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WO2024199111A1 - 靶向adam9的人源化抗体、其抗体药物偶联物及其应用 - Google Patents

靶向adam9的人源化抗体、其抗体药物偶联物及其应用 Download PDF

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Publication number
WO2024199111A1
WO2024199111A1 PCT/CN2024/083218 CN2024083218W WO2024199111A1 WO 2024199111 A1 WO2024199111 A1 WO 2024199111A1 CN 2024083218 W CN2024083218 W CN 2024083218W WO 2024199111 A1 WO2024199111 A1 WO 2024199111A1
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Prior art keywords
seq
amino acid
acid sequence
antibody
variable region
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PCT/CN2024/083218
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English (en)
French (fr)
Inventor
林圣超
花海清
朱忠远
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映恩生物制药（苏州）有限公司
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Publication of WO2024199111A1 publication Critical patent/WO2024199111A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • the present invention belongs to the technical field of biomedicine, and specifically relates to a humanized antibody targeting ADAM9, an antibody-drug conjugate thereof and applications thereof.
  • ADAM Disintegrin And Metalloproteinase
  • ADAM family members have a fairly conserved structure with 8 domains, among which there are metalloproteinase domains and integrin binding (disintegrin) domains.
  • ADAM metalloproteinase domains act as sheddases and have been reported to regulate a range of biological processes by cleaving transmembrane proteins, which in turn can act as soluble ligands and regulate cellular signaling.
  • ADAM9 is a member of the ADAM family of molecules. It is synthesized into an inactive form, which is proteolytically cleaved to produce an active enzyme. The processing at the upstream site is particularly important for the activation of the zymogen. ADAM9 is expressed in the following cells: fibroblasts, activated vascular smooth muscle cells, monocytes, activated macrophages.
  • ADAM9 The metalloproteinase activity of ADAM9 is involved in the degradation of matrix components, thereby enabling tumor cell migration.
  • the disintegrin domain of ADAM9 which is highly homologous to many snake venom disintegrins, allows interactions between ADAM9 and integrins and enables ADAM9 to positively or negatively regulate cell adhesion events.
  • the ADAM9 disintegrin domain has been shown to interact with ⁇ 6 ⁇ 1, ⁇ 6 ⁇ 4, ⁇ v ⁇ 5, and ⁇ 9 ⁇ 1 integrins.
  • ADAM9 is relevant to diseases, especially cancer. It has been found that ADAM9 cleaves and releases numerous molecules that play an important role in tumor formation and angiogenesis, such as TEK, KDR, EPHB4, CD40, VCAM1 and CDH5. ADAM9 is expressed by many types of tumor cells, including tumor cells of breast cancer, colon cancer, gastric cancer, glioma, liver cancer, non-small cell lung cancer, melanoma, myeloma, pancreatic cancer and prostate cancer.
  • ADAM9 has been found to be positively correlated with tumor malignancy and metastatic potential.
  • ADAM9 and its secreted soluble isoforms appear to be essential for the spread of cancer cells. Therefore, many studies have linked ADAM9 to Identified as a potential target for anticancer therapy.
  • Patent WO2018119196A1 discloses that the VH and VL domains of MAB-A are humanized on the basis of the mouse anti-human ADAM9 monoclonal antibody "MAB-A" (the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:7 in the patent, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:11 in the patent), and the CDR is optimized to improve affinity and/or remove potential unfavorable amino acid conditions.
  • MAB-A the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:7 in the patent, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:11 in the patent
  • hMAB-A (2I.2) (the amino acid sequence of its heavy chain is shown in SEQ ID NO:52 in the patent, and the amino acid sequence of its light chain is shown in SEQ ID NO:68 in the patent) has a strong hydrophobicity problem.
  • the present invention re-humanizes the mouse monoclonal antibody MAB-A disclosed in patent WO2018119196A1 to improve the solubility of its humanized version of the antibody, reduce the hydrophobicity of the antibody, thereby improving its coupling yield and drugability as an ADC drug, thereby providing a humanized antibody targeting ADAM9, its antibody-drug conjugate and its application.
  • the present invention solves the above technical problems through the following technical solutions.
  • the first aspect of the present invention provides an antibody or an antigen-binding fragment thereof targeting ADAM9, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3;
  • the HCDR1 comprises the amino acid sequence shown as LYWMX 1
  • the HCDR2 comprises the amino acid sequence shown as X 2 IIPIFGHTX 3 YX 4 EKFX 5 X 6
  • the HCDR3 comprises the amino acid sequence shown as SEQ ID NO: 11
  • the LCDR1 comprises the amino acid sequence shown as SEQ ID NO: 12
  • the LCDR2 comprises the amino acid sequence shown as SEQ ID NO: 16
  • the LCDR3 comprises the amino acid sequence shown as SEQ ID NO: 19;
  • X1 is N or H.
  • X2 is R; X3 is K; X4 is N; X5 is K; X6 is D or N.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:8
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:2
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 9
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:10
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:11
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:12
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19.
  • the amino acid sequence of the HCDR1 is shown as SEQ ID NO:3
  • the amino acid sequence of the HCDR2 is shown as SEQ ID NO:8
  • the amino acid sequence of the HCDR3 is shown as SEQ ID NO:11
  • the amino acid sequence of the LCDR1 is shown as SEQ ID NO:12
  • the amino acid sequence of the LCDR2 is shown as SEQ ID NO:16
  • the amino acid sequence of the LCDR3 is shown as SEQ ID NO:19.
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO:1
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO:5
  • the amino acid sequence of HCDR3 is shown as SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown as SEQ ID NO:12
  • the amino acid sequence of LCDR2 is shown as SEQ ID NO:16
  • the amino acid sequence of LCDR3 is shown as SEQ ID NO:19.
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO:2
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO:6
  • the amino acid sequence of HCDR3 is shown as SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown as SEQ ID NO:12
  • the amino acid sequence of LCDR2 is shown as SEQ ID NO:16
  • the amino acid sequence of LCDR3 is shown as SEQ ID NO:19.
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO:3
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO:7
  • the amino acid sequence of HCDR3 is shown as SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown as SEQ ID NO:12
  • the amino acid sequence of LCDR2 is shown as SEQ ID NO:16
  • the amino acid sequence of LCDR3 is shown as SEQ ID NO:19.
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO: 1
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO: 7
  • the amino acid sequence of HCDR3 is shown as SEQ ID NO: 11
  • the amino acid sequence of LCDR1 is shown as SEQ ID NO: 12
  • the amino acid sequence of LCDR2 is shown as SEQ ID NO: 16
  • the amino acid sequence of LCDR3 is shown as SEQ ID NO: 19.
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO:2
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO:9
  • the amino acid sequence of HCDR3 is shown as SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown as SEQ ID NO:12
  • the amino acid sequence of LCDR2 is shown as SEQ ID NO:16
  • the amino acid sequence of LCDR3 is shown as SEQ ID NO:19.
  • the amino acid sequence of HCDR1 is shown as SEQ ID NO:4
  • the amino acid sequence of HCDR2 is shown as SEQ ID NO:10
  • the amino acid sequence of HCDR3 is shown as SEQ ID NO:11
  • the amino acid sequence of LCDR1 is shown as SEQ ID NO:12
  • the amino acid sequence of LCDR2 is shown as SEQ ID NO:16
  • the amino acid sequence of LCDR3 is shown as SEQ ID NO:19.
  • the heavy chain variable region and/or light chain variable region further comprises a framework region, and the framework region is a human framework region.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the heavy chain variable region comprises at least 90%
  • the light chain variable region comprises an amino acid sequence having at least 95% or at least 99% sequence identity to SEQ ID NO:38
  • the light chain variable region comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:38.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:31; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:23.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:42; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:43.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:22; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:23.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:24; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:25.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:26; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:28; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:30.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:34; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:35.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:36; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:30.
  • 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.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:36; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:39; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:44; and/or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:45.
  • the ADAM9 is preferably human or monkey ADAM9; in some specific embodiments of the present invention, the ADAM9 is human ADAM9.
  • the antibody or antigen-binding fragment thereof (1) is a full-length antibody, Fab, Fab', F(ab') 2 , Fv, sdAb or scFv; and/or, (2) is a monoclonal antibody, a bispecific antibody or a multispecific antibody.
  • the antibody or its antigen-binding fragment when 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 the 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 ⁇ chain.
  • the amino acid sequence of the heavy chain constant region of the human antibody IgG1 is as shown in 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 ⁇ chain is as shown in SEQ ID NO:49, or has at least 90%, at least 95% or at least 99% sequence identity with SEQ ID NO:49.
  • amino acid sequence of the heavy chain constant region of the human antibody IgG1 is shown in SEQ ID NO:48
  • amino acid sequence of the light chain constant region of the human antibody ⁇ chain is shown in SEQ ID NO:49.
  • 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.
  • 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.
  • 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.
  • amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:50; and/or, the amino acid sequence of the light chain is as shown in SEQ ID NO:51.
  • amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:52; and/or, the amino acid sequence of the light chain is as shown in SEQ ID NO:53.
  • the second aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the first aspect.
  • the third aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid as described in the second aspect.
  • the backbone plasmid of the recombinant expression vector is PTT5.
  • the fourth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the third aspect.
  • the host cell of the transformant is a eukaryotic cell.
  • the eukaryotic cell is a CHO cell.
  • the fifth aspect of the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof targeting ADAM9, the method comprising the steps of culturing the transformant as described in the fourth aspect, and isolating and obtaining the antibody or an antigen-binding fragment thereof targeting ADAM9 from the culture.
  • the sixth aspect of the present invention provides a method for detecting ADAM9, comprising the step of contacting the antibody or antigen-binding fragment thereof as described in the first aspect with a sample to be tested.
  • the detection is for non-diagnostic and/or therapeutic purposes.
  • the seventh aspect of the present invention provides an antibody-drug conjugate, which comprises the antibody or antigen-binding fragment thereof as described above, a linker unit L, and a cytotoxic drug.
  • the cytotoxic drug is a structure represented by formula (A-1), its stereoisomers, pharmaceutically acceptable salts, solvates or solvates of its salts,
  • M is -L 2 -L 1 -C(O)-;
  • L2 is -O- or -S-, and L2 is connected to the linker unit L;
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 saturated cycloalkyl or 3-6 membered saturated heterocyclic group, and the C 3 -C 6 saturated cycloalkyl and 3-6 membered saturated heterocyclic group are each independently optionally substituted by one or more R 2a ;
  • n is selected from 1, 2, 3 or 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are independently N, O and S, and the number of heteroatoms is 1, 2 or 3;
  • each R 1a , R 1b and R 2a is independently hydrogen, halogen, hydroxy, amino or C 1 -C 6 alkyl, said C 1 -C 6 alkyl being optionally substituted with one or more R;
  • Each R is independently hydrogen or halogen.
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -; each R 1a is independently hydrogen, halogen or C 1 -C 6 alkyl; each R 1b is independently hydrogen, halogen or C 1 -C 6 alkyl.
  • L1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • L 1 is a C 3 -C 6 saturated cycloalkyl group, and the C 3 -C 6 saturated cycloalkyl group is optionally substituted by one or more R 2a , and each R 2a is independently hydrogen, halogen or C 1 -C 6 alkyl group.
  • the cytotoxic drug is any of the following structures:
  • -L a - is Best The a end is connected to Ab, and the b end is connected to L b .
  • -L b - is any of the following structures:
  • the c-end is connected to L a
  • the d-end is connected to L c .
  • the linker unit L is Preferably
  • the structure of the antibody-drug conjugate is as shown in formula (A-2): p represents the average number of connections or the number of connections, and p is any integer or decimal from 1 to 10;
  • Ab is the antibody or antigen-binding fragment thereof as described in the first aspect
  • L is a linker unit as described above.
  • p is an integer or decimal of 3-9.
  • p is an integer or decimal of 7 to 8, for example, 7.8 or 7.9.
  • the antibody drug conjugate is selected from the following structural formula:
  • p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9;
  • Ab is the antibody or antigen-binding fragment thereof as described above.
  • the antibody-drug conjugate is any of the following conjugates:
  • p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; more preferably any integer or decimal from 6 to 8.
  • p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9, more preferably any integer or decimal from 6 to 8; for example, p is 7.9;
  • p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9, more preferably any integer or decimal from 6 to 8; for example, p is 7.8;
  • Ab16 is an antibody or an antigen-binding fragment thereof targeting ADAM9, wherein 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 an antigen-binding fragment thereof targeting ADAM9, and 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.
  • the antibody-drug conjugate is the following conjugate:
  • p represents the number of connections, and p is any integer from 1 to 10, preferably any integer from 3 to 9, and more preferably any integer from 4 to 8; for example, p is 4, 5, 6, 7 or 8;
  • Ab16 is an antibody or an antigen-binding fragment thereof targeting ADAM9, and 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 eighth aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect and/or the antibody-drug conjugate as described in the seventh aspect, and a pharmaceutically acceptable carrier.
  • the ninth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect in the preparation of a drug for diagnosing, preventing and/or treating cancer with high ADAM9 expression.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the tenth aspect of the present invention provides a method for diagnosing, preventing and/or treating cancer with high ADAM9 expression, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the eleventh aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect for diagnosing, preventing and/or treating cancers with high ADAM9 expression.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the twelfth aspect of the present invention provides a combination therapy, which comprises administering the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect, and a second therapeutic agent to a patient in need thereof.
  • the second therapeutic agent comprises other anti-ADAM9 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates or pharmaceutical compositions comprising the other anti-ADAM9 antibodies or antigen-binding fragments thereof, and/or other drugs for treating cancers with high ADAM9 expression.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the thirteenth aspect of the present invention provides a method for diagnosing, preventing and/or treating cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the fourteenth aspect of the present invention provides an antibody or antigen-binding fragment thereof 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.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the fifteenth aspect of the present invention provides a combination therapy, which comprises administering the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect, and a second therapeutic agent to a patient in need thereof.
  • the second therapeutic agent comprises other anti-ADAM9 antibodies or antigen-binding fragments thereof, or antibody-drug conjugates or pharmaceutical compositions comprising the other anti-ADAM9 antibodies or antigen-binding fragments thereof, and/or other drugs for treating cancer.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the sixteenth aspect of the present invention provides a method for preparing the antibody-drug conjugate according to the seventh aspect, the preparation method comprising reacting the antibody or antigen-binding fragment thereof according to the first aspect with a compound as shown in formula II to obtain the antibody-drug conjugate,
  • L' forms the linker unit L as described in the seventh aspect with the antibody or antigen-binding fragment thereof;
  • the cytotoxic drug is as described in the seventh aspect.
  • the antibody drug conjugate satisfies one or more of the following conditions:
  • the antibody or antigen-binding fragment thereof is Ab16 or Ab15;
  • amino acid sequence of the heavy chain of Ab16 is preferably as shown in SEQ ID NO:50, and the amino acid sequence of the light chain is preferably as shown in SEQ ID NO:51;
  • the amino acid sequence of the heavy chain of Ab15 is preferably as shown in SEQ ID NO:52, and the amino acid sequence of the light chain is preferably as shown in SEQ ID NO:53.
  • the seventeenth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof as described in the first aspect, the antibody-drug conjugate as described in the seventh aspect and/or the pharmaceutical composition as described in the eighth aspect in the preparation of a drug for diagnosing, preventing and/or treating cancer.
  • the cancer is selected from lung cancer, prostate cancer, liver cancer, breast cancer, thyroid cancer, esophageal cancer, pancreatic cancer, gastric cancer, ovarian cancer and intestinal cancer.
  • the cancer is non-small cell lung cancer, such as lung adenocarcinoma.
  • the reagents and raw materials used in the present invention are commercially available.
  • the antibodies targeting ADAM9 of the present invention have one or more of the following advantages:
  • the antibody of the present invention has high hydrophilicity, and the antibody molecule has better stability and drugability;
  • the antibody expression level of the present invention is significantly improved, the proportion of high molecular weight polymers is low, and the drugability is better;
  • the antibodies of the present invention have good affinity for human and monkey ADAM9 antigens
  • the antibodies of the present invention have a very high internalization rate for cells expressing ADAM9.
  • the antibody-drug conjugate of the present invention has good inhibitory activity on the proliferation of ADAM9-positive human intestinal cancer cells LS174T, Colo205, and human lung adenocarcinoma cell Calu-3;
  • the antibody-drug conjugate of the present invention has significant anti-tumor activity against mice bearing human intestinal cancer cells DLD-1, mice bearing intestinal cancer cells LS174T, and mice bearing human lung cancer cells Calu-3.
  • FIG1 shows the in vivo tumor inhibition test results of ADAM9-ADC on human colon cancer cell DLD-1 tumor-bearing mice.
  • FIG. 2 shows the in vivo tumor inhibition test results of ADAM9-ADC on human colon cancer cell LS174T tumor-bearing mice.
  • FIG3 shows the results of the in vivo tumor inhibition test of ADAM9-ADC on human lung cancer cell Calu-3 tumor-bearing mice.
  • FIG. 4 a shows the results of FACS binding assay on LS174T cells.
  • FIG4b shows the results of the LS174T-ADC proliferation inhibition experiment.
  • FIG5a shows the results of FACS binding assay on HepG2 cells.
  • FIG5b shows the results of the HepG2-ADC proliferation inhibition experiment.
  • the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as described in J.Biol.Chem, 243, p3558 (1968):
  • the term "and/or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above.
  • “or” or “and/or” should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when the opposite term is clearly indicated, such as “only one” or “exactly one” or when “consisting of" is used in the claims, it will refer to only one number listed or one element of the list.
  • antibody drug conjugate generally refers to an antibody connected to a biologically active cytotoxic drug via a stable linker.
  • antibody drug conjugate may refer to an antibody or its antigen-binding fragment connected to a biologically active cytotoxic drug fragment via a stable linker.
  • cytotoxic drug generally refers to a toxic drug, which can have strong chemical molecules in tumor cells that can destroy their normal growth. Cytotoxic drugs can kill tumor cells at a sufficiently high concentration.
  • the "cytotoxic drug” may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics or nucleolytic enzymes, or their derivatives, for example, it can be a toxic drug, including but not limited to camptothecin derivatives, for example, it can be the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4
  • the term "antibody” generally refers to an immunoglobulin reactive to a specified protein or peptide or a fragment thereof.
  • the antibody may be an antibody from any class, including but not limited to IgG, IgA, IgM, IgD and IgE, and an antibody from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4).
  • the antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4.
  • the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies of the present application may be derived from any species.
  • antibody may include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, antibodies comprising Fusion proteins and any other modified immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity.
  • an antigen binding domain generally refers to a portion of an antibody molecule that contains amino acids responsible for the specific binding between the antibody and the antigen.
  • the portion of an antigen that is specifically recognized and bound by an antibody is called an "epitope".
  • an antigen binding domain may typically include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • a Fd fragment for example, has two VH regions and generally retains some antigen binding functions of a complete antigen binding domain.
  • antigen-binding fragments of antibodies include (1) a Fab fragment, a monovalent fragment having VL, VH, a constant light chain (CL) and a CH1 domain; (2) a F(ab′) 2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) a Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety), which has a VH domain; (6) isolated complementarity determining regions (CDRs); and (7) a single-chain Fv (scFv), for example derived from an scFV library.
  • a Fab fragment
  • the two domains of the Fv fragment, VL and VH are encoded by independent genes, they can be joined using recombinant methods by means of a synthetic linker, which enables them to be prepared as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions are paired to form a monovalent molecule (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci.
  • scFv single-chain Fv
  • VHH refers to the variable antigen-binding domain of a heavy chain antibody from the Camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J. Biotechnol., 74, 277-302 and reviewed in Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be referred to as Nanobody (Nb).
  • variable region or “variable domain” generally refers to the domain of the antibody heavy chain or light chain involved in the binding of the antibody to the antigen.
  • variable generally refers to that some parts of the sequence of the variable domain of the antibody change strongly, forming the binding and specificity of various specific antibodies to their specific antigens. Variability is not evenly distributed in the entire variable region of the antibody. It is concentrated in three segments in the light chain variable region and the heavy chain variable region, which are called complementary determining regions (CDRs) or hypervariable regions (HVRs), respectively LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved parts in the variable domain are called framework regions (FRs).
  • CDRs complementary determining regions
  • HVRs hypervariable regions
  • variable domains of natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a ⁇ -folded configuration and are connected by three CDR structural loop regions.
  • the CDRs in each chain are held together by the FR regions and, together with the CDRs from the other chain, form the antibody antigen-binding site.
  • the amino acid sequences of the listed CDRs are shown in accordance with the Kabat definition rules.
  • the CDRs of antibodies can be defined by a variety of methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al.
  • CDR complementarity determining region
  • the scope of the antibodies also covers antibodies whose variable region sequences comprise the specific CDR sequences but whose declared CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination).
  • sequence identity between sequences is carried out as follows. To determine the percent identity of two amino acid sequences, the sequences are compared for the best comparison purpose (e.g., spaces can be introduced in the first and second amino acid sequences for the best comparison or non-homologous sequences can be abandoned for comparison purposes).
  • the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues at the corresponding amino acid positions are then compared. When the position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at this position.
  • the sequence comparison and the calculation of percent identity between two sequences can be realized using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program of the GCG software package (available at http://www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
  • a particularly preferred set of parameters is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the protein sequence described in the present invention can be further used as a "query sequence" to perform a search against a public database to, for example, identify other family member sequences or related sequences.
  • the term "full-length antibody” is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH in the present 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 the present invention) and a light chain constant region (abbreviated as CL in the present invention).
  • the light chain constant region consists of one domain CL.
  • Mammalian heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ and ⁇ .
  • Mammalian light chains are classified as ⁇ or ⁇ .
  • Immunoglobulins comprising ⁇ , ⁇ , ⁇ , ⁇ and ⁇ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG and IgM.
  • Ig immunoglobulins
  • Complete antibodies form a "Y" shape.
  • the stem of the Y consists of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) bound together, and disulfide bonds (interchain) are formed in the hinge.
  • the heavy chains ⁇ , ⁇ and ⁇ have a constant region consisting of three tandem (in a row) Ig domains, and a hinge region for increased flexibility; the heavy chains ⁇ and ⁇ have a constant region consisting of four immunoglobulin domains.
  • the second and third constant regions are called "CH2 domains" and "CH3 domains", respectively.
  • Each arm of the Y includes the variable region of a single heavy chain bound to the variable and constant regions of a single light chain and the first constant region. The variable regions of the light and heavy chains are responsible for antigen binding.
  • Fab is composed of a light chain and the CH1 and variable region of a heavy chain.
  • the heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc” region contains two heavy chain fragments including the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.
  • "Fab'" contains a light chain and a portion of a heavy chain including 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' to form a F(ab') 2 molecule.
  • F(ab') 2 contains two light chains and two heavy chains including 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 held together by a disulfide bond between the two heavy chains.
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking a constant region.
  • the scFv single chain antibody fragment
  • the scFv single chain antibody fragment
  • the scFv single chain antibody fragment
  • the scFv single chain antibody fragment
  • the scFv single chain antibody fragment
  • the VL and VH domains are paired to form a monovalent molecule through a linker that enables them to be produced as a single polypeptide chain [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 may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers are composed of repeated G 4 S amino acid sequences or variants thereof.
  • a linker having an amino acid sequence (G 4 S) 4 or (G 4 S) 3 may be used, but variants thereof may also be used.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the character of the antibody obtained from a substantially homogeneous antibody population, and should not be construed as requiring the antibody to be produced by any particular method.
  • multispecific antibody is used in its broadest sense to cover antibodies with multiple epitope specificities.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies, antibody fragments covalently or non-covalently linked together, etc.
  • human antibody refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • a human antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences.
  • a human antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant ( KD ), which is the ratio of the dissociation rate constant and the association rate constant ( kdis and kon, respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between an antibody of the present invention and an antigen.
  • a specific method for measuring affinity is the BIAcore method herein.
  • halogen generally refers to fluorine, chlorine, bromine, and iodine, for example, it can be fluorine or chlorine.
  • alkyl generally refers to a residue derived from an alkane by removing a hydrogen atom.
  • the alkyl group may be substituted or non-substituted, substituted or non-substituted.
  • alkyl generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of the parent alkane by removing a hydrogen atom, which may be a straight or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, for example, a chain alkyl containing 1 to 6 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably Contains 3 to 8 carbon atoms, more preferably contains 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, cyclooctane, etc.; polycyclic cycloalkyl includes spirocyclic, condensed ring and bridged ring cycloalkyl.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic light substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or sulfur, and the remaining ring atoms are carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms, of which 1-3 are heteroatoms; more preferably, it contains 3 to 6 ring atoms, of which 1-3 are heteroatoms; most preferably, it contains 5 or 6 ring atoms, of which 1-3 are heteroatoms.
  • Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl and homopiperazinyl, etc.
  • Polycyclic heterocyclyls include spirocyclic, fused and bridged heterocyclyls.
  • the heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, and the ring connected to the parent structure is a heterocyclyl.
  • the term "independently” generally means that the variable applies to any case, regardless of whether the variable with the same or different definition exists in the same compound.
  • the variable may refer to the type, number of substituents of the compound, or the type of atoms in the compound.
  • R appears twice in the compound and R is defined as "independently carbon or nitrogen” both R can be carbon, both R can be nitrogen, or one R can be carbon and the other R can be nitrogen.
  • the term “optional” or “optionally” generally means that the event or circumstance described subsequently may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur.
  • a heterocyclic group optionally substituted with an alkyl group means that the alkyl group may but need not be present, and the description may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.
  • substituted generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are replaced independently of each other by a corresponding number of substituents.
  • the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.
  • alkyl alkenyl
  • cycloalkyl alkyl
  • terms such as “alkyl”, “alkenyl”, “cycloalkyl”, etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C 1 -C 4 alkyl, C 3 -C 7 cycloalkyloxy, C 1 -C 4 alkylcarbonylamino, etc., and the subscript number following "C” indicates the number of carbon atoms present in the group.
  • C 3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); in C 1-10 , the members of the group may have any number of carbon atoms falling within the range of 1-10.
  • the compound or antibody drug conjugate of the present invention includes its tautomer, mesomer, exomer, Racemate, enantiomer and/or diastereomer.
  • diastereomer generally refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as, melting point, boiling point, spectral properties and reactivity.
  • tautomer or “tautomeric form” is used interchangeably and generally refers to structural isomers of different energies that can be mutually converted by a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • proton tautomers include interconversions via migration of a proton, such as keto-enol isomerization and imine-enamine isomerization.
  • Valence tautomers include interconversions via reorganization of some of the bonding electrons.
  • the term “meso” generally refers to an asymmetric atom within a molecule, but has factors of symmetry such that the overall optical rotation within the molecule is zero.
  • racemate or “racemic mixture” refers to a composition consisting of equimolar amounts of two enantiomeric species.
  • linker unit or “linker structure” generally refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end, and may also be connected to a cytotoxic drug after being connected to other linkers.
  • the directly or indirectly connected ligand may refer to the group being directly connected to the ligand through a covalent bond, or may be connected to the ligand through a linker structure.
  • a chemical structure fragment or bond containing an acid-labile linker structure e.g., hydrazone
  • a protease-sensitive linker structure e.g., peptidase-sensitive linker structure
  • a photolabile linker structure e.g., a dimethyl linker structure
  • a disulfide-containing linker structure e.g., a linker structure having a hydrophilic property.
  • antibody drug conjugates refer to compositions comprising different DAR distributions thereof
  • drug loading generally refers to the average number of cytotoxic drugs loaded on each ligand, which can be referred to as the average number of connections, or can be expressed as the ratio of the amount of cytotoxic drugs to the amount of antibodies, and the range of cytotoxic drug loading can be 0-12, for example, 1-10 cytotoxic drugs connected to each ligand (Ab).
  • the drug loading of each ADC molecule after the coupling reaction can be identified by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics.
  • the average number of connections p can be an integer or decimal from 1 to 10.
  • the average number of connections p can be an integer or decimal from 2 to 8.
  • the average number of connections p can be an integer or decimal from 3 to 8.
  • the average number of connections p can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10.
  • the average number of connections p is 7.8 or 7.9.
  • antibody drug conjugates refer to compounds containing the same DAR distribution
  • drug loading refers to the number of cytotoxic drugs loaded on each ligand, which can be called the number of connections, or can be expressed as the ratio of the amount of cytotoxic drugs to the amount of antibodies.
  • the range of cytotoxic drug loading can be 0-12, such as 1-10 cytotoxic drugs connected to each ligand (Ab).
  • the number of connections p can be any integer from 1 to 10.
  • the number of connections p can be any integer from 3 to 9.
  • the number of connections p can be any integer from 4 to 8.
  • the number of connections p can be 4, 5, 6, 7 or 8.
  • some atoms of the compounds or antibody drug conjugates of the present invention may appear in more than one isotopic form.
  • hydrogen may exist in the form of protium ( 1H ), deuterium ( 2H ) and tritium ( 3H ), and carbon may exist naturally in three different isotopes ( 12C , 13C and 14C ).
  • isotopes that can be incorporated into the compounds of the present application also include, but are not limited to, 15N , 18O , 17O , 18F , 32P, 33P , 129I , 131I , 123I , 124I , 125I , or similar isotopes.
  • the compounds or antibody drug conjugates of the present invention may be enriched in one or more of these isotopes.
  • such isotope-enriched compounds can be used for a variety of purposes.
  • substitution with heavy isotopes such as deuterium ( 2H ) may provide certain therapeutic advantages, which may be due to higher metabolic stability.
  • the natural abundance of deuterium ( 2H ) is about 0.015%. Therefore, there is one deuterium atom for approximately every 6500 hydrogen atoms in nature. Therefore, the deuterium abundance of the deuterium-containing compound or antibody drug conjugate of the present invention at one or more positions (as the case may be) is greater than 0.015%.
  • the structures described in the present invention may also include compounds or antibody drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms.
  • compounds or antibody drug conjugates that are consistent with the structure of the present invention except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon 13 or carbon 14, are within the scope of the present invention.
  • nucleic acid in the present invention refers to a nucleotide chain of any length, and includes DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.
  • the recombinant expression vector of the present invention can be any suitable recombinant expression vector, which can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the gene or sequence in the host cell.
  • Suitable vectors include those designed for expansion and amplification or for expression or both, and examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors.
  • the term "host cell” refers to any type of cell that can contain a nucleic acid or vector described herein.
  • the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it can be a prokaryotic cell, such as a bacterium or a protozoan.
  • the host cell is a cell that originates from or is obtained from an individual.
  • the host cell is derived from or is obtained from a mammal.
  • the methods and conditions for culturing the produced transformants and for recovering the produced antibody molecules are known to those skilled in the art and can be varied or optimized according to the specific expression vector and mammalian host cell used based on this specification and methods known in the prior art.
  • the application scenarios of detection for non-diagnostic and/or therapeutic purposes are, for example: detecting the presence of ADAM9 protein in the laboratory; or using it as a positive antibody to screen other antibodies targeting ADAM9; or competing with other anti-ADAM9 antibodies to detect whether there is competition between antibodies, i.e., whether the antigenic epitopes are the same or similar, etc. Use scene.
  • the term "pharmaceutical composition” generally refers to a mixture containing one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical composition can be used to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • the preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.
  • the pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be prepared according to known techniques using the above-mentioned suitable dispersants or wetting agents and suspending agents.
  • the sterile injection preparation can also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol.
  • sterile fixed oils can be conveniently used as solvents or suspension media.
  • any blended fixed oils including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the term "pharmaceutically acceptable salt” or “pharmaceutically usable salt” generally refers 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 form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, 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.
  • a pharmaceutically acceptable carrier is any one of those conventionally used carriers, and is limited only by physical-chemical considerations (such as solubility and lack of reactivity with antibodies targeting ADAM9), and is limited by the route of administration.
  • Pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents are well known to those skilled in the art and are easily available to the public.
  • a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition, and is a carrier that does not have adverse side effects or toxicity under the conditions of use. In certain embodiments, when given to an animal or human, the carrier does not produce adverse, allergic or other inappropriate reactions.
  • the pharmaceutical composition does not contain pyrogens and other impurities that may be harmful to humans or animals.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc.; its use is well known in the art.
  • the term "effective amount” means an amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response of a tissue, system, animal or human that is being sought, for example, by a researcher or clinician.
  • therapeutically effective amount means an amount that results in improved treatment, cure, prevention or alleviation of a disease, condition or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject not receiving that amount.
  • the term also includes within its scope an amount that is effective to enhance The amount of normal physiological function.
  • the term “cancer” refers to a malignant tumor, which is a disease caused by abnormal control of the mechanism of cell growth and proliferation.
  • intestinal cancer generally refers to colorectal cancer, also known as “colorectal cancer”, which refers to cancers of large intestinal epithelial origin, including colon cancer and rectal cancer.
  • lung adenocarcinoma refers to a malignant tumor originating from the epithelial tissue of the lung, and lung adenocarcinoma belongs to the category of non-small cell lung cancer.
  • cancer with high expression of ADAM9 refers to a malignant tumor caused by abnormally high expression of ADAM9 in the body.
  • the purpose of re-humanization of the mouse anti-ADAM9 monoclonal antibody MAB-A antibody is to improve the solubility of its humanized version of the antibody, reduce the hydrophobicity of the antibody, and thus improve its coupling yield and drugability as an ADC drug.
  • the humanized version of MAB-A hMAB-A (2I.2) (sequence information of the heavy chain SEQ ID NO:52 and the light chain SEQ ID NO:68 disclosed in patent WO2018119196A1) that has entered the clinical research stage has the problem of strong hydrophobicity.
  • the murine antibody was re-humanized as disclosed in the literature in the art, i.e., the murine antibody constant domain was replaced with a human constant domain, and the CDR region was modified for the purpose of optimizing affinity and drugability.
  • the human antibody sequence was selected based on the homology between the hMAB-A (2I.2) parent murine MAB-A antibody and human antibodies, and the MAB-A antibody was re-humanized.
  • the specific method is to compare the heavy and light chain variable region sequences with the human antibody germline database on the basis of the typical structure of VH/VL CDR of the mouse antibody MAB-A to obtain a human germline template with high homology; transplant the mouse antibody CDR region to the selected corresponding humanized template; then, based on the three-dimensional structure of the mouse antibody, perform back mutations on the embedded residues, residues that directly interact with the CDR region, and residues that have an important influence on the conformation of VL and VH, and optimize the chemically unstable amino acid residues in the CDR region.
  • the antibody designed with a combination of humanized heavy chain variable region HCVR and light chain variable region LCVR sequences is selected.
  • the CDR region sequence number of the humanized antibody is shown in Table 1, the amino acid sequence of the CDR is shown in Table 2, and the heavy chain and light chain variable region sequences of the humanized antibody are shown in Table 3.
  • the CDR regions of the antibodies are defined according to the Kabat numbering system, wherein X1 may be an amino acid N, D, H or E; X2 may be an amino acid D or R; X3 may be an amino acid K or D; X4 may be an amino acid N or E; X5 may be an amino acid R or K; and X6 may be an amino acid D or N.
  • the designed heavy chain and light chain variable region sequences are connected to the heavy chain constant region and light chain constant region sequences of human antibodies, respectively.
  • the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO: 48; the antibody light chain constant region is selected from the constant region of the human ⁇ chain as shown in SEQ ID NO: 49, and the antibody constant region sequence is shown in Table 4.
  • the heavy chain amino acid sequence of Ab16 is (SEQ ID NO:50):
  • the light chain amino acid sequence of Ab16 is (SEQ ID NO:51):
  • the heavy chain amino acid sequence of Ab15 is (SEQ ID NO:52):
  • the light chain amino acid sequence of Ab15 is (SEQ ID NO:53):
  • the PTT5 plasmids corresponding to the heavy and light chains of each antibody were co-transfected into CHO cells using PEImax 40000; the cell culture supernatant was collected after 5 days of culture, the antibody components were purified by protein A (MabSelectTM PrismA), and the antibodies were quantified by OD280nm (commissioned by Nanjing GenScript Biotechnology Co., Ltd.).
  • Example 1 20 antibodies were expressed in a 4 ml system, and the expression level of each antibody is shown in Table 7 below.
  • Dilute human ADAM-His protein (AcroBiosystems, Cat#AD9-H52H7) to a concentration of 1 ⁇ g/ml with PBS at pH 7.4, add 100 ⁇ L/well to a 96-well high-affinity ELISA plate, and incubate overnight (16-20 hours) in a 4°C refrigerator. Wash the plate three times with PBST (pH 7.4 PBS containing 0.05% Tween-20), add 200 ⁇ L/well of 1% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate at 37°C for 1 hour for blocking. After blocking, discard the blocking solution and wash the plate once with PBST buffer.
  • BSA bovine serum albumin
  • Mouse anti-human IgG (Fc) antibody (Cat. No.: 29234600, Cytiva) was diluted to 25 ⁇ g/mL with a fixation reagent (10 mM sodium acetate, pH 5.0), i.e., 950 ⁇ L of the fixation reagent was added to 50 ⁇ L of mouse anti-human IgG (Fc) antibody for fixation of eight channels.
  • a fixation reagent 10 mM sodium acetate, pH 5.0
  • 950 ⁇ L of the fixation reagent was added to 50 ⁇ L of mouse anti-human IgG (Fc) antibody for fixation of eight channels.
  • the surface of the CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 ⁇ L/min for 420 s.
  • mouse anti-human IgG (Fc) antibody was injected into the experimental channel at a flow rate of 10 ⁇ L/min for about 360 s, and the fixed amount was about 7000 to 14000 RU.
  • the chip was blocked with 1 M ethanolamine at 10 ⁇ L/min for 420 s.
  • the reference channel was operated in the same way as the experimental channel.
  • Capture ligand Antibodies Ab10, Ab15, Ab16, and hMAB-A (2I.2) were diluted to 4 ⁇ g/mL with running reagent (containing 10 mM N-(2-hydroxyethyl)piperazine-N-2-sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20 (Tween-20), pH adjusted to 7.4) and injected into the human IgG (Fc) capture experimental channel at a flow rate of 10 ⁇ L/min, about 200 RU. The reference channel does not need to be captured by ligand.
  • running reagent containing 10 mM N-(2-hydroxyethyl)piperazine-N-2-sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20 (
  • the chip After each concentration analysis, the chip needs to be regenerated with 3M magnesium chloride at a flow rate of 20 ⁇ L/min for 30s to wash away the ligand and the undissociated analyte.
  • the experimental channel When performing the next concentration analysis, the experimental channel needs to recapture the same amount of ligand.
  • a binding plate was set up, and human lung cancer cells Calu-3 (purchased from the Chinese Academy of Sciences Cell Bank, catalog number TCHu157) with high ADAM9 expression were digested with trypsin, and the cells were collected by centrifugation.
  • the cell density was adjusted with FACS buffer (1 ⁇ PBS containing 2% FBS) and then spread on a 96-well U-bottom plate, 1 ⁇ 10 5 cells per well, centrifuged at 1200g for 5 minutes, the supernatant was discarded, and 100 ⁇ L of antibody solution diluted with FACS buffer was added (the initial working concentration of the antibody was 100nM, 10-fold dilution, 8 concentration points, and 0nM point was set), and incubated at 4°C for 1 hour.
  • an endocytosis plate was set up. After Calu-3 was digested with trypsin, the cells were collected by centrifugation, and the cell density was adjusted with FACS buffer and then spread on a 96-well U-bottom plate. 1 ⁇ 10 5 cells per well were centrifuged at 1200g for 5 minutes, the supernatant was discarded, and 100 ⁇ L of antibody solution that had been gradiently diluted with FACS buffer was added (the initial working concentration of the antibody was 100nM, 10-fold dilution, 8 concentration points, and a 0nM point was set), and incubated at 37°C for 16 hours.
  • benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO 3 (10.9 g, 129 mmol) in DMF (50 mL), and the mixture was reacted at 25° C. for 17 hours.
  • HATU 74 mg, 0.19 mmol
  • DMF 2.5 mL
  • 27f 90 mg, 0.13 mmol
  • KI-1 92 mg, 0.19 mmol
  • DIEA 50 mg, 0.39 mmol
  • LCMS showed that the basic reaction was complete.
  • MS-ESI m/z 503.3 [M+H]+.
  • MS-ESI m/z 830.3[M+H]+.
  • the reaction solution was purified by desalting with a G-25 gel column on AKTA (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration with a 30KD ultrafiltration tube to obtain a solution of the exemplary product Ab16-X1 (30 mM histidine-hydrochloric acid pH 5.5; 38 mg, 5.6 mg/mL, yield: 95%), which was stored at -80°C.
  • linker-cell Cytotoxin X2 (2.6 mg, 2.43 ⁇ mol) was dissolved in 0.24 mL DMA, added to the above solution, placed in a thermostatic stirrer, stirred at 60 rpm, and oscillated at 22 ° C for 2 hours to stop the reaction.
  • reaction solution was desalted and purified on an AKTA G-25 gel column (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product Ab16-X2 (30 mM histidine-hydrochloric acid pH 5.5; 37 mg, 5.6 mg/mL, yield: 92%), which was stored at -80 ° C.
  • the reaction solution was purified by desalting with a G-25 gel column on AKTA (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration with a 30KD ultrafiltration tube to obtain a solution of the exemplary product Ab15-X2 (30 mM histidine-hydrochloric acid pH 5.5; 32 mg, 5.2 mg/mL, yield: 80%), which was stored at -80°C.
  • the reaction solution was purified by desalting with a G-25 gel column on AKTA (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration with a 30KD ultrafiltration tube to obtain a solution of the exemplary product hMAB-A(2I.2)-X2 (30 mM histidine-hydrochloric acid pH 5.5; 29 mg, 3.2 mg/mL, yield: 72%), which was stored at -80°C.
  • Example 8 In vitro cell proliferation inhibition activity test of anti-ADAM9 antibody drug conjugate (ADAM9-ADC)
  • the chemiluminescent cell viability assay was used to evaluate the inhibitory effect of each anti-ADAM9 antibody-conjugated camptothecin toxin ADC drug on cell proliferation after incubation for 6 days in ADAM9-positive human colorectal cancer cells LS174T (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu32) or Colo205 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu102) and human lung adenocarcinoma cells Calu-3.
  • CCG method chemiluminescent cell viability assay
  • Cells in the logarithmic growth phase were collected and plated at a density of 5000 cells/well.
  • the cell plates were placed in a 37°C, 5% CO2 incubator for overnight culture.
  • each ADC drug was diluted 3 times with complete medium to obtain 9 concentration gradients (starting with the highest concentration of 300nM). After the drug, 100 ⁇ L/well was added to the cell culture plate, and the complete medium was used as a blank control. Three replicates were set up; continue to incubate in a 37°C, 5% CO2 incubator for 6 days.
  • the cell viability calculation formula (Lum test drug - Lum blank control ) / (Lum solvent blank control - Lum blank control ) ⁇ 100%.
  • Example 9 In vivo tumor inhibition test of anti-ADAM9 antibody drug conjugate (ADAM9-ADC) on human colorectal cancer cell DLD-1 tumor-bearing mice
  • ADAM9-ADC To evaluate the inhibitory effect of ADAM9-ADC on tumor formation in vivo, ADAM9 positive expression was used in mice. After forming transplanted tumors with human colorectal cancer cells DLD-1 (purchased from the cell bank of the Chinese Academy of Sciences, catalog number TCHu134), the anti-tumor effect of each ADAM9-ADC was evaluated.
  • Blank control group normal saline, tail vein injection, single dose
  • Ab15-X2 treatment group: 5 mg/kg, injected into the tail vein, once
  • Ab16-X2 (treatment group): 5 mg/kg, injected into the tail vein, once
  • hMAB-A(2I.2)-X2 treatment group: 5 mg/kg, injected into the tail vein, once
  • DLD-1 cells were inoculated subcutaneously at the right anterior scapula of 8-week-old female NOD SCID mice.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM and injected with ADC drugs intravenously (iv) starting on the same day (day 0) for a total of 1 injection at a dose of 5 mg/kg.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • Tumor inhibition rate 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).
  • Example 10 In vivo tumor inhibition test of anti-ADAM9 antibody drug conjugate (ADAM9-ADC) on human colon cancer cell LS174T tumor-bearing mice
  • ADAM9-positive human colon cancer cell LS174T was used to form transplanted tumors in mice, and the anti-tumor effect of each ADAM9-ADC was evaluated.
  • Blank control group physiological saline, intravenous injection (i.v.), 1 injection
  • Ab16-X2 (treatment group): 5 mg/kg, administered by tail vein injection (i.v.), 1 injection in total
  • DS-8201 (treatment group): 5 mg/kg, administered by tail vein injection (iv), 1 injection in total
  • DS-8201 (Enhertu, purchased from Daiichi Sankyo Company Limited)
  • LS174T cells were inoculated subcutaneously at the right anterior scapula of 8-week-old female BALB/c-Nu mice.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM and injected with ADC drugs intravenously (iv) starting on the same day (day 0) for a total of 1 injection at a dose of 5 mg/kg. Tumor volume and body weight were measured twice a week and the data were recorded.
  • Tumor inhibition rate 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).
  • Tumor inhibition rate TGI
  • Example 11 In vivo tumor inhibition test of anti-ADAM9 antibody drug conjugate (ADAM9-ADC) on human lung cancer cell Calu-3 tumor-bearing mice
  • ADAM9-positive human lung cancer Calu-3 cells were used to form xenografts in mice, and the anti-tumor effects were evaluated.
  • Blank control group Normal saline, injection into the tail vein, once every 7 days, a total of 2 injections
  • Ab16-X1 (treatment group): 5 mg/kg, injected into the tail vein, once every 7 days, for a total of 2 injections
  • Ab16-X2 (treatment group): 5 mg/kg, administered by tail vein injection, once every 7 days, for a total of 2 injections
  • 1 ⁇ 10 7 Calu-3 cells were inoculated subcutaneously at the right anterior scapula of 8-week-old female CB-17SCID mice.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM and ADC drugs were injected intravenously (iv) starting on the same day (day 0) once every 7 days for a total of 2 injections, with a dose of 5 mg/kg. Tumor volume and body weight were measured twice a week and the data were recorded.
  • Tumor inhibition rate 100% - (tumor volume of the treatment group on the day of measurement - tumor volume of the treatment group on day 0) / (tumor volume of the control group on the day of measurement - tumor volume of the control group on day 0).
  • the tumor inhibition rate [1-(tumor volume of the treatment group on the day of measurement ⁇ tumor volume of the treatment group on day 0) ⁇ (tumor volume of the control group on the day of measurement ⁇ tumor volume of the control group on day 0)] ⁇ [1-(tumor volume of the control group on day 0 ⁇ tumor volume of the control group on the day of measurement)] ⁇ 100%.
  • the experimental results are shown in FIG3 and Table 17.
  • Example 12 Cell proliferation inhibition of anti-ADAM9 antibody drug conjugate (ADAM9-ADC) at low cell binding activity
  • Tumor cells expressing both ADAM9 and Her2 were used to detect the binding activity of Ab16-X2 to cells.
  • DS-8201 (Enhertu, purchased from Daiichi Sankyo Company Limited) was used as a positive control, and the negative control was a human IgG antibody against HIV virus (iso-IgG1, a product of WuXi AppTec).
  • HepG2 cells purchased from the Chinese Academy of Sciences Cell Bank, catalog number TCHu72
  • LS174T cells with medium and low ADAM9 expression were added to a round-bottom 96-well plate at 1E5 cells per well, and then 100 ⁇ L of gradient diluted Ab16-X2, DS-8201 or iso-IgG1 were added and incubated at 4 degrees for 1 hour.
  • FACS buffer PBS containing 2% FBS
  • PE fluorescently labeled goat anti-human secondary antibody Abcam, ab98596
  • the chemiluminescent cell viability assay was used to evaluate the cell killing effect of Ab16-X2 and DS-8201 incubated with LS174T (incubated for 3 days) and HepG2 (incubated for 6 days) cells.
  • Cells in the logarithmic growth phase were collected and plated at a density of 3000 cells/well.
  • the cell plates were placed in a 37°C, 5% CO2 incubator for overnight culture.
  • the drug was diluted 3 times with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 1000nM).
  • the HepG2 cell binding activity and cell proliferation inhibition activity were detected with reference to Example 12.1, and the specific experimental steps were the same as those for LS174T cells.
  • Example 13 Pre-toxicity of anti-ADAM9 antibody drug conjugate (ADAM9-ADC) in cynomolgus monkeys
  • AB16-X2 was administered to cynomolgus monkeys by intravenous infusion once every 3 weeks for a total of 2 times. The nature, extent and time-effect relationship of the toxic reactions that may be caused by the drug conjugate were observed, and the toxic target organs or target tissues were preliminarily determined.
  • Two cynomolgus monkeys (from Suzhou Xishan Zhongke Experimental Animal Co., Ltd.), one male and one female, were intravenously injected with AB16-X2, with the first dose of 45 mg/kg; the second dose was given 3 weeks later, and the dose was increased to 80 mg/kg.
  • the autopsy was performed one week after the second dose.
  • the detection and observation indicators during the period include:
  • Toxicokinetics Gross anatomical observation and histopathological examination: The sternum (including bone marrow), heart, lung, kidney, bladder, duodenum, colon, cecum, eyeball, skin and grossly abnormal organs/tissues (spleen, rectum) of the animals to be dissected were routinely processed for histology and subjected to histopathological examination.
  • IMGC-936 developed by MacroGenics is an ADAM9-ADC coupled to the maytansine-type tubulin inhibitor DM21-C. It has entered the clinical research stage. In monkey toxicology studies, corneal lesions and ocular toxicity were observed after administration of 22.5 mg/kg IMGC-936 (Mol Cancer Ther 2022; 21:1047–59). In monkey toxicology studies, no ocular toxicity was observed at a dose of 80 mg/kg for AB16-X2.

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Abstract

一种靶向ADAM9的人源化抗体、其抗体药物偶联物及其应用。所述靶向ADAM9的人源化抗体包括重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3。所述抗体药物偶联物包含所述的人源化抗体、接头单元L和细胞毒性药物。所述人源化抗体具有良好的亲和力，亲水性、溶解度、稳定性更高，对表达ADAM9的细胞具有很高的内化率。所述抗体药物偶联物对ADAM9阳性表达细胞具有良好的增殖抑制作用，在体内能表现出显著的抗肿瘤药效。

Description

靶向ADAM9的人源化抗体、其抗体药物偶联物及其应用
本申请要求申请日为2023/3/24的中国专利申请2023103024365，以及申请日为2024/3/7的中国专利申请202410263320X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药技术领域，具体涉及一种靶向ADAM9的人源化抗体、其抗体药物偶联物及其应用。
背景技术
ADAM(A Disintegrin And Metalloproteinase，解联蛋白和金属蛋白酶结构域)为涉及各种生理和病理过程的蛋白质家族。已鉴别出该家族的至少40个基因成员，并且认为此类成员中的至少21个在人类中具有功能性。ADAM家族成员有具有8个结构域的相当保守的结构，在所述8个结构域中有金属蛋白酶结构域和整联蛋白结合(解联蛋白)结构域。ADAM金属蛋白酶结构域充当脱落酶(sheddase)且已报道通过裂解跨膜蛋白来调节一系列生物学过程，所述跨膜蛋白则可充当可溶性配体并调控细胞传讯。
ADAM9为ADAM分子家族的成员。其被合成为非活性形式，所述非活性形式被蛋白水解裂解，产生活性酶。在上游位点处的加工对酶原的活化特别重要。ADAM9在以下细胞中表达：成纤维细胞、活化的血管平滑肌细胞、单核细胞、活化的巨噬细胞。
ADAM9的金属蛋白酶活性参与基质组分的降解，从而使肿瘤细胞迁移。ADAM9的与许多蛇毒解联蛋白高度同源的解联蛋白结构域允许ADAM9与整联蛋白之间的相互作用，且使ADAM9能够正向或负向地调节细胞粘附事件。已显示ADAM9解联蛋白结构域与α6β1、α6β4、αvβ5及α9β1整联蛋白相互作用。
已发现ADAM9的表达与疾病(尤其是癌症)相关。已发现ADAM9裂解并释放众多在肿瘤形成和血管生成方面起重要作用的分子，如TEK、KDR、EPHB4、CD40、VCAM1及CDH5。ADAM9是由许多类型的肿瘤细胞表达，所述肿瘤细胞包括乳腺癌、结肠癌、胃癌、神经胶质瘤、肝癌、非小细胞肺癌、黑色素瘤、骨髓瘤、胰腺癌及前列腺癌的肿瘤细胞。
显著地，已发现ADAM9表达增加与肿瘤恶性及转移可能性正相关。另外，ADAM9及其分泌的可溶性同种型似乎对癌细胞的散布至关重要。因此，众多研究已将ADAM9 鉴别为抗癌疗法的潜在标靶。专利WO2018119196A1中披露了在鼠源的抗人ADAM9单克隆抗体“MAB-A”(其重链可变区的氨基酸序列在该专利中如SEQ ID NO:7所示，其轻链可变区的氨基酸序列在该专利中如SEQ ID NO:11所示)的基础上将MAB-A的VH和VL结构域人源化，并将CDR优化以改善亲和力和/或去除潜在的氨基酸不利条件。然而，在该专利中，MAB-A进入临床研究阶段的人源化版本抗体hMAB-A(2I.2)(其重链的氨基酸序列在该专利中如SEQ ID NO:52所示，其轻链的氨基酸序列在该专利中如SEQ ID NO:68所示)存在疏水性较强的问题。
因此，仍需要研发出疏水性更低、溶解度更高，且作为ADC(Antibody-Drug Conjugate，抗体药物偶联物)药物的偶联得率和成药性更好的抗ADAM9人源化抗体。
发明内容
本发明为了克服现有技术中的人源化的抗ADAM9抗体存在疏水性较强、溶解度过低，且其作为ADC药物的偶联得率和成药性较差的技术问题，对专利WO2018119196A1中所披露的鼠源单克隆抗体MAB-A重新人源化改造，以提高其人源化版本抗体的溶解度、降低抗体的疏水性、从而提高其作为ADC药物的偶联得率和成药性，从而提供了一种靶向ADAM9的人源化抗体、其抗体药物偶联物及其应用。
本发明通过以下技术方案解决上述技术问题。
本发明的第一方面提供一种靶向ADAM9的抗体或其抗原结合片段，所述抗体或其抗原结合片段包括重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3；
所述HCDR1包含如LYWMX1所示的氨基酸序列，所述HCDR2包含如X2IIPIFGHTX3YX4EKFX5X6所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；
其中，X1为N、D、H或E；X2为R或D；X3为D或K；X4为N或E；X5为K或R；X6为D或N。
本发明一些实施方案中，所述HCDR1中，X1为N或H。
本发明一些实施方案中，所述HCDR2中，X2为R；X3为K；X4为N；X5为K；X6为D或N。
本发明一些具体实施方案中：
当X1为N时，X2为R；X3为K；X4为N；X5为K；X6为D；
当X1为H时，X2为R；X3为K；X4为N；X5为K；X6为N。
本发明一些实施方案中，所述HCDR1包含如SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:8所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明另一些实施方案中，所述HCDR1包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明另一些实施方案中，所述HCDR1包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:5所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明另一些实施方案中，所述HCDR1包含如SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:6所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明另一些实施方案中，所述HCDR1包含如SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明另一些实施方案中，所述HCDR1包含如SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述 LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明另一些实施方案中，所述HCDR1包含如SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:10所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
本发明一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:8所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明另一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:5所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明另一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:2所示，所述HCDR2的氨基酸序列如SEQ ID NO:6所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明另一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:7所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明另一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:7所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明另一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:2所示，所述HCDR2的氨基酸序列如SEQ ID NO:9所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明另一些具体实施方案中，所述HCDR1的氨基酸序列如SEQ ID NO:4所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
本发明中，所述重链可变区和/或轻链可变区还包括框架区，所述框架区为人源框架区。
本发明一些实施方案中，所述重链可变区包含与SEQ ID NO:31具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:23具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:42具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:43具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:22具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:23具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:24具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:25具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:26具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:27具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:28具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:30具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:34具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:35具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:36具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:30具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:37具有至少90％、 至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:38具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:36具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:27具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:39具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:27具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明另一些实施方案中，所述重链可变区包含与SEQ ID NO:44具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:45具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明中，所述具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列的可变区维持与原序列至少同等的抗原结合功能。
本发明一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:31所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:23所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:42所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:43所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:22所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:23所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:24所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:25所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:26所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:27所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:28所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:30所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:34所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:35所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:36所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:30所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:37所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:38所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:36所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:27所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:39所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:27所示。
本发明另一些具体实施方案中，所述重链可变区的氨基酸序列如SEQ ID NO:44所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:45所示。
本发明中，所述ADAM9优选为人或猴的ADAM9；本发明一些具体实施方案中，所述ADAM9为人ADAM9。
本发明一些实施方案中，所述抗体或其抗原结合片段：(1)为全长抗体、Fab、Fab’、F(ab’)2、Fv、sdAb或scFv；和/或，(2)为单克隆抗体、双特异性抗体或多特异性抗体。
本发明中，当所述抗体或其抗原结合片段为全长抗体时，其包括人源抗体的重链的重链恒定区，优选人源抗体IgG1的重链恒定区；和/或，其包括人源抗体的轻链的轻链恒定区，优选人源抗体κ链的轻链恒定区。
本发明一些实施方案中，所述人源抗体IgG1的重链恒定区的氨基酸序列如SEQ ID NO:48所示，或与SEQ ID NO:48具有至少90％、至少95％或者至少99％序列同一性；和/或，所述人源抗体κ链的轻链恒定区的氨基酸序列如SEQ ID NO:49所示，或与SEQ ID NO:49具有至少90％、至少95％或者至少99％序列同一性。
本发明一些实施方案中，所述人源抗体IgG1的重链恒定区的氨基酸序列如SEQ ID NO:48所示，所述人源抗体κ链的轻链恒定区的氨基酸序列如SEQ ID NO:49所示。
本发明一些实施方案中，所述抗体或其抗原结合片段的重链包含与SEQ ID NO:50具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述抗体或其抗原结合片段的轻链包含与SEQ ID NO:51具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明一些实施方案中，所述抗体或其抗原结合片段的重链包含与SEQ ID NO:52具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述抗体或其抗原结合片段的轻链包含与SEQ ID NO:53具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列。
本发明中，所述具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列维持与原序列至少同等的抗原结合功能。
本发明一些具体实施方案中，所述抗体或其抗原结合片段重链的氨基酸序列如SEQ ID NO:50所示；和/或，轻链的氨基酸序列如SEQ ID NO:51所示。
本发明一些具体实施方案中，所述抗体或其抗原结合片段重链的氨基酸序列如SEQ ID NO:52所示；和/或，轻链的氨基酸序列如SEQ ID NO:53所示。
本发明的第二方面提供一种分离的核酸，所述核酸编码如第一方面所述的抗体或其抗原结合片段。
本发明的第三方面提供一种重组表达载体，所述重组表达载体包含如第二方面所述的分离的核酸。
本发明一些具体实施方案中，所述重组表达载体的骨架质粒为PTT5。
本发明的第四方面提供一种转化体，所述转化体包含如第三方面所述的重组表达载体。
本发明一些实施方案中，所述转化体的宿主细胞为真核细胞。
本发明一些具体实施方案中，所述真核细胞为CHO细胞。
本发明的第五方面提供一种靶向ADAM9的抗体或其抗原结合片段的制备方法，所述方法包含培养如第四方面所述的转化体，并从培养物中分离获得所述靶向ADAM9的抗体或其抗原结合片段的步骤。
本发明的第六方面提供一种检测ADAM9的方法，其包括使用如第一方面所述的抗体或其抗原结合片段与待测样品接触的步骤。
本发明一些实施方案中，所述检测为非诊断和/或治疗目的的。
本发明的第七方面提供一种抗体药物偶联物，所述抗体药物偶联物包含如前所述的抗体或其抗原结合片段、接头单元L和细胞毒性药物。
本发明一些实施方案中，所述细胞毒性药物为式(A-1)所示结构，其立体异构体、药学上可用的盐、溶剂化物或其盐的溶剂合物，
其中，
M为-L2-L1-C(O)-；
L2为-O-或-S-，且L2与所述的接头单元L连接；
L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代；
m选自1、2、3或4；所述的3-6元饱和的杂环基中的杂原子各自独立地为N、O和S，杂原子数为1、2或3个；
各个R1a、R1b和R2a独立地为氢、卤素、羟基、氨基或C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
各个R独立地为氢或卤素。
本发明一些实施方案中，L1为-(C(R1a)(R1b))m-CH2-；各个R1a独立地为氢、卤素或C1-C6烷基；各个R1b独立地为氢、卤素或C1-C6烷基。
本发明一些实施方案中，L1为
本发明一些实施方案中，L1为C3-C6饱和的环烷基，所述C3-C6饱和的环烷基任选被一个或多个R2a取代，各个R2a独立地为氢、卤素或C1-C6烷基。
本发明一些实施方案中，为
本发明一些实施方案中，所述细胞毒性药物为下任一结构：

本发明一些实施方案中，-La-为优选其中a端和Ab相连，b端与Lb相连。
本发明一些实施方案中，-Lb-为以下任一结构：
优选为更优选其中c端和La相连，d端与Lc相连。
本发明一些实施方案中，-Lc-为
本发明一些实施方案中，所述接头单元L为 优选为
本发明一些实施方案中，所述抗体药物偶联物结构如式(A-2)所示：p表示平均连接数或连接数，且p为1到10中任一整数或小数；
Ab为如第一方面所述的抗体或其抗原结合片段；
M如第七方面所述；
L为如前所述的接头单元。
本发明一些实施方案中，p为3-9的整数或小数。
本发明一些实施方案中，p为7～8的整数或小数，例如7.8或7.9。
本发明一些实施方案中，其中所述抗体药物偶联物选自以下结构式：


其中，
p为1到10中任一整数或小数，优选3到9中任一整数或小数；Ab为如前所述的抗体或其抗原结合片段。
本发明一些实施方案中，所述抗体药物偶联物为如下任一偶联物：
p为1到10中任一整数或小数，优选3到9中任一整数或小数；更优选6到8的任 一整数或小数；更优选地，p为7.8；
p为1到10中任一整数或小数，优选3到9中任一整数或小数，更优选6到8的任一整数或小数；例如，p为7.9；
p为1到10中任一整数或小数，优选3到9中任一整数或小数，更优选6到8的任一整数或小数；例如，p为7.8；
Ab16为靶向ADAM9的抗体或其抗原结合片段，所述Ab16的重链的氨基酸序列如SEQ ID NO:50所示，轻链的氨基酸序列如SEQ ID NO:51所示；
Ab15为靶向ADAM9的抗体或其抗原结合片段，所述Ab15的重链的氨基酸序列如SEQ ID NO:52所示，轻链的氨基酸序列如SEQ ID NO:53所示。
本发明一些实施方案中，所述抗体药物偶联物为如下偶联物：
p表示连接数，且p为1到10中任一整数，优选3到9中任一整数，更优选4到8的任一整数；例如，p为4、5、6、7或8；
Ab16为靶向ADAM9的抗体或其抗原结合片段，所述Ab16的重链的氨基酸序列如SEQ ID NO:50所示，轻链的氨基酸序列如SEQ ID NO:51所示。
本发明的第八方面提供一种药物组合物，其包含如第一方面所述的抗体或其抗原结合片段和/或如第七方面所述的抗体药物偶联物，以及药学上可接受的载体。
本发明的第九方面提供如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物在制备诊断、预防和/或治疗ADAM9高表达的癌症的药物中的应用。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十方面提供一种诊断、预防和/或治疗ADAM9高表达的癌症的方法，所述方法包括向有需要的患者施用治疗有效量的如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十一方面提供将如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物用于诊断、预防和/或治疗ADAM9高表达的癌症。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十二方面提供一种联合疗法，其包括分别向有需要的患者施用如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物，以及第二治疗剂。
本发明一些实施方案中，所述第二治疗剂包含其他抗ADAM9抗体或其抗原结合片段或者包含所述其他抗ADAM9抗体或其抗原结合片段的抗体药物偶联物或药物组合物，和/或其他治疗ADAM9高表达的癌症的药物。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十三方面提供一种诊断、预防和/或治疗癌症的方法，所述方法包括向有需要的患者施用治疗有效量的如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十四方面提供一种用于诊断、预防和/或治疗癌症的如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十五方面提供一种联合疗法，其包括分别向有需要的患者施用如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物，以及第二治疗剂。
本发明一些实施方案中，所述第二治疗剂包含其他抗ADAM9抗体或其抗原结合片段或者包含所述其他抗ADAM9抗体或其抗原结合片段的抗体药物偶联物或药物组合物，和/或其他治疗癌症的药物。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
本发明的第十六方面提供一种如第七方面所述的抗体药物偶联物的制备方法，所述制备方法包括将如第一方面所述的抗体或其抗原结合片段与如式II所示的化合物反应得到所述的抗体药物偶联物，
L’为与所述的抗体或其抗原结合片段形成如第七方面所述的接头单元L；
所述细胞毒性药物如第七方面所述。
本发明一些实施方案中，所述抗体药物偶联物满足如下条件一种或多种：
(1)所述如式II所示的化合物为
或者
(2)所述的抗体或其抗原结合片段为Ab16或Ab15；
所述Ab16的重链的氨基酸序列优选如SEQ ID NO:50所示，轻链的氨基酸序列优选如SEQ ID NO:51所示；
所述Ab15的重链的氨基酸序列优选如SEQ ID NO:52所示，轻链的氨基酸序列优选如SEQ ID NO:53所示。
本发明的第十七方面提供如第一方面所述的抗体或其抗原结合片段、如第七方面所述的抗体药物偶联物和/或如第八方面所述的药物组合物在制备诊断、预防和/或治疗癌症的药物中的应用。
本发明一些实施方案中，所述癌症选自肺癌、前列腺癌、肝癌、乳腺癌、甲状腺癌、食管癌、胰腺癌、胃癌、卵巢癌和肠癌。
本发明一些具体实施方案中，所述癌症为非小细胞肺癌，例如肺腺癌。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的靶向ADAM9的抗体具有以下一种或多种优势：
1.相比现有技术中的人源化的抗ADAM9抗体hMAB-A(2I.2)，本发明抗体亲水性高，抗体分子具有更好的稳定性和成药性；
2.相比现有技术中的人源化的抗ADAM9抗体hMAB-A(2I.2)，本发明抗体表达量显著提高，高分子量聚体比例低，成药性更好；
3.本发明抗体对人和猴ADAM9抗原具有良好的亲和力；
4.本发明抗体对表达ADAM9的细胞均具有很高的内化率。
本发明的抗体药物偶联物具有以下一种或两种优势：
1.本发明的抗体药物偶联物对ADAM9阳性表达的人肠癌细胞LS174T、Colo205、人肺腺癌细胞Calu-3增殖具有良好的抑制活性；
2.本发明的抗体药物偶联物对人肠癌细胞DLD-1荷瘤小鼠、肠癌细胞LS174T荷瘤小鼠、人肺癌细胞Calu-3荷瘤小鼠具有显著的抗肿瘤活性。
附图说明
图1为ADAM9-ADC对人肠癌细胞DLD-1荷瘤小鼠体内肿瘤抑制测试结果。
图2为ADAM9-ADC对人肠癌细胞LS174T荷瘤小鼠体内肿瘤抑制测试结果。
图3为ADAM9-ADC对人肺癌细胞Calu-3荷瘤小鼠体内肿瘤抑制测试结果。
图4a为LS174T细胞上的FACS结合检测结果。
图4b为LS174T-ADC增殖抑制实验结果。
图5a为HepG2细胞上的FACS结合检测结果。
图5b为HepG2-ADC增殖抑制实验结果。
具体实施方式
定义
本发明中，氨基酸序列中的字母表示本领域公知的氨基酸的单字母缩写，例如J.Biol.Chem,243,p3558(1968)中所述：
丙氨酸：Ala-A、精氨酸：Arg-R、天冬氨酸：Asp-D、半胱氨酸：Cys-C、谷氨酰胺：Gln-Q、谷氨酸：Glu-E、组氨酸：His-H、甘氨酸：Gly-G、天冬酰胺：Asn-N、酪氨酸：Tyr-Y、脯氨酸：Pro-P、丝氨酸：Ser-S、甲硫氨酸：Met-M、赖氨酸：Lys-K、缬氨酸：Val-V、异亮氨酸：Ile-I、苯丙氨酸：Phe-F、亮氨酸：Leu-L、色氨酸：Trp-W、苏氨酸：Thr-T。
在本发明中，术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
在本发明中，术语“或”应被理解为与如上定义的“和/或”具有相同的含义。例如，当分离列表中的项目时，“或”或“和/或”应被解释为包容性的，即，包括数量或元素列表中的至少一个，但也包括多于一个，以及任选地，额外的未列出的项目。只有明确指出相反的术语下，例如“只有一个”或“的确一个”或者在权利要求中使用“由...组成”时，将指的是仅列出的一个数字或列表的一个元素。
在本发明中，术语“抗体药物偶联物”通常是指抗体通过稳定的连接单元与具有生物活性的细胞毒性药物相连。在本申请中“抗体药物偶联物”可以是指把抗体或其抗原结合片段通过稳定的连接单元与具有生物活性的细胞毒性药物片段相连。
在本发明中，术语“细胞毒性药物”通常指毒性药物，所述细胞毒性药物可以在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物可以在足够高的浓度下杀死肿瘤细胞。所述“细胞毒性药物”可以包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32或Lu的放射性同位素)，毒性药物，化疗药物，抗生素或核溶酶，或它们的衍生物，例如，可以是毒性药物，包括但不限于喜树碱衍生物，例如，可以是喜树碱衍生物依沙替康(化学名：(1S,9S)-1-氨基-9-乙基-5-氟-2,3-二氢-9-羟基-4-甲基-1H,12H-苯并[de]吡喃并[3’,4’:6,7]咪唑并[1,2-b]喹啉-10,13(9H,15H)-二酮)。
在本发明中，术语“抗体”通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。抗体可以是来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如kappa(κ)或lambda(λ)的轻链。本申请的抗体可衍生自任何物种。术语“抗体”可包括完整的多克隆抗体、完整的单克隆抗体、多特异性抗体(例如双特异性抗体)、嵌合抗体、人源化抗体、人抗体、包含抗体的 融合蛋白和任何其他经修饰的免疫球蛋白分子，只要这些抗体展示出所需的生物活性即可。
在本发明中，术语“抗原结合片段”通常是指抗体分子的一部分，其包含负责抗体与抗原之间的特异性结合的氨基酸。抗原中由抗体特异性地识别和结合的部分是称作“表位”。如上文所述，抗原结合结构域可典型地包含抗体轻链可变区(VL)和抗体重链可变区(VH)；然而，其并非必须包含两者。Fd片段例如具有两个VH区并且通常保留完整抗原结合结构域的一些抗原结合功能。抗体的抗原结合片段的实例包括(1)Fab片段，具有VL、VH、恒定轻链(CL)和CH1结构域的单价片段；(2)F(ab′)2片段，具有由铰链区的二硫桥连接的两个Fab片段的二价片段；(3)具有两个VH和CH1结构域的Fd片段；(4)具有抗体单臂的VL和VH结构域的Fv片段，(5)dAb片段(Ward等人，“Binding Activities of a Repertoire of Single Immunoglobulin Variable DomainsSecreted From Escherichia coli”，Nature 341:544-546(1989)，其以引用的方式整体并入本申请)，其具有VH结构域；(6)分离的互补决定区(CDR)；(7)单链Fv(scFv)，例如源于scFV-文库。尽管Fv片段的两个结构域VL和VH是由独立基因编码，但其可通过合成连接子使用重组方法接合，合成连接子使得其被制备为其中VL和VH区配对以形成单价分子的单一蛋白链(称为单链Fv(scFv))(可参见例如Huston等人,“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”涉及来自骆驼科(骆驼、单峰骆驼、美洲驼、羊驼等)重链抗体的可变抗原结合结构域(参见Nguyen V.K.等人，2000，The EMBO Journal，19，921-930；Muyldermans S.，2001，J Biotechnol.，74，277-302以及综述Vanlandschoot P.等人，2011，Antiviral Research 92，389-407)。VHH也可称为纳米抗体(Nanobody)(Nb)。
在本发明中，术语“可变区”或“可变结构域”通常是指参与抗体与抗原的结合的抗体重链或轻链的结构域。在本申请中，术语“可变”通常是指，抗体的可变结构域的序列的某些部分变化强烈，形成各种特定抗体对其特定抗原的结合和特异性。变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链可变区和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)，分别为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3。可变域中更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDR通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。
在本发明中，所列CDR的氨基酸序列均是按照Kabat定义规则所示出的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明请求保护的范围是基于Kabat定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
如下进行序列之间序列同一性的计算。为确定两个氨基酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置处的氨基酸残基。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基占据时，则所述分子在这个位置处是相同的。可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12、空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W. Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列之间的同一性百分数。额外地或备选地，可以进一步使用本发明所述的蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
本发明中，术语“全长抗体”可互换地用来指包含由二硫键相互连接的至少两条重链(HC)和两条轻链(LC)的糖蛋白。每条重链由重链可变区(本发明中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本发明中缩写为VL)和轻链恒定区(本发明中缩写为CL)组成。轻链恒定区由一个结构域CL组成。哺乳动物重链分类为α、δ、ε、γ和μ。哺乳动物轻链分类为λ或κ。包含α、δ、ε、γ和μ重链的免疫球蛋白分类为免疫球蛋白(Ig)A、IgD、IgE、IgG和IgM。完全抗体形成“Y”形状。Y的茎由两条重链的第二和第三恒定区(并且对于IgE和IgM，第四恒定区)结合在一起组成，并且二硫键(链间)在铰链中形成。重链γ、α和δ具有由三个串联(成一行)Ig结构域构成的恒定区，和用于增加柔性的铰链区；重链μ和ε具有由四个免疫球蛋白结构域构成的恒定区。第二和第三恒定区分别称为“CH2结构域”和“CH3结构域”。Y的每个臂包括结合到单个轻链的可变和恒定区的单个重链的可变区和第一恒定区。轻链和重链的可变区负责抗原结合。
本发明中，“Fab”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。“Fab’”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’的两条重链之间形成链间二硫键以形成F(ab’)2分子。“F(ab’)2”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。术语“Fv”意指向抗体的单臂的VL和VH结构域组成的抗体片段，但缺少恒定区。
本发明中，所述的scFv(single chain antibody fragment，单链抗体)可为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子[参见，例如，Bird等人，Science 242:423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)]。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的G4S氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(G4S)4或(G4S)3接头，但也可使用其变体。
在本发明中，术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
在本发明中，术语“人源抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
在本发明中，“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。在本发明的一些实施方案中，利用表面等离子共振(SPR)技术测量亲和力，例如本发明的抗体与抗原之间的亲和力。在本发明的一些优选的实施方案中，用于测量亲和力的一个具体方法是本文中的BIAcore法。
在本发明中，术语“卤素”通常是指氟、氯、溴、碘，例如，可以是氟、氯。
在本发明中，术语“烷基”通常是指烷除去氢原子所衍生的残基。烷基可以是取代的或非取代的，替代或者非替代的。术语“烷基”通常指饱和的直链或支链脂肪族烃基，其具有从母体烷的相同碳原子或两个不同的碳原子上除去氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如含有1至12个碳原子，例如含有1至6个碳原子的链烷基。烷基的非限制性实例包括但不限于甲基、乙基、丙基、丙基、丁基等。
在本发明中，术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，优选包含3至10个碳原子，优选 包含3至8个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性示例包括环丙烷基、环丁烷基、环戊烷基、环戊烯基、环己烷基、环己烯基、环己二烯基、环庚烷基、环庚三烯基、环辛烷基等；多环环烷基包括螺环、稠环和桥环的环烷基。
在本发明中，术语“杂环基”指饱和或部分不饱和单环或多环环状轻取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或硫的杂原子，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至8个环原子，其中1-3是杂原子；更优选包含3至6个环原子，其中1-3个是杂原子；最优选包含5或6个环原子，其中1-3个是杂原子。单环杂环基的非限制性示例包括吡咯烷基、四氢吡喃基、哌啶基、吗啉基、硫代吗啉基和高哌嗪基的等。多环杂环基包括螺环、稠环和桥环的杂环基。所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其与母体结构连接在一起的环为杂环基。
在本发明中，术语“独立地”通常是指变量适用于任何一种情况，而不考虑在相同化合物中具有相同或不同定义的变量存在与否。例如其中的变量可以是指化合物的取代基种类、数量或化合物中原子的种类等。例如，在化合物中出现2次R并且R被定义为“独立地碳或氮”时，两个R可以均为碳，两个R可以均为氮，或一个R可以为碳而另一个R为氮。
在本发明中，术语“任选”或“任选地”通常意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明可以包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
在本发明中，术语“取代的”通常指基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本发明中，如本领域技术人员可知的，“烷基”、“烯基”、“环烷基”等之类的术语可以在名称前加一个标识表示在特定情况下基团中存在的原子数，例如，C1-C4烷基，C3-C7环烷氧基，C1-C4烷基羰基氨基等，“C”后所跟下标数字表示在基团中存在的碳原子数。例如，C3烷基是指具有三个碳原子的烷基(例如，正丙基，异丙基)；C1-10中，基团的成员可具有落入1-10范围内的任何数目的碳原子。
在本发明中，本发明的化合物或抗体药物偶联物包含其互变异构体、内消旋体、外 消旋体、对映异构体、和/或非对映异构体。在本申请中，术语“非对映异构体”通常是指具有两个或更多个手性中心并且其分子不是彼此的镜像的立体异构体。非对映异构体可以具有不同的物理性质，例如、熔点、沸点、波谱性质和反应性。在本申请中，术语“互变异构体”或“互变异构形式”可互换使用，通常是指可通过低能垒(low energy barrier)互相转化的不同能量的结构异构体。例如，质子互变异构体(protontautomer)(也称为质子移变互变异构体(prototropic tautomer)包括通过质子迁移进行的互相转化，诸如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组进行的互相转化。在本申请中，术语“内消旋体”通常是指分子内含有不对称性的原子，但具有对称因素而使分子内总旋光度为零。术语"外消旋体"或"外消旋混合物"是指由等摩尔量的两种对映异构体物质构成的组合物。
在本发明中，术语“接头单元”或“接头结构”通常指指一端与配体连接而另一端与细胞毒性药物相连的化学结构片段或键，也可以连接其他接头后再与细胞毒性药物相连。所述直接或间接连接配体可以是指所述基团通过共价键直接连接配体，也可以是通过接头结构连接配体。例如，可以使用包含酸不稳定接头结构(例如腙)、蛋白酶敏感(例如肽酶敏感)接头结构、光不稳定接头结构、二甲基接头结构、或含二硫化物接头结构的化学结构片段或键作为接头结构。
在本发明的某些实施例中，抗体药物偶联物指包含其不同DAR分布的组合物，术语“载药量”通常是指每个配体上加载的细胞毒性药物平均数量，可称为平均连接数，也可以表示为细胞毒性药物和抗体量的比值，细胞毒性药物载量的范围可以是每个配体(Ab)连接0-12个，例如1-10个细胞毒性药物。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的载药量。平均连接数p可以为1到10的整数或小数。例如，平均连接数p可以为2到8的整数或小数。例如，平均连接数p可以为3到8的整数或小数。例如，平均连接数p可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10的整数或小数。例如，平均连接数p为7.8或7.9。
在本发明的某些实施例中，抗体药物偶联物指包含相同DAR分布的化合物，术语“载药量”指每个配体上加载的细胞毒性药物数量，可称为连接数，也可以表示为细胞毒性药物和抗体量的比值，细胞毒性药物载量的范围可以是每个配体(Ab)连接0-12个，例如1-10个细胞毒性药物。连接数p可以为1到10的任一整数。例如，连接数p可以为3到9的任一整数。例如，连接数p可以为4到8的任一整数。例如，连接数p可以为4、5、6、7或8。
在本发明中，本发明的化合物或抗体药物偶联物的某些原子可能以一种以上的同位素形式出现。例如，氢可能以氕(1H)、氘(2H)和氚(3H)的形式存在，碳可能以三种不同的同位素(12C、13C和14C)自然存在。可并入本申请化合物中的同位素示例还包括但不限于15N、18O、17O、18F、32P、33P、129I、131I、123I、124I、125I，或者类似的同位素。因此，相对于这些同位素的自然丰度，本发明的化合物或抗体药物偶联物可富集在一种或多种这些同位素中。如本领域技术人员所知，此类同位素富集化合物可用于多种用途。例如，用重同位素如氘(2H)替代可能会提供某些治疗优势，这可以是由于更高的代谢稳定性。例如，氘(2H)的自然丰度约为0.015％。因此，自然界中大约每6500个氢原子，就有一个氘原子。因此，本发明的含氘化合物或抗体药物偶联物在一个或多个位置(视情况而定)的氘丰度大于0.015％。除非另有指明，否则本发明所述的结构还可以包括仅在是否存在一个或多个同位素富集原子方面存在差别的化合物或抗体药物偶联物。举例而言，除了氢原子被氘或氚所取代，或碳原子被碳13或碳14所取代之外，其余部分均与本发明结构一致的化合物或抗体药物偶联物均在本发明的范围之内。
如本领域已知，在本发明中“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
本发明的重组表达载体可以是任何合适的重组表达载体，其能够用于转化或转染将一种或多种所关注的基因或序列递送入任何合适的宿主细胞并且优选在宿主细胞中表达所述基因或序列。合适的载体包括经过设计用于扩展和扩增或用于表达或以上两项的那些载体，载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体。
在本发明中，术语“宿主细胞”是指可以含有本文所描述的核酸或载体的任何类型的细胞。在示范性方面中，宿主细胞是真核细胞，例如植物、动物、真菌或海藻；或可以是原核细胞，例如细菌或原生动物。在示范性方面中，如本文所描述，宿主细胞是起源于或获自个体的细胞。在示范性方面中，宿主细胞来源于或获自哺乳动物。
在本发明中，用于培养所产生的转化体和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
在本发明中，非诊断和/或治疗目的的的检测的应用场景例如：在实验室中检测ADAM9蛋白存在与否；或作为阳性抗体来筛选其它靶向ADAM9的抗体；或与其它抗ADAM9的抗体竞争结合，检测抗体之间是否存在竞争即抗原表位是否相同或相似等应 用场景。
在本发明中，术语“药物组合物”通常是指含有一种或多种本申请所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物可以是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。常规的药物组合物的制备可以见中国药典。药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。例如，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
在本发明中，术语“药学上可接受的盐”或“可药用的盐”通常是指本发明化合物或抗体药物偶联物的盐，或本发明中所述的化合物或抗体药物偶联物的盐，这类盐用于哺乳动物体内时可以具有安全性和/或有效性，且可以具有应有的生物活性，本发明的化合物或抗体药物偶联物可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
在本发明中，药学上可接受的载体是常规使用的那些载体中的任一种，并且仅受到物理-化学考虑因素(如溶解性和与靶向ADAM9的抗体的反应性的缺乏)限制，并且受给药途径限制。本文所描述的药学上可接受的载体，例如媒剂、佐剂、赋形剂和稀释剂为所属领域的技术人员所熟知并且公众可容易获得。在一个方面中，药学上可接受的载体是对医药组合物的活性成分具有化学惰性的载体，并且是在使用条件下不具有不利的副作用或毒性的载体。在一些实施例中，当向动物或人类给予时，载体不产生不良、过敏或其它不适当的反应。在一些方面中，药物组合物不含热原质以及会对人类或动物有害的其它杂质。药学上可接受的载体包括任何和所有溶剂、分散介质、涂料、抗细菌剂和抗真菌剂、等张剂和吸收延迟剂等等；其用途在所属领域中是众所周知。
如本发明所用，术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示，与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强 正常生理功能的量。
在本发明中，术语“癌症”指的是恶性肿瘤，为由控制细胞生长增殖机制失常而引起的疾病。术语“肠癌”一般指大肠癌，又称为“结直肠癌”，是指大肠上皮来源的癌症，包括结肠癌与直肠癌。术语“肺腺癌”是指发生于肺的上皮组织来源的恶性肿瘤，肺腺癌属于非小细胞肺癌的范畴。术语“ADAM9高表达的癌症”是指由于机体内ADAM9的异常高表达而引起的恶性肿瘤。
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1对MAB-A抗体重新人源化改造
对鼠抗ADAM9单克隆抗体MAB-A抗体(专利WO2018119196A1所披露的重链可变区SEQ ID NO:7和轻链可变区SEQ ID NO:11的序列)重新人源化的目的是为了提高其人源化版本抗体的溶解度、降低抗体的疏水性、从而提高其作为ADC药物的偶联得率和成药性，MAB-A进入临床研究阶段的人源化版本抗体hMAB-A(2I.2)(专利WO2018119196A1所披露的重链SEQ ID NO:52和轻链SEQ ID NO:68的序列信息)存在疏水性较强的问题。
对鼠源抗体重新人源化如本领域文献公示的方法进行，即使用人恒定结构域替换鼠源抗体恒定结构域，并对CDR区进行以亲和力和成药性优化为目的的改造。根据hMAB-A(2I.2)亲本鼠源MAB-A抗体和人抗体的同源性选择人种抗体序列，将MAB-A抗体进行重新人源化。具体方法为在鼠源抗体MAB-A的VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板；将鼠源抗体CDR区移植到选择好的相应人源化模板上；然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基、以及对VL和VH的构象有重要影响的残基进行回复突变、并对CDR区化学不稳定氨基酸残基优化，经表达测试和回复突变数量对比，选择出设计了人源化重链可变区HCVR和轻链可变区LCVR序列组合而成的抗体，人源化抗体的CDR区序列编号如表1所示，CDR的氨基酸序列如表2所示，人源化抗体的重链和轻链可变区序列如表3所示。
表1人源化抗体的CDR区序列编号

表2人源化抗体的CDR区的氨基酸序列

在表2中，抗体的CDR区依据Kabat编号系统定义，其中X1可为氨基酸N、D、H或E；X2可为氨基酸D或R；X3可为氨基酸K或D；X4可为氨基酸N或E；X5可为氨基酸R或K；X6可为氨基酸D或N。
表3人源化抗体的重链和轻链可变区序列


表3中的氨基酸序列下方的下划线表示依据Kabat编号系统定义的CDR区域。
将设计的重链和轻链可变区序列分别与人抗体的重链恒定区和轻链恒定区序列连接。示例性地，抗体重链恒定区选自序列如SEQ ID NO:48所示的人IgG1重链恒定区；抗体轻链恒定区选自序列如SEQ ID NO:49所示的人κ链的恒定区，抗体恒定区序列如表4所示。
表4恒定区序列编号
Ab16的重链氨基酸序列为(SEQ ID NO:50):

Ab16的轻链氨基酸序列为(SEQ ID NO:51):
Ab15的重链氨基酸序列为(SEQ ID NO:52):
Ab15的轻链氨基酸序列为(SEQ ID NO:53):
实施例2人源化Anti-ADAM9抗体的疏水性检测
实验步骤：不同抗体的亲疏水性采用HIC-HPLC分析方法进行评估，抗体亲水性越强，与HIC配基——丁基的疏水相互作用越弱，保留时间相对更短。将所有抗体置换到200mM Arg.SSC pH 5.5缓冲液中，浓度调至5mg/mL，采用如下色谱方法(表5)检测各重新人源化anti-ADAM9抗体和hMAB-A(2I.2)抗体的疏水性。实验结果如表6所示。
表5色谱检测方法
表6色谱检测实验结果
实验结论：根据上述实验结果可以看出，经过结构设计和改造，改造抗体的亲水性得到显著提高，改善了抗体分子的稳定性和成药性。
实施例3人源化anti-ADAM9人源化抗体生产表达
将实施例1中20条抗体，Ab1至Ab20，以及hMAB-A(2I.2)的轻链可变区氨基酸序列分别与轻链κ恒定区氨基酸序列(SEQ ID NO:49)连接，重链可变区氨基酸序列分别与IgG1重链恒定区氨基酸序列(SEQ ID NO:48)连接，通过密码子优化合成cDNA并连接入PTT5质粒(委托南京金斯瑞生物科技有限公司)，21条抗体均采用相同的信号肽 序列。使用PEImax 40000将每一条抗体的重链和轻链对应的PTT5质粒共转到CHO细胞中；培养5天后收集细胞培养上清，经protein A(MabSelectTM PrismA)纯化抗体成分，并通过OD280nm对抗体进行定量(委托南京金斯瑞生物科技有限公司进行)。
实施例1中20条抗体按4ml体系表达，每条抗体的表达量如下表7所示。
表7 4ml体系表达的抗体产量
实验结论：Ab06、Ab10、Ab15、Ab16的表达量约3倍高于hMAB-A(2I.2)。
Ab10、Ab15、Ab16和hMAB-A(2I.2)这4条抗体继续按500ml体系表达，每条抗体的表达量如下表8所示。
表8 500ml体系表达的抗体产量
实验结论：Ab15、Ab16的表达量远高于hMAB-A(2I.2)，且SEC-HPLC检测显示高分子量聚体比例低，提示成药性较好于hMAB-A(2I.2)。
实施例4人源化anti-ADAM9抗体的体外结合活性检测实验
4.1体外间接ELISA结合实验
用pH7.4的PBS将和人ADAM-His蛋白(AcroBiosystems，Cat#AD9-H52H7)稀释至1μg/ml浓度，以100μL/孔的体积加入96孔高亲和力酶标板中，于4℃冰箱孵育过夜(16-20小时)。用PBST(pH7.4 PBS含0.05％Tween-20)洗板3次后，加入用PBST稀释的1％牛血清白蛋白(BSA)封闭液200μL/孔，37℃孵育1小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板1次。用含1％BSA的PBST稀释待测抗体，15μg/mL起始，3倍梯度稀释，11个剂量，以100μL/孔加到酶标板中，放于37℃孵育1小时。孵育结束后用PBST洗板3次，加入100μL/孔用含1％BSA的PBST稀释的HRP标记羊抗人二抗(Rockland，cat#609-103-123)，室温孵育45分钟。用PBST洗板6次后，加入100μl/孔TMB显色底物(苏州亚科化学试剂股份有限公司，cat#S0025)，于室温避光孵育10分钟，加入50μl/孔1M HCl终止反应，用酶标仪(Thermo，Ascent)在450nm处读取吸收值，分析数据。做浓度-信号值曲线分析结果，如下表9所示。结果显示：本发明抗体对人ADAM9抗原的亲和力良好。
表9重新人源化抗体对人ADAM9抗原的亲和力(EC50值)
4.2 Biacore检测亲和力实验
芯片制备：将鼠抗人IgG(Fc)抗体(货号：29234600，Cytiva)用固定试剂(10mM醋酸钠，pH 5.0)稀释到25μg/mL，即将950μL固定试剂加入50μL鼠抗人IgG(Fc)抗体，用于固定八个通道。首先，CM5芯片的表面用400mM EDC和100mM NHS以10μL/min的流速进行420s的活化。其次，将25μg/mL的鼠抗人IgG(Fc)抗体以10μL/min的流速注入到实验通道约360s，固定量约为7000至14000RU。最后，芯片用1M乙醇胺以10μL/min进行420s封闭。参比通道与试验通道进行相同的操作。
捕获配体：将抗体Ab10、Ab15、Ab16、hMAB-A(2I.2)用运行试剂(含10mM N-(2-羟乙基)哌嗪-N-2磺酸(HEPES)，150mM氯化钠(NaCl)，3mM乙二胺四乙酸(EDTA)，0.005％吐温-20(Tween-20)，pH调节至7.4)稀释至4μg/mL并以10μL/min的流速注入到人IgG(Fc)捕获实验通道约200RU。参比通道不需要进行配体的捕获。
分析物多循环分析：将人、食蟹猴ADAM9蛋白(人ADAM9蛋白购自ACRO，货号AD9-H52H7、猴ADAM9蛋白购自ACRO，货号AD9-C52H7)用运行试剂进行2倍 倍比稀释(表10)。将稀释后的人或食蟹猴ADAM9蛋白依次以30μL/min的流速注入到实验通道与参比通道，结合和解离相应时间。结合解离步骤均在运行试剂中进行。每一个浓度分析后，芯片需要用3M氯化镁以20μL/min的流速再生30s，洗掉配体以及未解离的分析物。进行下一个浓度分析时，实验通道需要重新捕获相同量的配体。
表10 anti-ADAM9抗体与人、食蟹猴ADAM9蛋白亲和力测试的浓度梯度及结合解离时间
数据分析：使用Biacore 8K分析软件Biacore Insight Evaluation Software计算每个样品的KD值。参比通道用于背景的扣减。
体外间接ELISA结合实验和Biacore方法测定结果如表11和表12所示：
表11抗体对人ADAM9抗原的亲和力
表12抗体对猴ADAM9抗原的亲和力
ELISA和Biacore结果显示Ab15、Ab16抗体与人和猴ADAM9抗原具有良好的亲和力。
实施例5：人源化Anti-ADAM9抗体的内化能力
设置结合板，ADAM9高表达的人肺癌细胞Calu-3(购自中国科学院细胞库，货号TCHu157)，经胰酶消化后，离心收集细胞，用FACS缓冲液(含2％FBS的1×PBS)调节细胞密度后分铺于96孔U底板，每孔1×105个细胞，离心1200g、5分钟，弃上清，加入100μL已用FACS缓冲液梯度稀释的抗体溶液(抗体起始工作浓度为100nM，10倍稀释，8个浓度点，并设立0nM点)，4℃孵育1小时。离心1200g、5分钟，弃上清，PBS洗细胞2次后，添加FACS缓冲液配制的FITC荧光标记的羊抗人IgG H&L抗体(Abcam，Cat#ab6866)，100μL每孔重悬细胞，4℃孵育1小时。离心1200g、5分钟，弃上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞仪检测每个浓度抗体作用下细胞的平均荧光强度(MFI结合孔)。
同时设置内吞板，Calu-3经胰酶消化后，离心收集细胞，用FACS缓冲液调节细胞密度后分铺于96孔U底板，每孔1×105个细胞，离心1200g、5分钟，弃上清，加入100μL已用FACS缓冲液梯度稀释的抗体溶液(抗体起始工作浓度为100nM，10倍稀释，8个浓度点，并设立0nM点)，37℃孵育16小时。而后离心1200g、5分钟，弃上清，PBS洗细胞2次，添加FACS缓冲液配制的FITC荧光标记的羊抗人IgG H&L抗体，100μL每孔重悬细胞，4℃孵育1小时。离心1200g、5分钟，弃上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞仪检测每个浓度抗体作用下细胞的平均荧光强度(MFI内吞孔)。
各浓度抗体作用下的抗体内化率，用以下公式计算：内化率％＝(MFI结合孔-MFI内吞孔)/MFI结合孔×100％，并作浓度-曲线分析抗体内化的EC 50浓度，结果如表13所示。
表13抗体内化率
结果显示：Ab10、Ab15、Ab16抗体对表达ADAM9的细胞均具有很高的内化率。
实施例6：接头-细胞毒素的合成
接头-细胞毒素X1：
合成路线：
第一步：
氮气保护下，向27a(5.00g，43.0mmol)，NaHCO3(10.9g，129mmol)的DMF(50mL)溶液中滴加苄溴(11.0g，64.6mmol)，并在25℃反应17小时。TLC(PE/EA＝2/1)显示反应完全，将反应液加入到500mL水中，用EA(250mL)萃取两遍，分液后经饱和氯化钠水溶液(500mL)洗涤，无水Na2SO4干燥，浓缩过柱(PE:EA＝3:2)得5.1g无色液体，收率：57.1％。
第二步：
氮气保护下，向KI2(4.00g，10.9mmol)，TsOH(800mg，4.65mmol)的THF(30mL)溶液中，在0℃下，滴加27b(4.50g，21.8mmol)的THF(10mL)溶液，并在25℃反应2小时。TLC(PE/EA＝1/2)显示反应完全，将反应液加入到200mL水中，用EA(200mL) 萃取两次分液，无水Na2SO4干燥，浓缩过柱(PE/EA＝3/2)得白色固体1.56g，收率：26％。
第三步：
氢气环境下，在0℃下，向27c(800mg，1.55mmol)的EtOH(8mL)和EA(8mL)混合溶液中加入Pd/C(80mg)，在0℃搅拌2.5小时。LCMS显示反应完全。反应液经硅藻土过滤，用EA(200mL)洗涤滤饼浓缩后用THF(20mL)溶解旋干得白色固体600mg，收率：91％
第四步：
氮气保护下，在0℃下，向27d(220mg，0.515mmol)，HY-13631A(250mg，0.47mmol)和HATU(214mg，0.56mmol)的DMF(6mL)溶液中加入DIEA(152mg，1.18mmol)，并在0℃反应2小时。LCMS显示反应完全。将反应液加入柠檬酸水溶液(pH＝4)(150mL)中，过滤，并用175mL水洗涤滤饼，滤干，用油泵拉干得棕色固体260mg，收率：66％。
第五步：
氮气保护下，在0℃下，向27e(260mg，0.309mmol)的DCM(30mL)溶液中滴加二乙胺(8mL)，并在0℃反应3小时。LCMS显示反应完全。将反应液加入0℃的石油醚溶液(600mL)中，有固体析出，静置待固体吸附于瓶底后，倒出溶液，用油泵拉干，得棕色固体90mg，收率：47.1％。
第六步：
氮气保护下，在0℃下，向27f(90mg，0.13mmol)，KI-1(92mg，0.19mmol)和DIEA(50mg，0.39mmol)的DMF(2.5mL)溶液中加入HATU(74mg，0.19mmol)并在0℃反应2小时。LCMS显示基本反应结束。在0℃下，将反应液加入PH＝4的柠檬酸水溶液(30mL)中，有絮状固体析出，过滤，经制备板(DCM/MecOH＝10/1)得9.2mg淡黄色固体X1，收率：6％。
MS m/z(ESI)：1074[M+1]。
H-NMR(400MHz，MeOD)：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)。
接头-细胞毒素X2：
合成路线：
第一步
将34a(5g，48.0mmol)、K2CO3(19.9g，144.0mmol)溶于DMF(20mL)中，滴加苄溴(12.3g，72.0mmol)，25℃下反应17小时。TLC(PE/EA＝3/1)检测原料反应完全。将反应液加入水(200mL)中、用EA(250mL)萃取分液，用饱和NaCl洗涤，无水Na2SO4干燥后浓缩过柱(PE：EA＝2：1)得8.7g无色液体34b，收率93％。MS-ESI：m/z 195.1[M+H]+。
第二步
取34c(7.3g，19.8mmol)、TsOH(1.46g，8.5mmol)溶于THF(20mL)中，氮气保护并降温至0℃，滴加43b(7.7g，39.6mmol)的THF(10mL)溶液，加完后0℃反应2小时。TLC(PE/EA＝2/1)显示原料大部分反应。将反应液倒入100mL水中，DCM(100mL)萃取，分液并用饱和NaCl洗涤，无水Na2SO4干燥后过柱(PE/EA＝1/1)，得3.9g无色稠状物34d，收率：39％。MS-ESI：m/z 503.3[M+H]+。
第三步
氢气环境下，在0℃下，向34d(1.9g，3.78mmol)的EtOH(100mL)和EA(100mL)混合溶液中加入Pd/C(1g，10wt.％)，在0℃反应3小时。TLC(PE/EA＝2/1)显示反应完全。反应液经硅藻土过滤，用EA/EtOH(1：1，100mL×3)洗涤滤饼，滤液浓缩，并用THF(50mL×3)溶解旋干并重复三次得1g灰色固体34e，收率：64％。MS-ESI：m/z435.2[M+Na]+。
第四步
在氮气保护下，0℃下向34e(426mg，1.03mmol)，KI4(500mg，0.94mmol)和HATU(429mg，1.13mmol)的DMF(20mL)溶液中滴加DIEA(303mg，2.35mmol)，加完后在0℃反应2小时。LCMS显示反应结束。将反应液滴到300mL水中，搅拌后静置5分钟，过滤，滤饼用DCM/MeOH(10：1，100mL)的溶液溶解后，干燥旋干拌样，柱层析(EA：MeOH＝30：1)得600mg黄色固体34f，收率：77％。MS-ESI：m/z 830.3[M+H]+。
第五步
氮气保护下，在0℃下，向34f(150mg，0.18mmol)的DCM(5mL)溶液中滴加二乙胺(5mL)，并在0℃反应2小时。LCMS显示反应完全。将石油醚溶液(100mL×6)加入反应液中，有固体析出，静置待固体沉淀后，倒出溶液，再用油泵拉干，得120mg白色粉末34g，LCMS显示产物含量为70％，收率：76％。MS-ESI：m/z 608.3[M+H]+。
第六步
在氮气保护下，向34g(60mg，0.099mmol)、43h(51mg，0.108mmol)、和DIEA(32mg，0.25mmol)的DMF(1mL)溶液中在0℃下加入HATU(45mg，0.118mmol)的DMF(1mL)溶液，并在0℃下反应2小时。LCMS显示原料反应完全。将反应液直接过反相柱，洗脱剂(MeCN/MeOH＝1/1)：H2O＝60％：40％)，纯化得14.8mg黄色固体X2，收率14％。
MS-ESI：m/z 1062.4[M+H]+。
1H NMR(400MHz,Methanol-d4)δ7.69–7.61(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＝16.4Hz,1H),5.40–5.31(m,2H),5.26(d,J＝19.0Hz,1H),4.65–4.50(m,7H),4.25–4.16(m,1H),3.87(d,J＝16.7Hz,1H),3.83–3.76(m,3H),3.72(d,J＝17.0Hz,2H),3.44(t,J＝7.1Hz,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＝7.3Hz,3H)。
接头-细胞毒素X3：
合成路线：
第一步：
向32a(2.00g，6.6mmol)，K2CO3(1.82g，13.2mmol)的MeCN(20mL)中加入溴丙烯(960mg，7.92mmol)，在20℃下搅拌5小时。TLC(PE/EA＝1/2)显示反应结束。将反应液倒入水100mL中，将pH值调至5，用EA(100mL)萃取三次，无水硫酸钠干燥，旋干过柱纯化(PE/EA＝2/1)得1.83g白色固体32b，收率：81％。
第二步：
向32b(1.38g，4.02mmol)的DCM(10mL)中加入TFA(10mL)，在25℃下搅拌17小时。TLC(PE/EA＝1/3)显示反应结束。将反应液旋干得0.91g黄色粘状物32c，收率不计。
第三步：
向32c(910mg，4.87mmol)，NaHCO3(613mg，7.3mmol)的DME/H2O(20mL/10mL)中加入41d(1.92g，4.87mmol)，在25℃下搅拌3小时。TLC(DCM/MeOH＝1/1)显示反应结束。将反应液倒入水100mL中，用aq.HCl(1N)将pH值调至5，用EA(150mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(DCM/MeOH＝20/1)得1.53g白色固体32e，收率：67％。MS-ESI：m/z 467.4[M+H]+。
第四步：
向32f(3g，5.83mmol)的MeOH(50mL)中加入Pd/C(600mg)，在25℃在氢气球下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.9g白色固体32g，收率：77％。
第五步：
向32g(789mg，1.86mmol)，KI4(900mg，1.69mmol)，三乙胺(342mg，3.38mmol)的DMF(10mL)中加入HATU(707mg，1.86mmol)，在0℃下搅拌3.5小时。TLC(EA)显示反应结束。将反应液倒入H2O(80mL)，用EA(100mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(EA)得1.186g白色固体32h，收率：83％。MS-ESI：m/z 842.3[M+H]+。
第六步：
将32h(1.186g，1.41mmol)的DCM/二乙胺(20mL，20/1)在25℃下搅拌17小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液倒入石油醚(200mL)中过滤得768mg白色固体32i，收率：88％。MS-ESI：m/z 620.3[M+H]+。
第七步：
向32i(676mg，1.09mmol)，32e(508mg，1.09mmol)，DIEA(423mg，3.27mmol)的DMF(10mL)中加入HATU(414mg，1.09mmol)，在20℃下搅拌17小时。TLC(PE/EA＝1/5)显示反应结束。将反应液倒入水中(30mL)过滤，滤饼过柱纯化(DCM/MeOH＝50/1)511mg白色固体32j，收率：44％。MS-ESI：m/z 1068.3[M+H]+。
第八步：
将32j(482mg，0.451mmol)的二乙胺/DCM(10mL，1/5)的溶液在10℃下搅拌17小时。TLC(EA)显示反应结束。将反应液倒入PE(300mL)中过滤得301mg白色固体32k，收率不计。
第九步：
向32k(301mg，0.356mmol)，Pd(PPh3)4(82mg，0.071mmol)的THF(5mL)中加入吗啡啉(93mg，1.07mmol)，在25℃下搅拌5小时。LCMS显示反应结束。将反 应液制备得108mg白色固体32l，收率：38％。MS-ESI：m/z 806.3[M+H]+。
第十步：
向32l(108mg，0.134mmol)，三乙胺(41mg，0.402mmol)的THF(2mL)和DMF(2mL)中加入溴乙酰溴(27mg，0.134mmol)，在0℃下搅拌1小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液直接制备得15mg白色固体X3，收率：12％。
MS-ESI：m/z 926.3[M+H]+。
1H NMR(400MHz,DMSO-d6)δ12.11(s,1H),8.54–8.42(m,3H),8.27–8.16(m,2H),7.78(d,J＝11.0Hz,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＝5.6Hz,2H),3.70(d,J＝5.9Hz,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＝7.3Hz,3H)。
接头-细胞毒素X4：
合成路线：
第一步：
向33a(2.00g，2.58mmol)的MeOH(20mL)中加入Pd/C(400mg，10wt.％)，在 20℃下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.3g白色固体33b，收率：74％。
第二步：
向33b(0.55g，0.802mmol)，KI4(427mg，0.802mmol)和DIPEA(310mg，2.40mmol)的DMF(5mL)中加入HATU(305mg，0.802mmol)，在0℃下搅拌2小时。TLC(DCM/MeOH＝1/10)显示反应结束。将反应液倒入水(40mL)中，过滤得粗品，经柱纯化(DCM/MeOH＝20/1)得360mg黄色固体33c，收率41％。
第三步：
向33c(360mg，0.326mmol)的DCM(10mL)中加入二乙胺(2mL)。在25℃下搅拌17小时。TLC(DCM/MeOH＝5/1)显示反应结束。将反应液倒入PE(100Ml)中，过滤得205mg白色固体33d，收率：71％。MS-ESI：m/z 881.3[M+H]+。
第四步：
向33d(205mg，0.233mmol)和三乙胺(118mg，1.17mmol)的DMF(1mL)和水(1mL)中加入溴乙酰溴(94mg，0.446mmol)的THF(2mL)溶液，并在0度搅拌1小时，反应液直接制备得15mg白色固体X4，收率：6％。
MS-ESI：m/z 1001.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.57–8.50(m,1H),8.50–8.43(m,2H),8.35–8.29(m,1H),8.19–8.12(m,2H),7.80(d,J＝10.8Hz,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＝7.3Hz,3H)。
实施例7：Anti-ADAM9偶联喜树碱类接头-细胞毒素
抗体药物偶联物Ab16-X1的制备：
向抗体Ab16的缓冲液(PBS pH 7.2；40mg，8.7mg/mL，0.27μmol)加入2mM EDTA溶液(0.2mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(7.0mM，0.424mL，2.97μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应18小时，并停止反应；将接头-细胞毒素X1(2.6mg，2.43μmol)溶解于0.24mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10Desalting column,53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物Ab16-X1的溶液(30mM组氨酸-盐酸pH 5.5；38mg，5.6mg/mL，收率：95％)，于-80℃储存。HIC DAR分析检测并计算得DAR值p＝7.8。
抗体药物偶联物Ab16-X2的制备：
向抗体Ab16的缓冲液(PBS pH 7.2；40mg，8.7mg/mL，0.27μmol)加入2mM EDTA溶液(0.2mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(7.0mM，0.424mL，2.97μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应18小时，并停止反应；将接头-细 胞毒素X2(2.6mg，2.43μmol)溶解于0.24mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10Desalting column,53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物Ab16-X2的溶液(30mM组氨酸-盐酸pH 5.5；37mg，5.6mg/mL，收率：92％)，于-80℃储存。HIC DAR分析检测并计算得DAR值p＝7.9。
抗体药物偶联物Ab15-X2的制备：
向抗体Ab15的缓冲液(PBS pH 7.4；40mg，6.8mg/mL，0.27μmol)加入2mM EDTA溶液(0.2mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(7.0mM，0.471mL，3.29μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应18小时，并停止反应；将接头-细胞毒素X2(2.6mg，2.43μmol)溶解于0.24mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10Desalting column,53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物Ab15-X2的溶液(30mM组氨酸-盐酸pH 5.5；32mg，5.2mg/mL，收率：80％)，于-80℃储存。HIC DAR分析检测并计算得DAR值p＝7.8。
抗体药物偶联物hMAB-A(2I.2)-X2的制备：
向抗体hMAB-A(2I.2)的缓冲液(PBS pH 7.4；40mg，7.5mg/mL，0.27μmol)加入2mM EDTA溶液(0.2mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(7.0mM，0.379mL，2.65μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应18小时，并停止反应；将接头-细胞毒素X2(2.6mg，2.43μmol)溶解于0.24mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。在 AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10Desalting column，53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物hMAB-A(2I.2)-X2的溶液(30mM组氨酸-盐酸pH 5.5；29mg，3.2mg/mL，收率：72％)，于-80℃储存。HIC DAR分析检测并计算得DAR值p＝7.5。
实施例8：anti-ADAM9抗体药物偶联物(ADAM9-ADC)体外细胞增殖抑制活性测试
采用化学发光细胞活率检测法(即CTG方法)评估各anti-ADAM9抗体偶联喜树碱类毒素的ADC药物在ADAM9阳性表达的人肠癌细胞LS174T(购自中国科学院细胞库，货号TCHu32)或Colo205(购自中国科学院细胞库，货号TCHu102)、人肺腺癌细胞Calu-3中孵育处理6天，对细胞增殖的抑制作用。
收集对数生长期细胞，以5000个细胞/孔的密度铺板，细胞板放入37℃、5％CO2培养箱培养过夜。实验第二天，将各ADC药物用完全培养基按3倍稀释，获得9个浓度梯度(以300nM的最高浓度开始)药物后，100μL/孔加入细胞培养板中，完全培养基作为空白对照，设置3个复孔；继续于37℃、5％CO2培养箱内孵育6天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入50μL CTG检测试剂(Promega，Cat#:G7573)，震荡混匀后放置于暗处静置10分钟后，利用酶标仪检测读取信号值。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-反应曲线并计算IC50值。细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白对照-Lum空白对照)×100％。
实验结果如下表14所示，结果显示，Ab15-X2、Ab16-X1和Ab16-X2对人肠癌或人肺腺癌细胞的增殖都具有良好的抑制活性。
表14抗体偶联药物对人肠癌或人肺腺癌细胞的增殖抑制活性
实施例9：anti-ADAM9抗体药物偶联物(ADAM9-ADC)对人肠癌细胞DLD-1荷瘤小鼠体内肿瘤抑制测试
为评估ADAM9-ADC对体内形成肿瘤的抑制作用，在小鼠体内用ADAM9阳性表达 的人肠癌细胞DLD-1(购自中国科学院细胞库，货号TCHu134)形成移植瘤后，评价各ADAM9-ADC的抗肿瘤效果。
(1).受试药物及材料
·空白对照组(对照组)：生理盐水，尾静脉注射，单次给药
·Ab15-X2(治疗组)：5mg/kg，尾静脉注射给药，共注射1次
·Ab16-X2(治疗组)：5mg/kg，尾静脉注射给药，共注射1次
·hMAB-A(2I.2)-X2(治疗组)：5mg/kg，尾静脉注射给药，共注射1次
(2).配制方法：所有样品均用生理盐水稀释配制。
(3).试验动物：8周龄的雌性NOD SCID小鼠，购自北京维通利华实验动物技术有限公司。
(4).试验方法：
将5×106个DLD-1细胞接种于8周龄的雌性NOD SCID小鼠右前肩胛处皮下，当肿瘤长至约125mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射ADC药物，共注射1次，采用剂量分别为5mg/kg。每周测量2次瘤体积和体重，记录数据。
溶媒对照组或治疗组每组6只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。
实验结果如图1和表15所示，抗体药物偶联物Ab15-X2和Ab16-X2单次给药后表现出显著的抑瘤活性，且显著优于hMAB-A(2I.2)-X2。
表15抗体偶联药物对人肠癌移植瘤模型的体内抗肿瘤药效
实施例10：anti-ADAM9抗体药物偶联物(ADAM9-ADC)对人肠癌细胞LS174T荷瘤小鼠体内肿瘤抑制测试
为评估ADAM9-ADC对体内形成肿瘤的抑制作用，在小鼠体内用ADAM9阳性表达的人肠癌细胞LS174T形成移植瘤后，评价各ADAM9-ADC的抗肿瘤效果。
(1).受试药物及材料
·空白对照组(对照组)：生理盐水，尾静脉注射给药(i.v.)，共注射1次
·Ab16-X2(治疗组)：5mg/kg，尾静脉注射给药(i.v.)，共注射1次
·DS-8201(治疗组)：5mg/kg，尾静脉注射给药(i.v.)，共注射1次
其中，DS-8201(Enhertu，购自Daiichi Sankyo Company Limited)
(2).配制方法：所有样品均用生理盐水稀释配制。
(3).试验动物：8周龄的雌性BALB/c-Nu小鼠，购自北京维通利华实验动物技术有限公司。
(4).试验方法：
将5×106个LS174T细胞接种于8周龄的雌性BALB/c-Nu小鼠右前肩胛处皮下，当肿瘤长至约130mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射ADC药物，共注射1次，采用剂量为5mg/kg。每周测量2次瘤体积和体重，记录数据。
溶媒对照组或治疗组每组6只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。当对照组小鼠肿瘤体积超过3000mm3时，安乐死小鼠，之后该小鼠的肿瘤体积按处死时的肿瘤体积计算肿瘤抑制率(TGI)。
实验结果如图2和表16所示，抗体药物偶联物Ab16-X2单次给药后表现出显著的抑瘤活性。
表16抗体偶联药物对人肠癌移植瘤模型的体内抗肿瘤药效
实施例11：anti-ADAM9抗体药物偶联物(ADAM9-ADC)对人肺癌细胞Calu-3荷瘤小鼠体内肿瘤抑制测试
为评估Ab16-X1和Ab16-X2对体内形成肿瘤的抑制作用，在小鼠体内用ADAM9阳性表达的人肺癌细胞Calu-3形成移植瘤后，评价抗肿瘤效果。
(1).受试药物及材料
·空白对照组(对照组)：生理盐水，尾静脉注射给药，每7天一次，共注射2次
·Ab16-X1(治疗组)：5mg/kg，尾静脉注射给药，每7天一次，共注射2次
·Ab16-X2(治疗组)：5mg/kg，尾静脉注射给药，每7天一次，共注射2次
(2).配制方法：所有样品均用生理盐水稀释配制。
(3).试验动物：8周龄的雌性CB-17SCID小鼠，购自北京维通利华实验动物技术有限公司。
(4).试验方法：
将1×107个Calu-3细胞接种于8周龄的雌性CB-17SCID小鼠右前肩胛处皮下，当肿瘤长至约130mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射ADC药物，每7天一次，共注射2次，采用剂量分别为5mg/kg。 每周测量2次瘤体积和体重，记录数据。
溶媒对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。当肿瘤发生消退(测量时肿瘤体积小于初次给药时肿瘤体积)，抑瘤率＝[1-(治疗组测量当天肿瘤体积÷治疗组第0天肿瘤体积)÷(对照组测量当天肿瘤体积÷对照组肿第0天对瘤体积)]÷[1-(对照组第0天肿瘤体积÷对照组测量当天肿瘤体积)]×100％。
实验结果如图3和表17所示，抗体药物偶联物Ab16-X1和Ab16-X2给药后均表现出显著的抑瘤活性。
表17抗体偶联药物对人肠癌移植瘤模型的体内抗肿瘤药效
实施例12：anti-ADAM9抗体药物偶联物(ADAM9-ADC)在低细胞结合活性时的细胞增殖抑制作用
12.1 LS174T细胞的体外细胞增殖抑制活性测试
使用同时表达ADAM9和Her2的肿瘤细胞，检测Ab16-X2与细胞的结合活性，将DS-8201(Enhertu，购自Daiichi Sankyo Company Limited)作为阳性对照，阴性对照为抗HIV病毒的人IgG抗体(iso-IgG1，药明康德产品)。将中、低表达ADAM9的HepG2(购自中国科学院细胞库，货号TCHu72)和LS174T细胞按每孔1E5个细胞加入到圆底96孔板中，然后加入梯度稀释的Ab16-X2、DS-8201或iso-IgG1各100μL，置于4度孵育1小时。孵育完成后，用FACS buffer(含2％FBS的PBS)洗涤细胞，再加入PE荧光标记的羊抗人的二抗(Abcam，ab98596)，4℃孵育0.5小时。孵育完成后用FACS buffer洗涤细胞，用FACS buffer将细胞重悬后在流式细胞仪上进行检测。
采用化学发光细胞活率检测法(即CTG法)，评估Ab16-X2和DS-8201孵育LS174T(孵育3天)和HepG2(孵育6天)细胞，对细胞杀伤作用。收集对数生长期细胞，以3000个细胞/孔的密度铺板，细胞板放入37℃、5％CO2培养箱培养过夜。实验第二天，将药物用完全培养基按3倍稀释，获得9个浓度梯度(以1000nM的最高浓度开始)药物后，100μL/孔加入细胞培养板中，完全培养基作为空白对照，设置3个复孔；继续于37℃、5％CO2培养箱内孵育3天或6天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入50μL CTG检测试剂，震荡混匀后放置于暗处静置10分钟后， 利用酶标仪检测读取Luminescence信号值。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-反应曲线并计算IC50值。细胞存活率计算公式＝(Lum待测药-Lum空白对 照)/(Lum溶剂空白对照-Lum空白对照)×100％。增殖抑制率＝100％-细胞存活率％。
LS174T细胞上的FACS结合检测结果如表18和图4a所示。Ab16-X2对LS174T细胞有较低的结合能力(平均荧光强度)，而DS-8201对LS174T细胞有较高的结合能力，对于细胞最大结合水平，DS-8201约是Ab16-X2的2-3倍(0.976nM-1000nM浓度范围)，表明DS-8201结合LS174T的亲和力明显高于Ab16-X2。
CTG体外增殖抑制结果如表19和图4b。结果显示，当孵育浓度达到1.372nM，Ab16-X2的增殖抑制率与DS-8201相近；随着孵育浓度上升，在4.115nM-1000nM浓度范围内，Ab16-X2的增殖抑制率远大于DS-8201，以上结果表明，即使在较低细胞结合活性下，Ab16-X2仍有很强的细胞增殖抑制作用，但DS-8201没有这种效果。
表18 Ab16-X2与DS-8201对LS174T细胞的结合活性比较
表19 Ab16-X2与DS-8201对LS174T细胞的体外增殖抑制活性比较
12.2 HepG2细胞的体外细胞增殖抑制活性测试
HepG2细胞结合活性和细胞增殖抑制活性检测参照实施例12.1，具体实验步骤同LS174T细胞。
HepG2细胞上的FACS结合检测结果如表20和图5a所示。当孵育浓度为0.061035nM-62.5nM，Ab16-X1对HepG2细胞有较低的结合能力(平均荧光强度)，而相同浓度下，DS-8201对HepG2细胞有较高或的结合能力。实验表明，在此孵育浓度下，DS-8201结合HepG2的亲和力明显高于Ab16-X1。
CTG体外杀伤实验中，结果如表21和图5b所示。在0.051nM-333.33nM浓度范围内，Ab16-X1的增殖抑制率优于DS-8201。以上结果表明，即使在较低细胞结合活性下，Ab16-X1仍有很强的细胞增殖抑制作用，但DS-8201没有这种效果。
表20 Ab16-X1与DS-8201对HepG2细胞的结合活性比较
表21 Ab16-X1与DS-8201对HepG2细胞的体外增殖抑制活性比较
实施例13：anti-ADAM9抗体药物偶联物(ADAM9-ADC)食蟹猴预毒理
食蟹猴静脉输注给予AB16-X2，每3周给药1次，共给药2次。观察该药物偶联物可能引起的毒性反应的性质、程度及时效关系，初步判断毒性靶器官或靶组织。
方法
2只食蟹猴(来源于苏州西山中科实验动物有限公司)，雌、雄各一只，静脉注射给予AB16-X2，第1次给药剂量为45mg/kg；3周后予以第2次给药，给药剂量提高至80mg/kg，第2次给药后一周进行解剖。期间检测和观察指标包括：
(1)一般状态观察：试验期间，每日对外观体征、精神状态、行为活动、排泄物性状及给药部位等观察2～3次。(2)体重：适应期测定1次，给药期每周测定1次。(3)耗食量：每日测定1次。(4)眼科检查：适应期测定1次，D14、解剖前或给药期结束各检查1次。(5)心电图：适应期测定1次，第1、2次给药结束后6h和24h各测定1次。(6)临床病理：适应期测定1次，给药期D3、D7、D14、D21、D28各采集1次。(7)毒代动力学(8)大体解剖观察和组织病理学检查：对计划解剖动物胸骨(含骨髓)、心脏、肺脏、肾脏、膀胱、十二指肠、结肠、盲肠、眼球、皮肤及肉眼异常脏器/组织(脾脏、直肠)进行常规组织学处理并进行组织病理学检查。
结论
本试验条件下，食蟹猴静脉注射给予45～80mg/kg的AB16-X2，试验期间，所有动物均存活至给药期结束或计划解剖日，无动物濒死或死亡。AB16-X2给药后，仅观察到食蟹猴短暂性白细胞轻度减少和ALT、AST短暂性轻度升高，未见其他病理改变。
MacroGenics开发的IMGC-936是偶联了美登素类微管蛋白抑制剂DM21-C的ADAM9-ADC，目前已进入临床研究阶段，猴毒理研究中22.5mg/kg IMGC-936给药后观察到角膜病变眼毒性(Mol Cancer Ther 2022；21:1047–59)。AB16-X2在猴毒理研究中，80mg/kg的给药剂量下，未观察到眼毒性。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (36)

  1. 一种靶向ADAM9的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包括重链可变区和轻链可变区，所述重链可变区包含重链互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含轻链互补决定区LCDR1、LCDR2和LCDR3；
    所述HCDR1包含如LYWMX1所示的氨基酸序列，所述HCDR2包含如X2IIPIFGHTX3YX4EKFX5X6所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；
    其中，X1为N、D、H或E；X2为R或D；X3为D或K；X4为N或E；X5为K或R；X6为D或N。
  2. 如权利要求1所述的抗体或其抗原结合片段，其特征在于，所述HCDR1中，X1为N或H；所述HCDR2中，X2为R；X3为K；X4为N；X5为K；X6为D或N；较佳地：
    当X1为N时，X2为R；X3为K；X4为N；X5为K；X6为D；
    当X1为H时，X2为R；X3为K；X4为N；X5为K；X6为N。
  3. 如权利要求1或2所述的抗体或其抗原结合片段，其特征在于，所述HCDR1包含如SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:8所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；或者，
    所述HCDR1包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；或者，
    所述HCDR1包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:5所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；或者，
    所述HCDR1包含如SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含如SEQ ID  NO:6所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；或者，
    所述HCDR1包含如SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；或者，
    所述HCDR1包含如SEQ ID NO:2所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列；或者，
    所述HCDR1包含如SEQ ID NO:4所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:10所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:11所示的氨基酸序列，所述LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含如SEQ ID NO:19所示的氨基酸序列。
  4. 如权利要求3所述的抗体或其抗原结合片段，其特征在于，所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:8所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示；或者，
    所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:7所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示；或者，
    所述HCDR1的氨基酸序列如SEQ ID NO:1所示，所述HCDR2的氨基酸序列如SEQ ID NO:5所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示；或者，
    所述HCDR1的氨基酸序列如SEQ ID NO:2所示，所述HCDR2的氨基酸序列如SEQ ID NO:6所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所 述LCDR3的氨基酸序列如SEQ ID NO:19所示；或者，
    所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:7所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示；或者，
    所述HCDR1的氨基酸序列如SEQ ID NO:2所示，所述HCDR2的氨基酸序列如SEQ ID NO:9所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示；或者，
    所述HCDR1的氨基酸序列如SEQ ID NO:4所示，所述HCDR2的氨基酸序列如SEQ ID NO:10所示，所述HCDR3的氨基酸序列如SEQ ID NO:11所示，所述LCDR1的氨基酸序列如SEQ ID NO:12所示，所述LCDR2的氨基酸序列如SEQ ID NO:16所示，且所述LCDR3的氨基酸序列如SEQ ID NO:19所示。
  5. 如权利要求1～4任一项所述的抗体或其抗原结合片段，其特征在于，所述重链可变区和/或轻链可变区还包括框架区，所述框架区为人源框架区；优选地，
    所述重链可变区包含与SEQ ID NO:31具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:23具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:42具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:43具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:22具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:23具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:24具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:25具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:26具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:27具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:28具有至少90％、至少95％或者至少99％序列 同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:30具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:34具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:35具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:36具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:30具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:37具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:38具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:36具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:27具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:39具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:27具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，
    所述重链可变区包含与SEQ ID NO:44具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述轻链可变区包含与SEQ ID NO:45具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；
    所述具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列的可变区维持与原序列至少同等的抗原结合功能。
  6. 如权利要求5所述的抗体或其抗原结合片段，其特征在于，所述重链可变区的氨基酸序列如SEQ ID NO:31所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:23所示；
    所述重链可变区的氨基酸序列如SEQ ID NO:42所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:43所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:22所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:23所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:24所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:25所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:26所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:27所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:28所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:30所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:34所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:35所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:36所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:30所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:37所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:38所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:36所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:27所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:39所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:27所示；或，
    所述重链可变区的氨基酸序列如SEQ ID NO:44所示；和/或，所述轻链可变区的氨基酸序列如SEQ ID NO:45所示。
  7. 如权利要求1～6任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段：
    (1)为全长抗体、Fab、Fab’、F(ab’)2、Fv、sdAb或scFv；和/或，
    (2)为单克隆抗体、双特异性抗体或多特异性抗体；
    和/或，所述ADAM9为人或猴的ADAM9。
  8. 如权利要求7所述的抗体或其抗原结合片段，其特征在于，当所述抗体或其抗原结合片段为全长抗体时，其包括人源抗体的重链的重链恒定区，优选人源抗体IgG1的重链恒定区；和/或，其包括人源抗体的轻链的轻链恒定区，优选人源抗体κ链的轻链恒定区；
    较佳地，所述人源抗体IgG1的重链恒定区的氨基酸序列如SEQ ID NO:48所示，或与SEQ ID NO:48具有至少90％、至少95％或者至少99％序列同一性；和/或，所述人源抗体κ链的轻链恒定区的氨基酸序列如SEQ ID NO:49所示，或与SEQ ID NO:49具有至少90％、至少95％或者至少99％序列同一性；
    更佳地，所述抗体或其抗原结合片段的重链包含与SEQ ID NO:50具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述抗体或其抗原结合片段的 轻链包含与SEQ ID NO:51具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；或，所述抗体或其抗原结合片段的重链包含与SEQ ID NO:52具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列，和/或，所述抗体或其抗原结合片段的轻链包含与SEQ ID NO:53具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列；所述具有至少90％、至少95％或者至少99％序列同一性的氨基酸序列维持与原序列至少同等的抗原结合功能；
    进一步更佳地，所述抗体或其抗原结合片段重链的氨基酸序列如SEQ ID NO:50所示；和/或，轻链的氨基酸序列如SEQ ID NO:51所示；或，所述抗体或其抗原结合片段重链的氨基酸序列如SEQ ID NO:52所示；和/或，轻链的氨基酸序列如SEQ ID NO:53所示。
  9. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～8任一项所述的抗体或其抗原结合片段。
  10. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求9所述的分离的核酸；优选地，所述重组表达载体的骨架质粒为PTT5。
  11. 一种转化体，其特征在于，所述转化体包含如权利要求10所述的重组表达载体；较佳地，所述转化体的宿主细胞为真核细胞；更佳地，所述真核细胞为CHO细胞。
  12. 一种靶向ADAM9的抗体或其抗原结合片段的制备方法，其特征在于，所述方法包含培养如权利要求11所述的转化体，并从培养物中分离获得所述靶向ADAM9的抗体或其抗原结合片段的步骤。
  13. 一种检测ADAM9的方法，其特征在于，其包括使用如权利要求1～8任一项所述的抗体或其抗原结合片段与待测样品接触的步骤；优选地，所述检测为非诊断和/或治疗目的的。
  14. 一种抗体药物偶联物，其中，所述抗体药物偶联物包含权利要求1～8任一项所述的抗体或其抗原结合片段、接头单元L和细胞毒性药物。
  15. 如权利要求14所述的抗体药物偶联物，其特征在于，所述细胞毒性药物为式(A-1)所示结构，其立体异构体、药学上可用的盐、溶剂化物或其盐的溶剂合物，
    其中，
    M为-L2-L1-C(O)-；
    L2为-O-或-S-，且L2与所述的接头单元L连接；
    L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代；
    m选自1、2、3或4；所述的3-6元饱和的杂环基中的杂原子各自独立地为N、O和S，杂原子数为1、2或3个；
    各个R1a、R1b和R2a独立地为氢、卤素、羟基、氨基或C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    各个R独立地为氢或卤素。
  16. 如权利要求15所述的抗体药物偶联物，其中L1为-(C(R1a)(R1b))m-CH2-；各个R1a独立地为氢、卤素或C1-C6烷基；各个R1b独立地为氢、卤素或C1-C6烷基。
  17. 如权利要求16所述的抗体药物偶联物，其中L1为
  18. 如权利要求15所述的抗体药物偶联物，其中L1为C3-C6饱和的环烷基，所述C3-C6饱和的环烷基任选被一个或多个R2a取代，各个R2a独立地为氢、卤素或C1-C6烷基。
  19. 如权利要求18所述的抗体药物偶联物，其中L1为
  20. 如权利要求15所述的抗体药物偶联物，其中所述细胞毒性药物为下任一结构：
  21. 如权利要求14～20任一项所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述接头单元L为-La-Lb-Lc-；且所述Lc与所述的细胞毒性药物连接；
    -La-为优选其中a端和Ab相连，b端与Lb相连；
    -Lb-为以下任一结构：
    优选为更优选其中c端和La相连，d端与Lc相连；-Lc-为
  22. 如权利要求21所述的抗体药物偶联物，其中所述接头单元L为优选为
  23. 如权利要求14～22任一项所述的抗体药物偶联物，其中，所述抗体药物偶联物结构如式(A-2)所示：
    其中，p表示平均连接数或连接数，且p为1到10中任一整数或小数；优选为3到9中任一整数或小数；更优选为7～8的整数或小数，例如7.8或7.9；
    Ab为如权利要求1～8任一项所述的抗体或其抗原结合片段；
    M如权利要求14～20任一项所述；
    L为如权利要求21或22所述的接头单元。
  24. 如权利要求14～23任一项所述的抗体药物偶联物，其中所述抗体药物偶联物选自以下结构式：

    其中，
    p表示平均连接数或连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数；
    Ab为如权利要求1～8任一项所述的抗体或其抗原结合片段。
  25. 如权利要求14～24任一项所述的抗体药物偶联物，其特征在于，所述抗体药物偶 联物为如下任一偶联物：
    p为1到10中任一整数或小数，优选3到9中任一整数或小数；更优选6到8的任一整数或小数；更优选地，p为7.8；
    p为1到10中任一整数或小数，优选3到9中任一整数或小数；更优选6到8的任一整数或小数；例如，p为7.9；
    p为1到10中任 一整数或小数，优选3到9中任一整数或小数；更优选6到8的任一整数或小数；例如，p为7.8；
    Ab16为靶向ADAM9的抗体或其抗原结合片段，所述Ab16的重链的氨基酸序列如SEQ ID NO:50所示，轻链的氨基酸序列如SEQ ID NO:51所示；
    Ab15为靶向ADAM9的抗体或其抗原结合片段，所述Ab15的重链的氨基酸序列如SEQ ID NO:52所示，轻链的氨基酸序列如SEQ ID NO:53所示。
  26. 如权利要求14～25任一项所述的抗体药物偶联物，其特征在于，所述抗体药物偶联物为如下偶联物：
    p表示连接数，且p为1到10中任一整数，优选3到9中任一整数，更优选4到8的任一整数；例如，p为4、5、6、7或8；
    Ab16为靶向ADAM9的抗体或其抗原结合片段，所述Ab16的重链的氨基酸序列如SEQ ID NO:50所示，轻链的氨基酸序列如SEQ ID NO:51所示。
  27. 一种如权利要求14～26任一项所述的抗体药物偶联物的制备方法，其特征在于，所述制备方法包括将如权利要求1～8任一项所述的抗体或其抗原结合片段与如式II所示的化合物反应得到所述的抗体药物偶联物，
    L'-细胞毒性药物
    II；
    L’为与所述的抗体或其抗原结合片段形成如权利要求14～26任一项所述的接头单元L；
    所述细胞毒性药物如权利要求14～26任一项所述；
    较佳地，所述抗体药物偶联物满足如下条件一种或多种：
    (1)所述如式II所示的化合物为
    (2)所述的抗体或其抗原结合片段为Ab16或Ab15；
    所述Ab16的重链的氨基酸序列优选如SEQ ID NO:50所示，轻链的氨基酸序列优选如SEQ ID NO:51所示；
    所述Ab15的重链的氨基酸序列优选如SEQ ID NO:52所示，轻链的氨基酸序列优选如SEQ ID NO:53所示。
  28. 一种药物组合物，其特征在于，其包含如权利要求1～8任一项所述的抗体或其抗原结合片段和/或如权利要求14～26任一项所述的抗体药物偶联物，以及药学上可接受的载体。
  29. 如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物在制备诊断、预防和/或治疗ADAM9高表达的癌症的药物中的应用；
    优选地，所述ADAM9高表达的癌症为肠癌或肺腺癌。
  30. 如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物在制备诊断、预防和/或治疗癌症的药物中的应用；
    优选地，所述癌症为肠癌或肺腺癌。
  31. 一种诊断、预防和/或治疗ADAM9高表达的癌症的方法，其特征在于，所述方 法包括向有需要的患者施用治疗有效量的如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物；
    优选地，所述ADAM9高表达的癌症为肠癌或肺腺癌。
  32. 一种诊断、预防和/或治疗癌症的方法，其特征在于，所述方法包括向有需要的患者施用治疗有效量的如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物；
    优选地，所述癌症为肠癌或肺腺癌。
  33. 一种用于诊断、预防和/或治疗ADAM9高表达的癌症的如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物；
    优选地，所述ADAM9高表达的癌症为肠癌或肺腺癌。
  34. 一种用于诊断、预防和/或治疗癌症的如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物；
    优选地，所述癌症为肠癌或肺腺癌。
  35. 一种联合疗法，其特征在于，其包括分别向有需要的患者施用如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物，以及第二治疗剂；
    较佳地，所述第二治疗剂包含其他抗ADAM9抗体或其抗原结合片段或者包含所述其他抗ADAM9抗体或其抗原结合片段的抗体药物偶联物或药物组合物，和/或其他治疗ADAM9高表达的癌症的药物；
    更佳地，所述ADAM9高表达的癌症为肠癌或肺腺癌。
  36. 一种联合疗法，其特征在于，其包括分别向有需要的患者施用如权利要求1～8任一项所述的抗体或其抗原结合片段、如权利要求14～26任一项所述的抗体药物偶联物和/或如权利要求28所述的药物组合物，以及第二治疗剂；
    较佳地，所述第二治疗剂包含其他抗ADAM9抗体或其抗原结合片段或者包含所述其他抗ADAM9抗体或其抗原结合片段的抗体药物偶联物或药物组合物，和/或其他治疗癌症的药物；
    更佳地，所述癌症为肠癌或肺腺癌。
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