ring can be condensed with an aryl, heteroaryl, or cycloalkyl ring, and the ring linked to its parent structure is a heterocyclyl.

[0120] In this invention, the term “independently” generally means that the variable applies in all cases, regardless of whether the variable exists in the same compound under the same or different definitions. For example, the variable may refer to the type, number, or type of substituent in the compound or the type of atom in the compound. For example, if R appears twice in a compound and R is defined as “independently carbon or nitrogen,” both Rs may be carbon, both Rs may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0121] In the present invention, "optional" or "optionally" generally means that the matters or circumstances described below may occur but do not necessarily occur, and such description includes cases where the matters or circumstances described therein occur and cases where they do not occur. For example, "optionally alkyl-substituted multiple ring groups" means that alkyl may be present but is not necessarily required, and such description may include situations where multiple ring groups are substituted with alkyl and situations where multiple ring groups are not substituted with alkyl.

[0122] In the present invention, "substituted" generally refers to the substitution of one or more hydrogen atoms of a group, for example, up to five, and for example, one to three hydrogen atoms, independently with a corresponding number of substituents. The substituents are only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitutions with little effort (through experiment and theory). For example, when an amino or hydroxyl group containing free hydrogen is bonded to a carbon atom having an unsaturated (e.g., olefin) bond, it may be unstable.

[0123] In the present invention, as is known to those skilled in the art, terms such as "alkyl," "alkenyl," and "cycloalkyl" may be preceded by an identifier indicating the number of atoms present in the group in specific cases, such as C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc., and the subscript following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group with three carbon atoms (e.g., n-propyl, isopropyl), and C 1-10 Therefore, the members of the group can have any number of carbon atoms in the range of 1 to 10.

[0124] In the present invention, the compounds or antibody-drug conjugates of the present invention include their tautomers, mesomers, racemates, enantiomers, and / or diastereomers. In this application, the term “diastereomer” generally refers to stereoisomers having two or more chiral centers and whose molecules are non-mirror images of each other. Diastereomers may have different physical properties such as melting point, boiling point, spectral properties, and reactivity. In this application, the terms “tautomer” and “tautomer morph” are used interchangeably and generally refer to structural isomers of different energies that are interconvertible over a low energy barrier. For example, proton tautomers (also called prototropic tautomers) include interconversion via proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversion by rearrangement of some bonding electrons. In this application, the term "racemic compound" generally refers to a molecule that contains an asymmetric atom but has a symmetry factor such that the total optical rotation of the molecule is zero. The term "racemic mixture" or "racemic composition" refers to a composition consisting of equimolar amounts of two enantiomers.

[0125] In the present invention, the terms “linker unit” or “linker structure” generally refer to a chemical structural fragment or bond in which one end is linked to a ligand and the other end is linked to a cytotoxic drug, or a linker that is linked to another linker and then further linked to a cytotoxic drug. The directly or indirectly linked ligand refers to a group that is directly linked to the ligand via a covalent bond or linked to the ligand via a linker structure. For example, chemical structural fragments or bonds including acid-unstable linker structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linker structures, photo-unstable linker structures, dimethyl linker structures, or disulfide-containing linker structures can be used as linker structures.

[0126] In some embodiments of the present invention, the antibody-drug conjugate refers to a composition containing different DAR distributions, the term “drug loading capacity” generally refers to the average number of cytotoxic drugs loaded per ligand, also called the average number of conjugates, and is also expressed as the ratio of cytotoxic drugs to antibody amounts, and the range of cytotoxic drug loading is such that 0 to 12, for example 1 to 10 cytotoxic drugs can be conjugated per ligand (Ab). The drug loading capacity of each ADC molecule after the coupling reaction can be determined by conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA, and HPLC characterization. The average number of conjugates p may be an integer or decimal between 1 and 10. For example, the average number of conjugates p may be an integer or decimal between 2 and 8. For example, the average number of conjugates p may be an integer or decimal between 3 and 8. For example, the average number of conjugates p may be an integer or decimal between 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, or 9 and 10. For example, the average number of conjugates p is 7.8 or 7.9.

[0127] In some embodiments of the present invention, an antibody-drug conjugate refers to a compound containing the same DAR distribution, the term “drug loading capacity” refers to the number of cytotoxic drugs loaded per ligand, also called the number of conjugates, and is expressed as the ratio of cytotoxic drugs to antibody amount, and the range of cytotoxic drug loading is such that 0 to 12, for example 1 to 10 cytotoxic drugs can be conjugated per ligand (Ab). The number of conjugates p may be any integer from 1 to 10. For example, the number of conjugates p may be any integer from 3 to 9. For example, the number of conjugates p may be any integer from 4 to 8. For example, the number of conjugates p may be 4, 5, 6, 7, or 8.

[0128] In the present invention, specific atoms of the compound or antibody-drug conjugate of the present invention may exist in one or more isotopic forms. For example, hydrogen may be a hydrogen atom ( 1 H), deuterium ( 2 H) and tritium ( 3 It exists as H, and carbon has three different isotopes ( 12 C, 13 C, 14 It exists naturally as C). Examples of isotopes that can be incorporated into the compound of this application include: 15 N, 18 O, 17 O, 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125 This includes, but is not limited to, isotopes I or similar isotopes. Therefore, the compounds or antibody-drug conjugates of the present invention may be rich in one or more of these isotopes compared to their natural abundances. As isotopic-rich compounds are known to those skilled in the art, such isotopic-rich compounds can be used in a variety of applications. For example, deuterium ( 2 Substitution with heavier isotopes such as H) may offer certain therapeutic benefits due to improved metabolic stability. For example, deuterium ( 2The natural abundance of H) is approximately 0.015%. Therefore, in nature, there is approximately one deuterium atom for every 6500 hydrogen atoms. Accordingly, the deuterium-containing compounds or antibody-drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more positions (as may be determined). Unless otherwise defined, the structures described herein may include compounds or antibody-drug conjugates that differ only in the presence or absence of one or more isotope-rich atoms. For example, all compounds or antibody-drug conjugates having the same structure as the present invention except that a hydrogen atom is replaced with deuterium or tritium, or a carbon atom is replaced with carbon-13 or carbon-14 are within the scope of the present invention.

[0129] As is known to those skilled in the art, in the present invention, “nucleic acid” means a nucleotide chain of any length, including DNA and RNA. The nucleotide may be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0130] The recombinant expression vector of the present invention may be any suitable recombinant expression vector that can be used to deliver one or more target genes or sequences to any suitable host cell, and preferably to express said genes or sequences in the host cell. Suitable vectors include, but are not limited to, vectors designed for amplification and expansion, or expression, or both, and include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors.

[0131] In the present invention, the term “host cell” refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell may be a eukaryotic cell such as a plant, animal, fungus, or algae, or a prokaryotic cell such as a bacterium or protist. In exemplary aspects, as described herein, the host cell is a cell derived from or obtained from an individual. In exemplary aspects, the host cell is a cell derived from or obtained from a mammal.

[0132] In the present invention, the methods and conditions for culturing the generated transformants and recovering the generated antibody molecules are known to those skilled in the art and can be modified or optimized according to methods known herein and in the prior art, based on the specific expression vector and mammalian host cells used.

[0133] In the present invention, application scenarios for detection for non-diagnostic and / or non-therapeutic purposes include, for example, detecting the presence or absence of ADAM9 protein in a laboratory, or screening other ADAM9-targeting antibodies as positive antibodies, or detecting whether competition exists between antibodies by competitively binding with other anti-ADAM9 antibodies, i.e., whether the antigen epitopes are identical or similar.

[0134] In the present invention, the term “pharmaceutical composition” generally refers to a mixture of one or more compounds described in this application or their physiologically / pharmacologically acceptable salts or prodrugs with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to a living organism, enhance the absorption of the active ingredient, and thereby exert biological activity. Conventional methods for the manufacture of pharmaceutical compositions are described in the Chinese Pharmacopoeia. Pharmaceutical compositions may also be in the form of sterile water for injection or oily suspensions for intramuscular and subcutaneous administration. These suspensions can be manufactured according to known techniques using suitable dispersants or wetting agents and suspending agents as described above. Sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as 1,3-butanediol solution. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For example, any mixed fixative oil containing synthetic monoglycerides or diglycerides can be used. Furthermore, fatty acids such as oleic acid can also be used in the manufacture of injectable preparations.

[0135] In the present invention, the terms “pharmaceutically acceptable salt” or “pharmaceutically usable salt” generally refer to a salt of the compound or antibody-drug conjugate of the present invention, or a salt of the compound or antibody-drug conjugate described in the present invention. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The compound or antibody-drug conjugate of the present invention may also form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0136] In the present invention, a pharmaceutically acceptable carrier is any conventionally used carrier, limited only by physicochemical considerations (e.g., solubility and lack of reactivity with antibodies targeting ADAM9) and by the route of administration. The pharmaceutically acceptable carriers described herein, such as carriers, adjuvants, excipients, and diluents, are well known to those skilled in the art and readily available to the public. In one respect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of the pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some examples, when administered to animals or humans, the carrier does not cause adverse, allergic, or other inappropriate reactions. In some respects, the pharmaceutical composition does not contain pyrogens or other impurities harmful to humans or animals. Pharmaceutically acceptable carriers include all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, absorption retarders, etc., whose use is well known in the art.

[0137] As used in this invention, the term "effective dose" means the amount of drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human as desired by the researcher or clinician. Furthermore, the term "therapeutic effective dose" means the amount that, compared to a corresponding subject that has not received that dose, causes treatment, cure, prevention, or reduction of a disease, symptom, or side effect, or slows the rate of progression of the disease or condition. This term also includes the amount that effectively enhances normal physiological function.

[0138] In this invention, the term "cancer" refers to a malignant tumor, a disease caused by abnormalities in the mechanisms that control cell proliferation and growth. The term "colorectal cancer" generally refers to colorectal cancer, also called "linear colorectal cancer," and refers to cancers originating from colorectal epithelium, including colorectal cancer and rectal colorectal cancer. The term "lung adenocarcinoma" refers to a malignant tumor originating from the epithelial tissue of the lung, and is classified as non-small cell lung cancer. The term "cancer with high ADAM9 expression" refers to a malignant tumor caused by abnormally high expression of ADAM9 in vivo.

[0139] The present invention will be further described below with reference to embodiments, but this does not limit the present invention to the scope of the above embodiments. In the following embodiments, experimental methods for which specific conditions are not described are selected according to conventional methods and conditions or according to the product description.

[0140] Example 1: Re-humanization modification of MAB-A antibody The purpose of re-humanizing the mouse anti-ADAM9 monoclonal antibody MAB-A antibody (the sequence disclosed in patent WO2018119196A1, where the heavy chain variable region is sequence number 7 and the light chain variable region is sequence number 11) is to improve the solubility of the humanized version antibody, reduce the hydrophobicity of the antibody, and thereby increase the coupling yield and drug potential as an ADC drug. The humanized version antibody hMAB-A(2I.2), which is in the clinical research stage of MAB-A (sequence information of heavy chain sequence number 52 and light chain sequence number 68 disclosed in patent WO2018119196A1), has the problem of being highly hydrophobic.

[0141] The re-humanization of mouse antibodies was carried out according to methods known in the literature in the field, namely, the constant region of the mouse antibody was replaced with the constant region of the human antibody, and modifications were made to the CDR region with the aim of optimizing affinity and drug potential. Based on the homology between the parent mouse antibody MAB-A of hMAB-A(2I.2) and the human antibody, a human species antibody sequence was selected, and the MAB-A antibody was re-humanized. As a specific method, based on the typical VH / VL CDR structure of the mouse-derived antibody MAB-A, the heavy chain and light chain variable region sequences were compared with a human antibody germline database to obtain highly homologous human germline templates. The mouse antibody CDR region was then transplanted into the selected corresponding humanized template. Subsequently, based on the three-dimensional structure of the mouse antibody, reverse mutations were performed on buried residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH. Chemically unstable amino acid residues in the CDR region were optimized, and after expression tests and comparison of the number of reverse mutations, antibodies combining the designed humanized heavy chain variable region HCVR and light chain variable region LCVR sequences were selected. The CDR region sequence numbers of the humanized antibodies are shown in Table 1, the amino acid sequences of the CDRs are shown in Table 2, and the heavy chain and light chain variable region sequences of the humanized antibodies are shown in Table 3.

[0142] Table 1. CDR region sequence numbers of humanized antibodies [Table 1]

[0143] Table 2 Amino acid sequences of the CDR region of humanized antibodies [Table 2]

[0144] In Table 2, the CDR region of the antibody is defined based on the Kabat numbering system, where X1 may be amino acid N, D, H, or E; X2 may be amino acid D or R; X3 may be amino acid K or D; X4 may be amino acid N or E; X5 may be amino acid R or K; and X6 may be amino acid D or N.

[0145] Table 3 Variable region sequences of heavy and light chains of humanized antibodies [Table 3-1]

[0146] [Table 3-2]

[0147] [Table 3-3]

[0148] [Table 3-4]

[0149] [Table 3-5]

[0150] In Table 3, underlined amino acid sequences indicate CDR regions defined based on the Kabat numbering system. The designed heavy chain and light chain variable region sequences were ligated to the heavy chain constant region and light chain constant region sequences of a human antibody, respectively. For example, the antibody heavy chain constant region was selected from the human IgG1 heavy chain constant region, the sequence of which is shown in SEQ ID NO: 48; the antibody light chain constant region was selected from the human κ chain constant region, the sequence of which is shown in SEQ ID NO: 49; and the antibody constant region is indicated by 4.

[0151] Table 4 Steady-state region sequence numbers [Table 4]

[0152] The heavy chain amino acid sequence of Ab16 is (SEQ ID NO: 50): QVQLQQPGTEVVKPGASVKLSCKASGFTFPLYWMHWVRQRPGRGLEWIGRIIPIFGHTKYNEKFKNKATLTADESSSTAYMELRSLTSDDSAVYYCARGGYYYYPREGFLDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain amino acid sequence of Ab16 is (SEQ ID NO: 51): DIVMTQSPASLSVSPGERASISCRSSQSVDYSGDSYMNWYQQKPGQSPRLLIYAASDRESGVPDRFSGSGSGTDFTLKISRVEEEDAGTYYCQQSREDPFTFGQGTKVD IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The heavy chain amino acid sequence of Ab15 is (SEQ ID NO: 52): QVQLQQPGAELKKPGASVKVSCKASGFTFPLYWMNWVRQRPGRGLEWIGRIIPIFGHTKYNEKFKDRATMTADESSSTAYMELSSLTSEDSAVYYCARGGYYYYPREGFLDYW GQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The light chain amino acid sequence of Ab15 is (SEQ ID NO: 53): DIVMTQSPDSLSVSPGERATINCRSSQSVDYSGDSYMNWYQQKPGQSPRLLIYAASDRESGVPDRFSGSGSGTDFTLKISNVEEEDAATYYCQQSREDPFTFGQGTRVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0153] Example 2: Hydrophobic detection of humanized anti-ADAM9 antibody Experimental steps: The hydrophilicity and hydrophobicity of different antibodies were evaluated by HIC-HPLC analysis. The stronger the hydrophilicity of the antibody, the weaker the hydrophobic interaction with the HIC ligand butyl, and the shorter the retention time. All antibodies were replaced with 200 mM Arg. SSC pH 5.5 buffer, adjusted to a concentration of 5 mg / mL, and the hydrophobicity of each re-humanized anti-ADAM9 antibody and hMAB-A(2I.2) antibody was detected using the chromatography method described below (Table 5). The experimental results are shown in Table 6.

[0154] Table 5 Chromatography detection method [Table 5]

[0155] Table 6 Results of Chromatography Detection Experiments [Table 6]

[0156] Experimental conclusion: The above experimental results confirm that, after structural design and modification, the hydrophilicity of the modified antibody was significantly improved, and the stability and drug potential of the antibody molecule were also improved.

[0157] Example 3: Production and expression of humanized anti-ADAM9 antibody The light chain variable region amino acid sequences of the 20 antibodies from Example 1, Ab1-Ab20, and hMAB-A(2I.2), were each ligated to the light chain κ constant region amino acid sequence (SEQ ID NO: 49), and the heavy chain variable region amino acid sequences were each ligated to the IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 48). cDNA was synthesized by codon optimization and ligated to a PTT5 plasmid (commissioned to Nanjingjinsirui Science & Technology Biology Corp.), using the same signal peptide sequence for all 21 antibodies. The PTT5 plasmids corresponding to the heavy and light chains of each antibody were co-introduced into CHO cells using PEImax 40000 and cultured for 5 days. After collecting the cell culture supernatant, the antibody components were purified with Protein A (MabSelect™ PrismA), and the antibodies were quantified at OD280nm (commissioned to Nanjingjinsirui Science & Technology Biology Corp.). The 20 antibodies in Example 1 were expressed in a 4 ml reaction system, and the expression levels of each antibody are shown in Table 7 below.

[0158] Table 7: Antibody production volume expressed in 4 ml reaction systems [Table 7]

[0159] Experimental conclusion: The expression levels of Ab06, Ab10, Ab15, and Ab16 were approximately three times higher than those of hMAB-A(2I.2). Four antibodies, Ab10, Ab15, Ab16, and hMAB-A(2I.2), were expressed in a 500 ml reaction system, and the expression levels of each antibody are shown in Table 8 below.

[0160] Table 8 Antibody production expressed in a 500 ml reaction system [Table 8]

[0161] Experimental conclusion: The expression levels of Ab15 and Ab16 were much higher than those of hMAB-A(2I.2), and SEC-HPLC detection showed a lower proportion of high molecular weight polymers, suggesting superior drug potential compared to hMAB-A(2I.2).

[0162] Example 4: Experiment to detect in vitro binding activity of humanized anti-ADAM9 antibody 4.1 In vitro indirect ELISA binding experiment Human ADAM-His protein (AcroBiosystems, Cat#AD9-H52H7) was diluted to 1 μg / ml in pH 7.4 PBS and added to a 96-well high-affinity ELISA plate at a volume of 100 μL / well. The plates were incubated overnight (16-20 hours) at 4°C. After washing the plates three times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 200 μL / well of 1% bovine serum albumin (BSA) blocking solution diluted with PBST was added, and the plates were incubated at 37°C for 1 hour to perform blocking. After blocking was complete, the blocking solution was removed, and the plates were washed once with PBST buffer. A PBST test antibody containing 1% BSA was diluted, starting at 15 μg / mL, and then diluted 3-fold in 11 fractions. The diluted antibody was added to the ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. After culturing, the plates were washed three times with PBST, and 100 μL / well of HRP-labeled goat anti-human secondary antibody (Rockland, cat#609-103-123) diluted in PBST containing 1% BSA was added and incubated at room temperature for 45 minutes. After washing the plates six times with PBST, 100 μL / well of TMB chromogenic substrate (Suzhou Yacoo Chemical Reagent Corporation, cat#S0025) was added and incubated at room temperature under light-shielding conditions for 10 minutes. The reaction was stopped by adding 50 μL / well of 1 M HCl, and the absorbance at 450 nm was measured using a microplate reader (Thermo, Ascent), and the data were analyzed. Concentration-signal curves were created and the results were analyzed, as shown in Table 9 below. The results showed that the antibody of the present invention showed good affinity for the human ADAM9 antigen.

[0163] Table 9 Affinity of re-humanized antibodies against human ADAM9 antigen (EC 50 value) [Table 9]

[0164] 4.2 Biacore affinity measurement experiment Chip preparation: Mouse anti-human IgG(Fc) antibody (catalog number: 29234600, Cytiva) was diluted to 25 μg / mL with fixation reagent (10 mM sodium acetate, pH 5.0). Specifically, 50 μL of mouse anti-human IgG(Fc) antibody was added to 950 μL of fixation reagent and used for fixing 8 channels. First, the surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 seconds. Next, 25 μg / mL of mouse anti-human IgG(Fc) antibody was injected into the experimental channel at a flow rate of 10 μL / min for approximately 360 seconds, resulting in a fixation volume of 7000-14000 RU. Finally, the chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds. The reference channel was operated in the same manner as the experimental channel.

[0165] Capture ligands: Antibodies Ab10, Ab15, Ab16, and hMAB-A(2I.2) were diluted to 4 μg / mL with a running reagent (10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20, adjusted to pH 7.4) and injected into the human IgG(Fc) capture experimental channel at a flow rate of 10 μL / min and approximately 200 RU. A reference channel is not required for ligand capture.

[0166] Multicycle analysis of analytes: Human and cynomolgus monkey ADAM9 proteins (human ADAM9 protein purchased from ACRO, catalog number: AD9-H52H7 / monkey ADAM9 protein purchased from ACRO, catalog number: AD9-C52H7) were serially diluted 2-fold with operating buffer (Table 10). The diluted human or cynomolgus monkey ADAM9 proteins were sequentially injected into the experimental channel and reference channel at a flow rate of 30 μL / min and bound and dissociated for appropriate time. All binding and dissociation steps were performed in the operating reagent. After each concentration analysis, the tip was regenerated with 3M magnesium chloride at a flow rate of 20 μL / min for 30 seconds to wash away ligand and undissociated analytes. When performing the next concentration analysis, the same amount of ligand needed to be recaptured in the experimental channel.

[0167] Table 10 Concentration gradient and binding / dissociation time in affinity studies of anti-ADAM9 antibody against human and cynomolgus monkey ADAM9 protein. [Table 10]

[0168] Data Analysis: The KD value for each sample was calculated using Biacore 8K analysis software, Biacore Insight Evaluation Software. The reference channel was used for background correction. The results of the in vitro indirect ELISA binding experiment and the Biacore method are shown in Tables 11 and 12.

[0169] Table 11 Affinity of antibodies against human ADAM9 antigen [Table 11]

[0170] Table 12 Affinity of antibodies against monkey ADAM9 antigen [Table 12]

[0171] From the results of ELISA and Biacore, it was shown that the Ab15 and Ab16 antibodies have good affinity for human and monkey ADAM9 antigens.

[0172] Example 5: Internalization ability of humanized Anti-ADAM9 antibody The binding plate was set up. After trypsin-digesting ADAM9 highly-expressing human lung cancer cells Calu-3 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu157), the cells were collected by centrifugation. After adjusting the cell density with FACS buffer (1×PBS containing 2% FBS), they were dispensed into a 96-well U-bottom plate (1×105 cells / well), centrifuged at 1200 g for 5 minutes, the supernatant was removed, and an antibody solution serially diluted with 100 μL of FACS buffer (the working concentration of the antibody was 100 nM, 10-fold dilution, 8 concentration points, and a 0 nM point was set) was added, and cultured at 4°C for 1 hour. Centrifuged at 1200 g for 5 minutes, the supernatant was removed, the cells were washed twice with PBS, and FITC-fluorescently labeled goat anti-human IgG H&L antibody (Abcam, Cat#ab6866) prepared with FACS buffer was added. After resuspending the cells at 100 μL / well, they were cultured at 4°C for 1 hour. Centrifuged at 1200 g for 5 minutes, the supernatant was removed. After washing the cells twice with PBS, they were resuspended in PBS, and the mean fluorescence intensity (MFI 結合ウェル ) of the cells under the action of antibodies at each concentration was measured with a flow cytometer.

[0173] At the same time, an endocytosis plate was set up. After trypsinizing Calu-3 cells, they were centrifuged to collect the cells. The cell density was adjusted with FACS buffer, and then dispensed into a 96-well U-bottom plate (1×105 cells / well). It was centrifuged at 1200 g for 5 minutes, the supernatant was removed, and an antibody solution serially diluted with 100 μL of FACS buffer (initial working concentration of the antibody was 100 nM, 10-fold dilution, 8 concentration points, and a 0 nM point was set) was added. The cells were cultured at 37 °C for 16 hours. Then, it was centrifuged at 1200 g for 5 minutes, the supernatant was removed, the cells were washed twice with PBS, and a FITC-fluorescently labeled goat anti-human IgG H&L antibody prepared with FACS buffer was added. The cells were resuspended at 100 μL / well and cultured at 4 °C for 1 hour. It was centrifuged at 1200 g for 5 minutes, and the supernatant was removed. After washing the cells twice with PBS, they were resuspended in PBS, and the mean fluorescence intensity (MFI エンドサイトーシスウェル ) of the cells under the action of each concentration of antibody was measured with a flow cytometer.

[0174] The antibody internalization rate under the action of each concentration of antibody was calculated by the following formula: Internalization rate % = (MFI 結合ウェル - MFI エンドサイトーシスウェル ) / MFI 結合ウェル ×100%. The EC 50 concentration of antibody internalization was determined by concentration-curve analysis, and the results are shown in Table 13.

[0175] Table 13 Antibody internalization rate

Table 13

[0176] From the results, all of the Ab10, Ab15, and Ab-16 antibodies showed a high internalization rate against cells expressing ADAM9.

[0177] Example 6: Synthesis of linker-cytotoxin [[ID=​​​​​​​​​​​ [ka]

[0179] Step 1: Under the protection of nitrogen gas, benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol) in DMF (50 mL), and the mixture was reacted at 25°C for 17 hours. After confirming the completion of the reaction by TLC (PE / EA=2 / 1), the reaction mixture was added to 500 mL of water, extracted twice with EA (250 mL), separated, washed with saturated sodium chloride aqueous solution (500 mL), dried over anhydrous Na2SO4, concentrated, and then extracted by column (PE:EA=3:2) to obtain 5.1 g of colorless liquid, with a yield of 57.1%.

[0180] Step 2: Under the protection of nitrogen gas, a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) was added dropwise to a solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) at 0 °C, and the mixture was reacted at 25 °C for 2 hours. After confirming the completion of the reaction by TLC (PE / EA=1 / 2), the reaction mixture was added to 200 mL of water, extracted twice with EA (200 mL), separated, dried over anhydrous Na2SO4, concentrated, and then analyzed by column (PE / EA=3 / 2) to obtain 1.56 g of a white solid, with a yield of 26%.

[0181] Step 3: Under a hydrogen gas atmosphere, at 0°C, Pd / C (80 mg) was added to a mixed solution of 27c (800 mg, 1.55 mmol) EtOH (8 mL) and EA (8 mL), and the mixture was stirred at 0°C for 2.5 hours. LC-MS indicated that the reaction was complete. The reaction mixture was filtered through diatomaceous earth, the cake was washed with EA (200 mL), concentrated, dissolved in THF (20 mL), and spin-dried to obtain 600 mg of a white solid with a yield of 91%.

[0182] Step 4: Under the protection of nitrogen gas, at 0°C, DIEA (152 mg, 1.18 mmol) was added to a solution of 27d (220 mg, 0.515 mmol), HY-13631A (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol) in DMF (6 mL), and the mixture was reacted at 0°C for 2 hours. LC-MS indicated that the reaction was complete. The reaction mixture was added to an aqueous citric acid solution (pH=4) (150 mL), filtered, the cake was washed with 175 mL of water, filtered and dried, and then dried by suction using an oil pump to obtain 260 mg of brown solid, with a yield of 66%.

[0183] Step 5: Under the protection of nitrogen gas, at 0°C, 8 ml of diethylamine was added dropwise to a 30 mL solution of 27 e (260 mg, 0.309 mmol) of DCM and the mixture was allowed to react at 0°C for 3 hours. LC-MS indicated that the reaction was complete. The reaction mixture was added to a 600 mL solution of petroleum ether at 0°C, and a solid precipitated. After standing until the solid was adsorbed to the bottom of the bottle, the solution was removed and dried with an oil pump to obtain 90 mg of brown solid, with a yield of 47.1%.

[0184] Step 6: Under the protection of nitrogen gas, at 0°C, HATU (74 mg, 0.19 mmol) was added to a 2.5 mL solution of 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL), and the mixture was reacted at 0°C for 2 hours. LC-MS indicated that the reaction was basically complete. At 0°C, the reaction mixture was added to a pH=4 aqueous citric acid solution (30 mL), and a cotton-like solid precipitated. After filtration, the mixture was treated on a manufacturing plate (DCM / MeOH = 10 / 1) to obtain 9.2 mg of pale yellow solid X1, with a yield of 6%.

[0185] MS m / z (ESI): 1074 [M+1]. H-NMR (400 MHz, MeOD): δ 7.65 (d, 1H), 7.62 (s, 1H), 7.30 - 7.21 (m, 5H), 6.79 (s, 2H), 5.69 - 5.65 (m, 1H), 5.57 (d, 1H), 5.43 - 5.10 (m, 3H), 4.70 (d, 2H), 4.48 - 4.39 (m, 2H), 4.10 - 4.05 (m, 1H), 4.01 - 3.75 (m, 5H), 3.46 (t, 2H), 3.22 - 3.15 (m, 2H), 3.07 - 3.00 (m, 1H), 2.75 (m, 1H), 2.62 (m, 1H), 2.45 (s, 3H), 2.37 - 2.20 (m, 6H), 2.10 - 2.02 (m, 2H), 2.00 - 1.92 (m, 2H), 1.68 - 1.57 (m, 6H), 1.01 (t, 3H).

[0186] Linker-Cytotoxin X2:

Chem.

[0190] Step 3 Under a hydrogen gas atmosphere, at 0°C, a mixed solution of 34d (1.9 g, 3.78 mmol) of EtOH (100 mL) and EA (100 mL) was added to Pd / C (1 g, 10 wt.%) and reacted at 0°C for 3 hours. TLC (PE / EA = 2 / 1) indicated the completion of the reaction. The reaction mixture was filtered through diatomaceous earth, the cake was washed with EA / EtOH (1:1, 100 mL x 3), the filtrate was concentrated, dissolved in THF (50 mL x 3), and spin-dried. This process was repeated three times to obtain 1 g of gray solid 34e, with a yield of 64%. MS-ESI: m / z 435.2[M+Na]+.

[0191] Step 4 Under the protection of nitrogen gas, at 0°C, DIEA (303 mg, 2.35 mmol) was added dropwise to a 20 mL solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) in DMF (20 mL). After the addition was complete, the mixture was allowed to react at 0°C for 2 hours. LC-MS indicated that the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, allowed to stand for 5 minutes, filtered, and the cake was dissolved in a DCM / MeOH (10:1, 100 mL) solution. After drying and spin-drying, the sample was mixed, and 600 mg of yellow solid 34f was obtained by column chromatography (EA:MeOH = 30:1) with a yield of 77%. MS-ESI: m / z 830.3[M+H]+.

[0192] Step 5 Under the protection of nitrogen gas, at 0°C, 5 mL of diethylamine was added dropwise to a 34 f (150 mg, 0.18 mmol) DCM (5 mL) solution and the mixture was reacted at 0°C for 2 hours. LC-MS indicated that the reaction was complete. Six 100 mL packets of petroleum ether solution were added to the reaction mixture, and after allowing the mixture to stand until the solid precipitated, the solution was removed and the mixture was further dried with an oil pump to obtain 34 g of 120 mg white powder. LC-MS analysis showed a product content of 70% and a yield of 76%. MS-ESI: m / z 608.3[M+H]+.

[0193] Step 6 Under the protection of nitrogen gas, 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) in DMF (1 mL) solutions were added to HATU (45 mg, 0.118 mmol) in DMF (1 mL) at 0°C, and the mixture was reacted at 0°C for 2 hours. LC-MS showed that the starting materials had reacted completely. The reaction mixture was passed directly through a reversed-phase column, and the eluate (MeCN / MeOH = 1 / 1):H2O = 60%:40%) was purified to obtain 14.8 mg of yellow solid X2, with a yield of 14%.

[0194] MS-ESI:m / z 1062.4 [M+H]+. 1HNMR (400 MHz, Methanol-d4) δ 7.69 - 7.61 (m, 2H), 7.22 - 7.16 (m, 2H), 7.16 - 7.09 (m, 3H), 6.76 (s, 2H), 5.70 - 5.64 (m, 1H), 5.60 (d, J = 16.4 Hz, 1H), 5.40 - 5.31 (m, 2H), 5.26 (d, J = 19.0 Hz, 1H), 4.65 - 4.50 (m, 7H), 4.25 - 4.16 (m, 1H), 3.87 (d, J = 16.7 Hz, 1H), 3.83 - 3.76 (m, 3H), 3.72 (d, J = 17.0 Hz, 2H), 3.44 (t, J = 7.1 Hz, 2H), 3.25 - 3.17 (m, 2H), 3.10 - 3.02 (m, 1H), 2.92 - 2.83 (m, 1H), 2.45 - 2.39 (m, 5H), 2.32 - 2.20 (m, 5H), 1.97 - 1.89 (m, 2H), 1.63 - 1.50 (m, 4H), 1.34 - 1.20 (m, 6H), 0.99 (t, J = 7.3 Hz, 3H).

[0195] Rinkar-Cytotoxin X3:

change

[0196] Synthetic road:

change

[0197] ステップ1: Allyl bromide (960 mg, 7.92 mmol) was added to MeCN (20 mL) containing 32a (2.00 g, 6.6 mmol) and K2CO3 (1.82 g, 13.2 mmol), and the mixture was stirred at 20°C for 5 hours. TLC (PE / EA=1 / 2) indicated that the reaction was complete. The reaction mixture was poured into 100 mL of water, the pH was adjusted to 5, and the solution was extracted three times with EA (100 mL). After drying over anhydrous sodium sulfate, the solution was spin-dried and purified by column chromatography (PE / EA=2 / 1) to obtain 1.83 g of white solid 32b, with a yield of 81%.

[0198] Step 2: 10 mL of TFA was added to 10 mL of DCM containing 32b (1.38 g, 4.02 mmol), and the mixture was stirred at 25°C for 17 hours. TLC (PE / EA = 1 / 3) indicated that the reaction was complete. The reaction mixture was spin-dried to obtain 0.91 g of yellow viscous substance 32c, and the yield was not calculated.

[0199] Step 3: 32c (910 mg, 4.87 mmol) and NaHCO3 (613 mg, 7.3 mmol) were mixed in DME / H2O (20 mL / 10 mL) with 41d (1.92 g, 4.87 mmol) and stirred at 25°C for 3 hours. TLC (DCM / MeOH = 1 / 1) indicated the completion of the reaction. The reaction mixture was poured into 100 mL of water, the pH was adjusted to 5 with aq.HCl (1N), extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, then spin-dried, and purified by column (DCM / MeOH = 20 / 1) to obtain 1.53 g of white solid 32e, yielding 67%. MS-ESI: m / z 467.4 [M+H]+.

[0200] Step 4: Pd / C (600 mg) was added to 32f (3 g, 5.83 mmol) MeOH (50 mL) and stirred at 25°C under hydrogen balloon for 5 hours. TLC (EA) indicated that the reaction was complete. The reaction mixture was filtered and spin-dried to obtain 32 g of 1.9 g of white solid, with a yield of 77%.

[0201] Step 5: 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol) were added to DMF (10 mL), and HATU (707 mg, 1.86 mmol) was added. The mixture was stirred at 0°C for 3.5 hours. TLC (EA) indicated that the reaction was complete. The reaction mixture was poured into H2O (80 mL), extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, and then spin-dried. Purification by column (EA) yielded 1.186 g of a white solid 32 h, with a yield of 83%. MS-ESI: m / z 842.3 [M+H]+.

[0202] Step 6: A 32-hour (1.186 g, 1.41 mmol) solution of DCM / diethylamine (20 mL, 20 / 1) was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 10 / 1) indicated the completion of the reaction. The reaction mixture was poured into petroleum ether (200 mL), filtered, and yielded 768 mg of a white solid 32i with a yield of 88%. MS-ESI: m / z 620.3 [M+H]+.

[0203] Step 7: HATU (414 mg, 1.09 mmol) was added to DMF (10 mL) containing 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), and DIEA (423 mg, 3.27 mmol), and the mixture was stirred at 20°C for 17 hours. TLC (PE / EA = 1 / 5) indicated the completion of the reaction. The reaction mixture was poured into water (30 mL), filtered, and the cake was purified by column chromatography (DCM / MeOH = 50 / 1) to obtain 511 mg of white solid 32j, with a yield of 44%. MS-ESI: m / z 1068.3 [M+H]+.

[0204] Step 8: A solution of 32j (482 mg, 0.451 mmol) diethylamine / DCM (10 mL, 1 / 5) was stirred at 10°C for 17 hours. TLC(EA) indicated that the reaction was complete. The reaction mixture was poured into PE (300 mL) and filtered to obtain 301 mg of a white solid 32k. The yield was not calculated.

[0205] Step 9: Morpholine (93 mg, 1.07 mmol) was added to 5 mL of THF containing 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol), and the mixture was stirred at 25°C for 5 hours. LC-MS indicated that the reaction was complete. The reaction solution was prepared to obtain 32 L of a 108 mg white solid, with a yield of 38%. MS-ESI: m / z 806.3 [M+H]+.

[0206] Step 10: Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32 L (108 mg, 0.134 mmol) of triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL), and the mixture was stirred at 0°C for 1 hour. TLC (DCM / MeOH = 10 / 1) indicated that the reaction was complete. The reaction solution was directly prepared to obtain 15 mg of white solid X3, with a yield of 12%.

[0207] MS-ESI:m / z 926.3 [M+H]+. 1HNMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.54 - 8.42 (m, 3H), 8.27 - 8.16 (m, 2H), 7.78 (d, J = 11.0 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.61 - 5.51 (m, 1H), 5.42 (s, 2H), 5.20 - 5.05 (m, 2H), 4.56 - 4.42 (m, 2H), 4.32 - 4.22 (m, 1H), 3.96 - 3.87 (m, 3H), 3.79 (d, J = 5.6 Hz, 2H), 3.70 (d, J = 5.9 Hz, 2H), 3.25 - 3.08 (m, 2H), 2.61 - 2.53 (m, 2H), 2.45 - 2.36 (m, 4H), 2.36 - 2.22 (m, 3H), 2.20 - 2.03 (m, 4H), 1.99 - 1.68 (m, 4H), 0.87 (t, J = 7.3 Hz, 3H).

[0208] Linker-Cytotoxin X4: [ka]

[0209] Synthesis pathway: [ka]

[0210] Step 1: 33a (2.00 g, 2.58 mmol) was mixed with MeOH (20 mL) and Pd / C (400 mg, 10 wt.%), and the mixture was stirred at 20°C for 5 hours. TLC (EA) indicated that the reaction was complete. The reaction mixture was filtered and spin-dried to obtain 1.3 g of white solid 33b, with a yield of 74%.

[0211] Step 2: HATU (305 mg, 0.802 mmol) was added to DMF (5 mL) containing 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol), and DIPEA (310 mg, 2.40 mmol), and the mixture was stirred at 0°C for 2 hours. TLC (DCM / MeOH = 1 / 10) indicated that the reaction was complete. The reaction mixture was poured into water (40 mL), filtered to obtain the crude product, and purified by column (DCM / MeOH = 20 / 1) to obtain 360 mg of yellow solid 33c, with a yield of 41%.

[0212] Step 3: Diethylamine (2 mL) was added to 33c (360 mg, 0.326 mmol) in DCM (10 mL). The mixture was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 5 / 1) indicated that the reaction was complete. The reaction mixture was poured into PE (100 mL) and filtered to obtain 205 mg of a white solid 33d, with a yield of 71%. MS-ESI: m / z 881.3 [M+H]+.

[0213] Step 4: A solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) was added to 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL). The mixture was stirred at 0°C for 1 hour to directly prepare the reaction solution, yielding 15 mg of white solid X4 with a yield of 6%.

[0214] MS-ESI:m / z 1001.2 [M+H]+. 1HNMR (400 MHz, DMSO-d6) δ 8.57 - 8.50 (m, 1H), 8.50 - 8.43 (m, 2H), 8.35 - 8.29 (m, 1H), 8.19 - 8.12 (m, 2H), 7.80 (d, J = 10.8 Hz, 1H), 7.27 - 7.14 (m, 7H), 6.53 (s, 1H), 5.59 - 5.51 (m, 1H), 5.44 - 5.39 (m, 2H), 5.20 - 5.07 (m, 2H), 4.56 - 4.44 (m, 3H), 3.92 (s, 3H), 3.80 - 3.68 (m, 5H), 3.41 (s, 1H), 3.21 - 3.12 (m, 2H), 2.83 - 2.74 (m, 1H), 2.58 - 2.55 (m, 3H), 2.39 (s, 4H), 2.18 - 2.03 (m, 4H), 1.93 - 1.78 (m, 2H), 0.87 (t, J = 7.3 Hz, 3H).

[0215] Example 7: Anti-ADAM9-conjugated camptothecin-linker-cytotoxin Manufacturing of the antibody-drug conjugate Ab16-X1: [ka]

[0216] To a buffer solution of antibody Ab16 (PBS pH 7.2, 40 mg, 8.7 mg / mL, 0.27 μmol), 0.2 mL of 2 mM EDTA solution and 7.0 mM, 0.424 mL, 2.97 μmol of prepared tris(2-carbonylethyl)phosphine hydrochloride solution were added. The mixture was placed in a thermostatic stirrer and reacted at 60 rpm and 22°C for 18 hours, after which the reaction was stopped. Linker-cytotoxin X1 (2.6 mg, 2.43 μmol) was dissolved in 0.24 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer and reacted with shaking at 60 rpm and 22°C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using an AKTA system with a G-25 gel column (desalting column: HiPrep 26 / 10 desalting column, 53 mL, elution phase: 30 mM histidine hydrochloride, pH 5.5). After concentration in a 30 KD ultrafiltration tube, the exemplary product Ab16-X1 solution (30 mM histidine hydrochloride, pH 5.5, 38 mg, 5.6 mg / mL, yield: 95%) was obtained and stored at -80°C. The DAR value p = 7.8 was calculated by HIC DAR analysis and detection.

[0217] Manufacturing of the antibody-drug conjugate Ab16-X2: [ka]

[0218] To a buffer solution of antibody Ab16 (PBS pH 7.2, 40 mg, 8.7 mg / mL, 0.27 μmol), 0.2 mL of 2 mM EDTA solution and 7.0 mM, 0.424 mL, 2.97 μmol of prepared tris(2-carbonylethyl)phosphine hydrochloride solution were added. The mixture was placed in a thermostatic stirrer and reacted at 60 rpm and 22°C for 18 hours, after which the reaction was stopped. Linker-cytotoxin X2 (2.6 mg, 2.43 μmol) was dissolved in 0.24 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer and reacted with shaking at 60 rpm and 22°C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using an AKTA system with a G-25 gel column (desalting column: HiPrep 26 / 10 desalting column, 53 mL, elution phase: 30 mM histidine hydrochloride, pH 5.5). After concentration in a 30 KD ultrafiltration tube, the exemplary product Ab16-X2 solution (30 mM histidine hydrochloride, pH 5.5, 37 mg, 5.6 mg / mL, yield: 92%) was obtained and stored at -80°C. HIC DAR analysis detected and calculated a DAR value of p=7.9.

[0219] Manufacturing of the antibody-drug conjugate Ab15-X2: [ka]

[0220] To a buffer solution of antibody Ab15 (PBS pH 7.4, 40 mg, 6.8 mg / mL, 0.27 μmol), 0.2 mL of 2 mM EDTA solution and 7.0 mM, 0.471 mL, 3.29 μmol of prepared tris(2-carbonylethyl)phosphine hydrochloride solution were added. The mixture was placed in a thermostatic stirrer and reacted at 60 rpm and 22°C for 18 hours, after which the reaction was stopped. Linker-cytotoxin X2 (2.6 mg, 2.43 μmol) was dissolved in 0.24 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer and reacted with shaking at 60 rpm and 22°C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using an AKTA system with a G-25 gel column (desalting column: HiPrep 26 / 10 desalting column, 53 mL, elution phase: 30 mM histidine hydrochloride, pH 5.5). After concentration in a 30 KD ultrafiltration tube, the exemplary product Ab15-X2 solution (30 mM histidine hydrochloride, pH 5.5, 32 mg, 5.2 mg / mL, yield: 80%) was obtained and stored at -80°C. HIC DAR analysis detected and calculated a DAR value of p=7.8.

[0221] Manufacturing of the antibody-drug conjugate hMAB-A(2I.2)-X2: To a buffer solution of antibody hMAB-A(2I.2) (PBS pH 7.4, 40 mg, 7.5 mg / mL, 0.27 μmol), 0.2 mL of 2 mM EDTA solution and 7.0 mM, 0.379 mL, 2.65 μmol of prepared tris(2-carbonylethyl)phosphine hydrochloride solution were added. The mixture was placed in a thermostatic stirrer and reacted at 60 rpm and 22°C for 18 hours, after which the reaction was stopped. Linker-cytotoxin X2 (2.6 mg, 2.43 μmol) was dissolved in 0.24 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer and reacted with shaking at 60 rpm and 22°C for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using an AKTA system with a G-25 gel column (desalting column: HiPrep 26 / 10 desalting column, 53 mL, elution phase: 30 mM histidine hydrochloride, pH 5.5). After concentration in a 30 KD ultrafiltration tube, the exemplary product hMAB-A(2I.2)-X2 solution (30 mM histidine hydrochloride, pH 5.5, 29 mg, 3.2 mg / mL, yield: 72%) was obtained and stored at -80°C. HIC DAR analysis detected and calculated a DAR value of p=7.5.

[0222] Example 8: Extracorporeal cell proliferation inhibitory activity test of anti-ADAM9 antibody-drug conjugate (ADAM9-ADC) Using the CellTiter-Glo® chemiluminescent cell viability measurement method (CTG method), the cell proliferation inhibitory effect of ADC drugs (anti-ADAM9 antibodies conjugated with camptothecin toxins) was evaluated in ADAM9-positive expressing human colorectal cancer cells LS174T (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu32) or Colo205 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu102), and human lung adenocarcinoma cells Calu-3, after 6 days of culture treatment.

[0223] Cells in the logarithmic growth phase were collected, seeded at a density of 5000 cells / well, and the cell plates were cultured overnight in a 37°C, 5% CO2 incubator. On day 2 of the experiment, each ADC drug was diluted 3-fold in complete medium to obtain nine concentration gradients (starting from the highest concentration of 300 nM). 100 μL / well of each drug was added to the cell culture plate, and three replicates were set up using complete medium as a blank control. These were cultured for 6 days in a 37°C, 5% CO2 incubator. After culturing, the cell culture plates were removed and allowed to equilibrate at room temperature. 50 μL of CTG detection reagent (Promega, Cat#: G7573) was added to each well, and after shaking to mix uniformly, the plates were allowed to stand in the dark for 10 minutes. Signal values ​​were detected and read using a microplate reader. An S-type dose-response curve was plotted using a nonlinear regression model with GraphPad Prism software, and IC was calculated. 50 The value was calculated. Cell viability calculation formula = (Lum 試験薬 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 ) × 100%.

[0224] The experimental results are shown in Table 14 below. The results indicate that Ab15-X2, Ab16-X1, and Ab16-X2 showed good inhibitory activity against the proliferation of human colorectal cancer or lung adenocarcinoma cells.

[0225] Table 14 Inhibitory activity of antibody-drug conjugates against human colorectal cancer or human lung adenocarcinoma cells [Table 14]

[0226] Example 9: In vivo tumor inhibition study of anti-ADAM9 antibody-drug conjugate (ADAM9-ADC) in human colon cancer cell DLD-1 tumor-bearing mice. To evaluate the in vivo tumor formation inhibitory effect of ADAM9-ADCs, transplanted tumors were induced in mice using ADAM9-positive expressing human colorectal cancer cells DLD-1 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number: TCHu134), and the antitumor effects of each ADAM9-ADC were evaluated.

[0227] (1) Test drugs and materials Blank control group (control group): Saline solution, tail vein injection, single dose Ab15-X2 (treatment group): 5 mg / kg, administered via tail vein injection, single dose. Ab16-X2 (treatment group): 5 mg / kg, administered via tail vein injection, single dose. hMAB-A(2I.2)-X2 (treatment group): 5 mg / kg, tail vein injection, one dose

[0228] (2) Preparation method: All samples were prepared by diluting them with physiological saline.

[0229] (3) Test animals: 8-week-old female NOD SCID mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0230] (4) Test method: 5 x 10 6 Individual DLD-1 cells were inoculated subcutaneously into the right anterior scapula of 8-week-old female NOD SCID mice, resulting in a tumor volume of approximately 125 mm². 3 Upon reaching a certain stage, tumor-bearing mice were randomly assigned to groups using StudyDirector™, and on the same day (day 0), they received a single intravenous dose of ADC at a dose of 5 mg / kg. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0231] Six mice were assigned to either a solvent control group or a treatment group. Tumor volume was measured, and the tumor inhibition rate was calculated. Inhibition rate (TGI%) = 100% - (Treatment group tumor volume on measurement day - Treatment group tumor volume on day 0) / (Control group tumor volume on measurement day - Control group tumor volume on day 0).

[0232] The experimental results are shown in Figure 1 and Table 15. The antibody-drug conjugates Ab15-X2 and Ab16-X2 showed significant tumor inhibitory activity after a single dose, and were significantly superior to hMAB-A(2I.2)-X2.

[0233] Table 15. In vivo antitumor effects of antibody-drug conjugates in a human colorectal cancer xenograft model. [Table 15]

[0234] Example 10: In vivo tumor inhibition study of anti-ADAM9 antibody-drug conjugate (ADAM9-ADC) in human colorectal cancer cell LS174T tumor-bearing mice To evaluate the in vivo tumor formation inhibitory effect of ADAM9-ADCs, transplanted tumors were induced in mice using ADAM9-positive human colorectal cancer cells LS174T, and the antitumor effects of each ADAM9-ADC were assessed.

[0235] (1) Test drugs and materials Blank control group (control group): Saline solution, tail vein injection (IV), one dose. Ab16-X2 (treatment group): 5 mg / kg, tail vein injection (iv), single dose DS-8201 (treatment group): 5 mg / kg, administered intravenously (iv), once. Here, I purchased the DS-8201 (Enhertu, from Daiichi Sankyo Company Limited).

[0236] (2) Preparation method: All samples were prepared by diluting them with physiological saline.

[0237] (3) Test animals: 8-week-old female BALB / c-Nu mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0238] (4) Test method: 5 x 10 6 Individual LS174T cells were inoculated subcutaneously into the right anterior scapula of 8-week-old female BALB / c-Nu mice, resulting in a tumor volume of approximately 130 mm². 3Upon reaching a certain stage, tumor-bearing mice were randomly assigned to groups using StudyDirector™, and on the same day (day 0), they received a single intravenous dose of ADC at a dose of 5 mg / kg. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0239] Six mice were assigned to either a solvent control group or a treatment group. Tumor volume was measured to calculate the tumor inhibition rate. Inhibition rate (TGI%) = 100% - (Treatment group tumor volume on measurement day - Treatment group tumor volume on day 0) / (Control group tumor volume on measurement day - Control group tumor volume on day 0). The tumor volume of the control group mice was 3000 mm³. 3 The mice were euthanized once the tumor volume exceeded a certain threshold, and the tumor growth index (TGI) was calculated based on the tumor volume at the time of euthanasia.

[0240] The experimental results are shown in Figure 2 and Table 16, where the antibody-drug conjugate Ab16-X2 showed significant tumor inhibitory activity after a single dose.

[0241] Table 16. In vivo antitumor effects of antibody-drug conjugates in a human colorectal cancer xenograft model. [Table 16]

[0242] Example 11: In vivo tumor inhibition study of anti-ADAM9 antibody-drug conjugate (ADAM9-ADC) in human lung cancer cell Calu-3-bearing mice To evaluate the in vivo tumor formation inhibitory effects of Ab16-X1 and Ab16-X2, ADAM9-positive human lung cancer cells (Calu-3) were used to induce transplanted tumors in mice, and their antitumor effects were assessed.

[0243] (1) Test drugs and materials Blank control group (control group): Saline solution, administered via tail vein injection, once every 7 days, twice. Ab16-X1 (treatment group): 5 mg / kg, administered via tail vein injection, once every 7 days for two doses. Ab16-X2 (treatment group): 5 mg / kg, administered via tail vein injection, once every 7 days for two doses.

[0244] (2) Preparation method: All samples were prepared by diluting them with physiological saline.

[0245] (3) Test animals: 8-week-old female CB-17 SCID mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0246] (4) Test method: 1 x 10 7 Individual Calu-3 cells were inoculated subcutaneously into the right anterior scapula of 8-week-old female CB-17 SCID mice, resulting in a tumor volume of approximately 130 mm². 3 Upon reaching a certain stage, tumor-bearing mice were randomly assigned to groups using StudyDirector™, and ADC drugs were administered intravenously (iv) at a dose of 5 mg / kg once every 7 days for a total of two doses, starting on the same day (day 0). Tumor volume and body weight were measured twice a week, and the data were recorded.

[0247] Five mice were assigned to either a solvent control group or a treatment group. Tumor volume was measured and the tumor inhibition rate was calculated. Inhibition rate (TGI%) = 100% - (Treatment group tumor volume on measurement day - Treatment group tumor volume on day 0) / (Control group tumor volume on measurement day - Control group tumor volume on day 0). If the tumor regressed (tumor volume at measurement was smaller than tumor volume at the time of initial administration), the inhibition rate = [1 - (Treatment group tumor volume on measurement day ÷ Treatment group tumor volume on day 0) ÷ (Control group tumor volume on measurement day ÷ Control group tumor volume on day 0)] ÷ [1 - (Control group tumor volume on day 0 ÷ Control group tumor volume on measurement day)] × 100%.

[0248] The experimental results are shown in Figure 3 and Table 17, where both the antibody-drug conjugates Ab16-X1 and Ab16-X2 showed significant tumor inhibitory activity after administration.

[0249] Table 17 In vivo antitumor effects of antibody-drug conjugates in a human colorectal cancer xenograft model [Table 17]

[0250] Example 12: Cell proliferation inhibitory effect of anti-ADAM9 antibody-drug conjugate (ADAM9-ADC) on low cell binding activity 12.1 Extracorporeal cell proliferation inhibitory activity test of LS174T cells Tumor cells co-expressing ADAM9 and Her2 were used to detect the cell-binding activity of Ab16-X2. DS-8201 (Enhertu, purchased from Daiichi Sankyo Company Limited) was used as a positive control, and anti-HIV human IgG antibody (iso-IgG1, WuXi AppTec product) was used as a negative control. HepG2 (Chinese Academy of Sciences Cell Bank, catalog number: TCHu72) and LS174T cells expressing moderate to low levels of ADAM9 were seeded at 1E5 cells / well in 96-well round-bottom plates. 100 μL of gradient-diluted Ab16-X2, DS-8201, or iso-IgG1 was added, and the cells were cultured at 4°C for 1 hour. After culturing, the cells were washed with FACS buffer (PBS containing 2% FBS), and PE fluorescently labeled goat anti-human secondary antibody (Abcam, ab98596) was added. The cells were cultured at 4°C for 0.5 hours. After culturing was complete, the cells were washed with FACS buffer, resuspended in FACS buffer, and then detected using a flow cytometer.

[0251] The cytotoxic effects of Ab16-X2 and DS-8201 on LS174T (cultured for 3 days) and HepG2 (cultured for 6 days) cells were evaluated using the CellTiter-Glo® chemiluminescent cell viability measurement method (CTG method). Cells in the logarithmic growth phase were collected, seeded at a density of 3000 cells / well, and cultured overnight in a 37°C, 5% CO2 incubator. On the second day of the experiment, the drugs were diluted 3-fold in complete medium to obtain nine concentration gradients (starting from the highest concentration of 1000 nM). 100 μL / well of each gradient was added to the cell culture plate, and three replicates were set up with complete medium as a blank control. These were then cultured for 3 or 6 days in a 37°C, 5% CO2 incubator. After culturing was complete, the cell culture plate was removed and allowed to equilibrate at room temperature. 50 μL of CTG detection reagent was added to each well, and after shaking to mix uniformly, the plate was left to stand in the dark for 10 minutes. Luminescence signal values ​​were detected and read using a microplate reader. An S-type dose-response curve was plotted using a nonlinear regression model with GraphPad Prism software, and IC was calculated. 50 The value was calculated. Cell viability calculation formula = (Lum 試験薬 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 ) × 100%. Proliferation inhibition rate = 100% - cell viability %.

[0252] The results of FACS binding detection in LS174T cells are shown in Table 18 and Figure 4a. Ab16-X2 showed low binding affinity (average fluorescence intensity) to LS174T cells, while DS-8201 showed high binding affinity to LS174T cells. At the maximum cell binding level, DS-8201 was approximately 2 to 3 times higher than Ab16-X2 (concentration range of 0.976 nM to 1000 nM), indicating that DS-8201 has significantly higher affinity for LS174T than Ab16-X2.

[0253] The results of CTG in vitro proliferation inhibition are shown in Table 19 and Figure 4b. The results show that when the culture concentration reached 1.372 nM, the proliferation inhibition rate of Ab16-X2 was similar to that of DS-8201. As the culture concentration increased, in the concentration range of 4.115 nM to 1000 nM, the proliferation inhibition rate of Ab16-X2 significantly exceeded that of DS-8201. These results indicate that Ab16-X2 showed a strong cell proliferation inhibitory effect even under conditions of low cell binding activity, whereas this effect was not observed with DS-8201.

[0254] Table 18 Comparison of binding activity of Ab16-X2 and DS-8201 to LS174T cells. [Table 18]

[0255] Table 19 Comparison of in vitro proliferation inhibitory activity of Ab16-X2 and DS-8201 against LS174T cells. [Table 19]

[0256] 12.2 Inhibitory activity of HepG2 cells in extracorporeal cell proliferation For measuring HepG2 cell binding activity and cell proliferation inhibitory activity, refer to Example 12.1; the specific experimental steps are the same as for LS174T cells.

[0257] The results of FACS binding detection in HepG2 cells are shown in Table 20 and Figure 5a. At culture concentrations of 0.061035 nM to 62.5 nM, Ab16-X1 showed low binding affinity (average fluorescence intensity) to HepG2 cells, while DS-8201 showed higher binding affinity under the same concentration conditions. This experiment confirmed that the HepG2 binding affinity of DS-8201 was significantly higher than that of Ab16-X1 within this culture concentration range.

[0258] The results of the hand CTG in vitro toxicization experiment are shown in Table 21 and Figure 5b. In the concentration range of 0.051 nM to 333.33 nM, the proliferation inhibition rate of Ab16-X1 was superior to that of DS-8201. From these results, it became clear that Ab16-X1 still showed a strong cell proliferation inhibitory effect even under conditions of low cell binding activity, but this effect was not observed with DS-8201.

[0259] Table 20 Comparison of binding activity of Ab16-X1 and DS-8201 to HepG2 cells [Table 20]

[0260] Table 21 Comparison of in vitro proliferation inhibitory activity of Ab16-X1 and DS-8201 against HepG2 cells [Table 21]

[0261] Example 13: Pre-toxicity study of cynomolgus monkeys with anti-ADAM9 antibody-drug conjugate (ADAM9-ADC). Cynomolgus monkeys were administered AB16-X2 intravenously once every three weeks for a total of two doses. The nature, extent, and temporal relationship of potential toxic reactions caused by the antibody-drug conjugate were observed, and target organs or tissues were preliminaryly identified.

[0262] method Two cynomolgus monkeys (provided by Suzhou Xishan Zhongke Experimental Animal Co., Ltd), one female and one male, were intravenously injected with AB16-X2. The first dose was 45 mg / kg, and the dose was increased to 80 mg / kg for the second dose three weeks later. Dissection was performed one week after the second dose. The following detection and observation indicators were included during the period.

[0263] (1) General condition observation: During the study period, external signs, mental state, behavioral activity, excretory material characteristics, and administration site were observed 2-3 times a day. (2) Body weight: Measured once during the acclimatization period and once a week during the administration period. (3) Food intake: Measured once daily. (4) Ophthalmic examination: Measured once during the acclimatization period and once each on D14, before dissection, or at the end of the administration period. (5) Electrocardiogram: Measured once during the acclimatization period and once each 6 hours and 24 hours after the completion of the first and second doses. (6) Clinical pathology: Measured once during the acclimatization period and collected once each on D3, D7, D14, D21, and D28 of the administration period. (7) Toxicity (8) Gross anatomical observation and histopathological examination: The sternum (including bone marrow), heart, lungs, kidneys, bladder, duodenum, colon, cecum, eyeballs, skin, and macroscopically abnormal organs / tissues (spleen, rectum) of the planned dissection animals were subjected to standard histological processing and histopathological examination.

[0264] conclusion Under the conditions of this study, when 45-80 mg / kg of AB16-X2 was administered intravenously to cynomolgus monkeys, all animals survived throughout the study period, either until the end of the administration period or until the planned dissection day, and no animals were morbid or died. After administration of AB16-X2, only transient mild leukopenia and transient mild elevations of ALT and AST were observed in cynomolgus monkeys; no other pathological changes were observed.

[0265] IMGC-936, developed by MacroGenics, is an ADAM9-ADC coupled with the meitansine-type microtubule inhibitor DM21-C and is currently in clinical research. In a monkey toxicity study, ocular toxicity, specifically corneal lesions, was observed after administration of IMGC-936 at a dose of 22.5 mg / kg (Mol Cancer Ther 2022;21:1047-59). In a monkey toxicity study, AB16-X2 did not show ocular toxicity even at a dose of 80 mg / kg.

[0266] Although specific embodiments of the present invention have been described above, those skilled in the art will understand that these are merely illustrative descriptions, and various changes and modifications can be made to these embodiments, provided that they do not contradict the principles of the present invention in substance. Therefore, the scope of protection of the present invention is limited by the appended claims.

Claims

1. It includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes heavy chain complementarity determination regions HCDR1, HCDR2 and HCDR3, and the light chain variable region includes light chain complementarity determination regions LCDR1, LCDR2 and LCDR3, The HCDR1 comprises the amino acid sequence represented by LYWMX1, and the HCDR2 comprises X ２ IIPIFGHTX ３ YX ４ EKFX ５ X ６ The HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:

19. However, X １ is N, D, H or E, and X ２ is R or D, and X ３ is D or K, and X ４ is N or E, and X ５ is K or R, and X ６ is D or N. An antibody targeting ADAM9 or its antigen-binding fragment.

2. In the HCDR1, X １ is N or H, and in the HCDR2, X ２ R is X ３ is K, and X ４ N is X ５ is K, and X ６ is D or N, preferably, X １ However, if N, then X ２ R is X ３ is K, and X ４ N is X ５ is K, and X ６ It is D, X １ However, if H, then X ２ R is X ３ is K, and X ４ N is X ５ is K, and X ６ The antibody or antigen-binding fragment thereof according to claim 1, characterized in that is N.

3. The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 8, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, or The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, or The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 5, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, or The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 6, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, or The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, or The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 9, the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 19, or The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that HCDR1 includes the amino acid sequence shown in SEQ ID NO: 4, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 10, HCDR3 includes the amino acid sequence shown in SEQ ID NO: 11, LCDR1 includes the amino acid sequence shown in SEQ ID NO: 12, LCDR2 includes the amino acid sequence shown in SEQ ID NO: 16, and LCDR3 includes the amino acid sequence shown in SEQ ID NO:

19.

4. The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 5, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 6, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 3, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, or The amino acid sequence of HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 9, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 19, or The antibody or antigen-binding fragment according to claim 3, characterized in that the amino acid sequence of HCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 10, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 12, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

19.

5. The heavy chain variable region and / or light chain variable region further include a framework region, the framework region being a human framework region, preferably The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 31, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 23, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 42, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 43, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 22, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 23, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 24, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 25, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 26, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 27, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 28, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 30, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 34, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 35, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 36, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 30, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 37, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 38, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 36, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 27, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 39, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 27, or The heavy chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 44, and / or the light chain variable region includes an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:

45. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that the variable region of the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same antigen-binding function as the original sequence.

6. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 31, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

23. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 42, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 43, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 25, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 34, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 35, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 30, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 37, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 38, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 36, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27, or The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 39, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27, or The antibody or antigen-binding fragment according to claim 5, characterized in that the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 44, and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

45.

7. The antibody or its antigen-binding fragment is (1) Full-length antibody, Fab, Fab', F(ab') ２ , Fv, sdAb or scFv, and / or, (2) Monoclonal antibody, bispecific antibody or multispecific antibody, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that the ADAM9 is human or monkey ADAM9.

8. If the antibody or its antigen-binding fragment is a full-length antibody, it includes the heavy chain constant region of the heavy chain of the human antibody, preferably the heavy chain constant region of the human antibody IgG1, and / or the light chain constant region of the light chain of the human antibody, preferably the light chain constant region of the human antibody κ chain. Preferably, the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is represented by SEQ ID NO: 48, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 48, and / or the amino acid sequence of the light chain constant region of the human antibody κ chain is represented by SEQ ID NO: 49, or has at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO:

49. More preferably, the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 50, and / or the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 51, or the heavy chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 52, and / or the light chain of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 53, and the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least equivalent antigen-binding function to the original sequence. More preferably, the antibody or antigen-binding fragment according to claim 7, characterized in that the amino acid sequence of the antibody or its antigen-binding fragment heavy chain is shown in SEQ ID NO: 50 and / or the amino acid sequence of the light chain is shown in SEQ ID NO: 51, or the amino acid sequence of the antibody or its antigen-binding fragment heavy chain is shown in SEQ ID NO: 52 and / or the amino acid sequence of the light chain is shown in SEQ ID NO:

53.

9. An isolated nucleic acid characterized by encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A recombinant expression vector comprising the isolated nucleic acid described in claim 9, preferably characterized in that the backbone plasmid is PTT5.

11. A transformant comprising the recombinant expression vector described in claim 10, preferably characterized in that the host cell is a eukaryotic cell, and more preferably the eukaryotic cell is a CHO cell.

12. A method for producing an antibody targeting ADAM9 or an antigen-binding fragment thereof, comprising the steps of culturing the transformant described in claim 11, and separating from the culture to obtain an antibody targeting ADAM9 or an antigen-binding fragment thereof.

13. A method for detecting ADAM9, comprising the step of contacting an antibody or antigen-binding fragment thereof described in any one of claims 1 to 8 with a test sample, preferably for non-diagnostic and / or therapeutic purposes.

14. An antibody-drug conjugate comprising an antibody or its antigen-binding fragment, a linker unit L, and a cytotoxic drug according to any one of claims 1 to 8.

15. The antibody-drug conjugate according to claim 14, characterized in that the cytotoxic drug is a structure represented by formula (A-1), a stereoisomer thereof, a pharmaceutically acceptable salt, a solvate, or a solvate of a salt thereof. 【Chemistry 1】 (however, M is -L ２ -L １ -C(O)-, L ２ is -O- or -S- and L ２ It is connected to the linker unit L, L １ is -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ -, C ３ ~C ６ The C is a saturated cycloalkyl or a 3-6 member saturated heterocycline. ３ ~C ６ Saturated cycloalkyls and 3- to 6-membered saturated heterocyclines each independently contain one or more R ２ａ Replaced by choice, m is selected from 1, 2, 3, or 4, and the heteroatoms in the 3-6 member saturated heterocycline are independently N, O, and S, and the number of heteroatoms is 1, 2, or 3. Each R １ａ , R １ｂ and R ２ａ These are independently hydrogen, halogen, hydroxyl, amino, or C １ ~C ６ Alkyl, and the C １ ~C ６ Alkyl is optionally substituted with one or more R groups. Each R is independently either hydrogen or a halogen.

16. L １ is -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - and each R １ａ These are independently hydrogen, halogen, or C １ ~C ６ It is alkyl, and each R １ｂ These are independently hydrogen, halogen, or C １ ~C ６ The antibody-drug conjugate according to claim 15, wherein the conjugate is alkyl.

17. L １ teeth, 【Chemistry 2】 The antibody-drug conjugate according to claim 16.

18. L １ C ３ ~C ６ It is a saturated cycloalkyl, and the C ３ ~C ６ Saturated cycloalkyl is one or more R ２ａ This is optionally replaced by each R ２ａ These are independently hydrogen, halogen, or C １ ~C ６ The antibody-drug conjugate according to claim 15, wherein the conjugate is alkyl.

19. L １ teeth, 【Transformation 3】 The antibody-drug conjugate according to claim 18.

20. The antibody-drug conjugate according to claim 15, wherein the cytotoxic drug has one of the following structures. 【Chemistry 4】

21. The linker unit L is -L ａ -L ｂ -L ｃ - and the L ｃ This is linked to the cytotoxic drug, -L ａ -teeth, 【Transformation 5】 Preferably, 【Transformation 6】 Here, the a-terminus is connected to Ab, and the b-terminus is connected to L. ｂ It is connected to, -L ｂ - is one of the following structures: 【Transformation 7】 Preferably 【Transformation 8】 and, more 【Chemistry 9】 And here, the c-terminus is L ａ It is connected to the d-terminus, and the d-terminus is L ｃ It is connected to -L ｃ -teeth 【Chemistry 10】 The antibody-drug conjugate according to any one of claims 14 to 20, its isomer, a pharmaceutically acceptable salt thereof, or a mixture thereof.

22. The linker unit L is 【Chemistry 11】 Preferably 【Chemistry 12】 The antibody-drug conjugate according to claim 21.

23. An antibody-drug conjugate according to any one of claims 14 to 22, wherein the structure is represented by formula (A-2). 【Chemistry 13】 (wherein p represents the average number of connections or the number of connections, and p is one integer or decimal number from 1 to 10, preferably one integer or decimal number from 3 to 9, more preferably an integer or decimal number from 7 to 8, for example, 7.8 or 7.9.) Ab is an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, M is as described in any one of claims 14 to 20, L is the linker unit described in claim 21 or 22.

24. An antibody-drug conjugate according to any one of claims 14 to 23, selected from the following structural formulas. 【Chemistry 14】 【Chemistry 15】 (however, p represents the average number of connections or the number of connections, and p is one integer or decimal number from 1 to 10, preferably one integer or decimal number from 3 to 9. Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

25. The antibody-drug conjugate is one of the following conjugates: 【Chemistry 16】 p is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 3 to 9, more preferably an integer or decimal number from 6 to 8, and more preferably p is 7.

8. 【Chemistry 17】 p is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 3 to 9, more preferably an integer or decimal number from 6 to 8, for example, p is 7.

9. [Chemistry 18] p is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 3 to 9, more preferably an integer or decimal number from 6 to 8, for example, p is 7.

8. Ab16 is an antibody or antigen-binding fragment thereof that targets ADAM9, the amino acid sequence of the heavy chain of Ab16 is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain is shown in SEQ ID NO:

51. The antibody-drug conjugate according to any one of claims 14 to 24, characterized in that Ab15 is an antibody or antigen-binding fragment thereof that targets ADAM9, the amino acid sequence of the heavy chain of Ab15 is shown in SEQ ID NO: 52, and the amino acid sequence of the light chain is shown in SEQ ID NO:

53.

26. The antibody-drug conjugate is the following conjugate: 【Chemistry 19】 , where p represents the number of connections, p is one integer from 1 to 10, preferably one integer from 3 to 9, more preferably one integer from 4 to 8, for example, p is 4, 5, 6, 7, or 8. The antibody-drug conjugate according to any one of claims 14 to 25, characterized in that Ab16 is an antibody or antigen-binding fragment thereof that targets ADAM9, the amino acid sequence of the heavy chain of Ab16 is shown in SEQ ID NO: 50, and the amino acid sequence of the light chain is shown in SEQ ID NO:

51.

27. The step of reacting an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 with a compound represented by formula II to obtain the antibody-drug conjugate, 【Chemistry 20】 L' forms a linker unit L according to any one of claims 14 to 26 with the antibody or its antigen-binding fragment. The cytotoxic drug is as described in any one of claims 14 to 26, Preferably, the antibody-drug conjugate is (1) The compound represented by formula II is 【Chemistry 21】 The condition, (2) The antibody or its antigen-binding fragment is Ab16 or Ab15, The amino acid sequence of the heavy chain of Ab16 is preferably shown in SEQ ID NO: 50, and the amino acid sequence of the light chain is preferably shown in SEQ ID NO:

51. The amino acid sequence of the heavy chain of Ab15 is preferably shown in SEQ ID NO: 52, and the amino acid sequence of the light chain is preferably shown in SEQ ID NO:

53. A method for producing an antibody-drug conjugate according to any one of claims 14 to 26, characterized by satisfying one or more of the following conditions.

28. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and / or an antibody-drug conjugate according to any one of claims 14 to 26, and a pharmaceutically acceptable carrier.

29. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an antibody-drug conjugate according to any one of claims 14 to 26, and / or a pharmaceutical composition according to claim 28 in the manufacture of a pharmaceutical for diagnosing, preventing and / or treating cancers that highly express ADAM9, Preferably, the cancer that highly expresses ADAM9 is colorectal cancer or lung adenocarcinoma.

30. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an antibody-drug conjugate according to any one of claims 14 to 26, and / or a pharmaceutical composition according to claim 28 in the manufacture of a pharmaceutical for diagnosing, preventing and / or treating cancer, Preferably, the cancer is colorectal cancer or lung adenocarcinoma.

31. A method for diagnosing, preventing and / or treating cancer that highly expresses ADAM9, comprising the step of administering to a patient in need a therapeutically effective amount of an antibody or its antigen-binding fragment according to any one of claims 1 to 8, an antibody-drug conjugate according to any one of claims 14 to 26, and / or a pharmaceutical composition according to claim 28, Preferably, the cancer that highly expresses ADAM9 is colorectal cancer or lung adenocarcinoma.

32. A method for diagnosing, preventing and / or treating cancer, comprising the step of administering to a patient in need a therapeutically effective amount of an antibody or its antigen-binding fragment according to any one of claims 1 to 8, an antibody-drug conjugate according to any one of claims 14 to 26, and / or a pharmaceutical composition according to claim 28, Preferably, the cancer is colorectal cancer or lung adenocarcinoma.

33. It is used in the diagnosis, prevention and / or treatment of cancers that highly express ADAM9. Preferably, the cancer that highly expresses ADAM9 is colorectal cancer or lung adenocarcinoma, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody-drug conjugate according to any one of claims 14 to 26 and / or the pharmaceutical composition according to claim 28.

34. Used in the diagnosis, prevention and / or treatment of cancer, Preferably, the cancer is colorectal cancer or lung adenocarcinoma, the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the antibody-drug conjugate according to any one of claims 14 to 26 and / or the pharmaceutical composition according to claim 28.

35. A combination therapy comprising the step of administering to a patient in need an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an antibody-drug conjugate according to any one of claims 14 to 26 and / or a pharmaceutical composition according to claim 28, and a second therapeutic agent, Preferably, the second therapeutic agent comprises another anti-ADAM9 antibody or its antigen-binding fragment, or an antibody-drug conjugate or pharmaceutical composition of the other anti-ADAM9 antibody or its antigen-binding fragment, and / or a pharmaceutical agent for treating other cancers that highly express ADAM9. More preferably, the cancer that highly expresses ADAM9 is colorectal cancer or lung adenocarcinoma, in combination therapy.

36. A combination therapy comprising the step of administering to a patient in need an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, an antibody-drug conjugate according to any one of claims 14 to 26 and / or a pharmaceutical composition according to claim 28, and a second therapeutic agent, Preferably, the second therapeutic agent comprises another anti-ADAM9 antibody or its antigen-binding fragment, or an antibody-drug conjugate or pharmaceutical composition comprising the other anti-ADAM9 antibody or its antigen-binding fragment, and / or a pharmaceutical for treating other cancers. More preferably, the cancer is colorectal cancer or lung adenocarcinoma, in combination therapy.

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