WO2022171134A1 - 包含抗cldn18.2的抗体或其抗原结合片段的抗体药物偶联物及其用途 - Google Patents

包含抗cldn18.2的抗体或其抗原结合片段的抗体药物偶联物及其用途 Download PDF

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WO2022171134A1
WO2022171134A1 PCT/CN2022/075689 CN2022075689W WO2022171134A1 WO 2022171134 A1 WO2022171134 A1 WO 2022171134A1 CN 2022075689 W CN2022075689 W CN 2022075689W WO 2022171134 A1 WO2022171134 A1 WO 2022171134A1
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antibody
seq
cdr
amino acid
drug
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French (fr)
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周伟
徐辉
王珍珍
朱会凯
殷龙
谭小钉
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江苏迈威康新药研发有限公司
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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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention belongs to the field of biotechnology, and in particular, the present invention relates to an antibody-drug conjugate targeting CLDN18.2 and its application.
  • Gastric cancer is the fifth most common cancer and the third leading cause of cancer death globally. According to statistics, in 2018, there were more than 1 million new cases of gastric cancer and 783,000 deaths. At present, the clinical treatment of gastric cancer is mainly surgery, chemotherapy and radiotherapy. Although there are various treatment options, the 5-year survival rate is still very low.
  • trastuzumab which has been successful in Her2-positive breast cancer, is not very effective in gastric cancer; similarly, it targets epidermal growth factor receptor (EGFR) and mammalian rapamycin Inhibitors of mammalian target of rapamycin (mTOR) have also been shown to be ineffective in phase III clinical trials in gastric tumors.
  • EGFR epidermal growth factor receptor
  • mTOR mammalian rapamycin Inhibitors of mammalian target of rapamycin
  • Ccludin is a protein family first discovered in 1998 by Shorichiro Tsukita et al. Members of this family have important physiological roles, such as participating in paracellular permeability, conductance regulation, maintaining molecules between epithelial and endothelial cells Exchanged tight junctions, etc.
  • CLDN18 is one of at least 24 members of the CLDN family and has two isoforms, CLDN18.1 and CLDN18.2, of which CLDN18.2 is a gastric-specific isoform that is usually buried in differentiated epithelial cells of the gastric mucosa. , which is rarely or not expressed in normal tissues and widely expressed in cancer cells, is a highly selective molecule. When malignant tumors occur, the tight junctions of molecular exchange between cells are destroyed, and the epitope of CLDN18.2 on the surface of tumor cells is exposed, making CLDN18.2 a specific site for targeted therapy.
  • CLDN18.2 has been found to be expressed in primary gastric cancer and its metastases.
  • gastric cancer patients 53% of patients have high-abundance expression of CLDN18.2, and 20% of patients have medium-abundant expression; while in gastric cancer brain metastases
  • 45.16% of the patients were found to have high-abundance expression of CLDN18.2 in the metastases.
  • studies have shown that CLDN18.2 is ectopically activated in a variety of human cancers and is highly selectively and stably expressed in specific tumor tissues, such as pancreatic, esophageal, ovarian and lung tumors.
  • CLDN18.2 It is the highly selective expression of CLDN18.2 in multiple cancer tissues and the high positive rate in patients with specific cancer types such as gastric cancer and pancreatic cancer that make CLDN18.2 a potential target therapy and tumor immunity. New therapeutic targets have attracted the attention of researchers at home and abroad.
  • the Japanese pharmaceutical company Astellas has developed the antibody drug Claudiximab (IMAB362) against this target, which has shown good therapeutic effects in clinical phase I/II, and has started the phase III clinical trial of the drug in July 2018 test.
  • the main clinical program is the combination therapy of EOX+Claudiximab for gastric cancer, and the single drug use of Claudiximab has poor results.
  • gastric cancer especially advanced gastric cancer
  • Immunotherapy can only benefit some patients, and the number of available immunotherapy inhibitors is also limited. It is extremely limited.
  • the technical problem to be solved by the present invention is to obtain a high-affinity antibody that specifically binds to human CLDN18.2 through hybridoma screening and humanization technology, wherein the antibody is designed to minimize the number of mouse-derived amino acids through humanization design. , in order to have better in vivo safety and application prospects; on this basis, further screen antibodies with stronger endocytosis effect, and prepare them with small molecule chemical drugs into antibody-drug conjugates (Antibody-Drug Conjugate, ADC), using the targeting of the antibody to CLDN18.2 expressing cells and the strong endocytosis effect through the target, combined with the effect of the small molecule chemical drug, to enhance the therapeutic effect of the antibody drug against CLDN18.2.
  • ADC Antibody-Drug Conjugate
  • an object of the present invention is to provide antibodies or fragments thereof that specifically bind to human CLDN18.2; wherein, the fragments of the antibody cover various functional fragments of the antibody, such as antigen-binding parts thereof, such as Fab, F( ab') 2 or scFv fragment.
  • Another object of the present invention is to provide an antibody-drug conjugate prepared by using the antibody or its fragment.
  • the present invention provides an antibody-drug conjugate comprising an anti-CLDN18.2 antibody or a fragment thereof covalently linked to a drug.
  • the anti-CLDN18.2 antibody or its fragment comprises a heavy chain and a light chain
  • the heavy chain and the light chain respectively comprise the heavy chain complementarity determining regions 1 to 1 as shown below 3 (CDR-H1, CDR-H2 and CDR-H3) and light chain complementarity determining regions 1 to 3 (CDR-L1, CDR-L2 and CDR-L3):
  • amino acid sequences are respectively CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:25; and, the amino acid sequences are respectively shown in SEQ ID NO:32 , CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NO: 30 and SEQ ID NO: 33;
  • the heavy chain of the anti-CLDN18.2 antibody or its fragment comprises a heavy chain variable region (VH), and the heavy chain variable region (VH) comprises The amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8 or a variant thereof; and/or, the light chain of the anti-CLDN18.2 antibody or fragment thereof comprises a light chain variable region (VL), so
  • the light chain variable region (VL) comprises: an amino acid sequence selected from the group consisting of SEQ ID NO: 11 or SEQ ID NO: 12 or a variant thereof.
  • the heavy chain of the anti-CLDN18.2 antibody or its fragment comprises a heavy chain variable region (VH), the heavy chain variable region (VH) ) comprising the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16 or a variant thereof; and/or, the light chain of the anti-CLDN18.2 antibody or fragment thereof comprises a light chain variable region (VL) , the light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 or SEQ ID NO: 22 or a variant thereof.
  • the heavy chain and light chain of the anti-CLDN18.2 antibody or its fragment respectively comprise the variable region (VH) of the heavy chain and the light chain as shown below Variable Region (VL):
  • a "variant of an amino acid sequence” means at least 75% sequence identity to said amino acid sequence (eg at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity, etc. any percent identity > 75%).
  • the anti-CLDN18.2 antibody or its fragment can be a monoclonal antibody, single chain antibody, diabody, single domain antibody, nanobody, complete antibody against CLDN18.2 Or partially humanized antibody or chimeric antibody in any form, or the antibody or its fragment is a half-antibody against CLDN18.2 or an antigen-binding fragment of a half-antibody, such as scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv; with regard to the fragment of the antibody provided by the present invention, preferably, the fragment is any fragment of the antibody that can specifically bind to CLDN18.2. And, as for the anti-CLDN18.2 antibody or fragment thereof, preferably, the CLDN18.2 is human CLDN18.2.
  • the anti-CLDN18.2 antibody or fragment thereof comprises at least the variable region of the heavy chain and the variable region of the light chain of the antibody, and both of them may respectively include
  • the above-mentioned CDR1 to CDR13 and the spaced framework regions FR1 to FR4 are arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the "at least 75% sequence identity" between the variant of the amino acid sequence described above and the amino acid sequence may be determined by any of the framework regions FR1 to FR4 in the variable heavy and light chain regions
  • Such differences can be amino acid deletions, additions or substitutions at any position, where the substitutions can be conservative or non-conservative.
  • the anti-CLDN18.2 antibody or its fragment further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or or a heavy chain constant region (CH); more preferably, the antibody or fragment thereof comprises a heavy chain constant region and/or a kappa or lambda type light chain constant region selected from IgG, IgA, IgM, IgD or IgE.
  • a human or murine constant region preferably a human or murine light chain constant region (CL) and/or or a heavy chain constant region (CH); more preferably, the antibody or fragment thereof comprises a heavy chain constant region and/or a kappa or lambda type light chain constant region selected from IgG, IgA, IgM, IgD or IgE.
  • the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain The constant region is of the kappa type.
  • the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 48 or a variant thereof.
  • the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 49 or a variant thereof.
  • the antibody drug conjugate provided by the present invention has the molecular formula Ab-[L-D]n, wherein Ab represents the anti-CLDN18.2 antibody or fragment thereof provided by the present invention, L represents the linker, D represents the drug, and n represents relative to Average number of drug attachments (DAR) per molecule of Ab.
  • the drug D is a cytotoxic small-molecule drug and/or an immunotherapeutic-based small-molecule drug.
  • the cytotoxic small molecule drug can be a tubulin inhibitor and/or a topoisomerase I inhibitor, and the tubulin inhibitor can be an auristatin compound, such as MMAE, MMAF, etc.; topoisomerase Enzyme I inhibitors can be camptothecins such as SN38, Exatecan and Dxd. Examples of the structures of these compounds are shown below:
  • the immunotherapeutic small molecule drug can be a STING agonist and/or a TLR7/8 agonist.
  • the STING agonist can be a cyclic dinucleotide compound, such as compounds 10 and 11 shown below;
  • the TLR7/8 agonist can be an imidazoquinoline compound and/or a benzoazepine compound, such as Compounds 1, 2, 3, 4, 5, 6, 7, 8 and 9, etc. are shown below.
  • the linker L can be a cysteine-coupling linker or a lysine-coupling linker, and its release mechanism is cleavable or non-cleavable.
  • Cleavable linkers such as can be selected from: MC-Val-Cit-PAB, MC-Val-Ala and MC-Gly-Gly-Phe-Gly etc.; non-cleavable linkers such as can be selected from MCC, PEG 4 Mal and mc etc. .
  • the cytotoxic drug-like linker ie the "[L-D]” moiety, “drug linker”, “[L-D]” and “drug-containing linker” in the context of the present invention " are used interchangeably
  • the cytotoxic drug-like linker ie the "[L-D]” moiety, “drug linker”, “[L-D]” and “drug-containing linker” in the context of the present invention " are used interchangeably
  • Camptothecin-like topoisomerase I inhibitor-ADC linker MC-GGFG-Dxd (abbreviated as EX) and CL2A-SN38 (abbreviated as EX2):
  • the immunotherapeutic drug linker (ie the "[L-D]" moiety) can be:
  • the cyclic dinucleotide STING agonist-ADC linker can be:
  • [L-D]” in the antibody drug conjugate provided by the present invention is the specific compound used in the Examples, wherein MC-GGFG-Dxd is abbreviated as “EX”, and CL2A-SN38 is abbreviated as “EX”.
  • EX2 MC-GGFG-Dxd
  • CL2A-SN38 is abbreviated as "EX”.
  • EX2 MC-VC-MMAE is abbreviated as "M”
  • L-A is abbreviated as "TLR8”
  • L-B is abbreviated as "STING”.
  • the antibody drug conjugate provided by the present invention can be named as "abbreviation for antibody-[L-D] abbreviation”.
  • n is 1 to 12, preferably 1 to 8, more preferably 3 to 8, such as 4 to 8, 5 to 8, 6 to 8, or even 7 to 8.
  • the present invention also provides a method for preparing the antibody-drug conjugate, the method comprising the following steps:
  • step (2) Cross-linking the reduced antibody obtained in step (1) with a drug linker (linker-drug conjugate) in a mixed solution of a buffer and an organic solvent to obtain an antibody-drug conjugate.
  • a drug linker linker-drug conjugate
  • the method includes the following steps:
  • Reduction dilute the antibody stock solution with reaction buffer, add a reducing agent such as TCEP in a molar ratio of 1.5-2.5 times the antibody, and stir the reaction solution at room temperature for 1-4 hours;
  • a reducing agent such as TCEP
  • the present invention provides a preparation method of an antibody drug conjugate with a DAR of 4, including:
  • Reduction Dilute the antibody stock solution with reaction buffer to about 10 mg/mL, add tris(2-carboxyethyl)phosphonium hydrochloride (TCEP) in a molar ratio of 1.5-2.5 times the antibody, and stir the reaction solution at 25°C for 2 Hour;
  • TCEP tris(2-carboxyethyl)phosphonium hydrochloride
  • the present invention provides a preparation method of an antibody drug conjugate with a DAR of 8, including:
  • Reduction Dilute the antibody stock solution with reaction buffer to about 10 mg/mL, add tris(2-carboxyethyl) phosphine hydrochloride (TCEP) in a molar ratio of 9.0-11 times the antibody, and stir the reaction solution at 25°C for 2 Hour;
  • TCEP tris(2-carboxyethyl) phosphine hydrochloride
  • the present invention provides an anti-CLDN18.2 antibody or a fragment thereof.
  • the above descriptions and definitions for the anti-CLDN18.2 antibody or fragment thereof contained in the antibody drug conjugate are applicable to the anti-CLDN18.2 antibody or fragment thereof in this regard.
  • the anti-CLDN18.2 antibody or fragment thereof provided by the present invention comprises a heavy chain and a light chain, and the heavy chain and the light chain respectively comprise the heavy chain complementarity determining regions 1 to 3 (CDRs 1 to 3) shown below -H1, CDR-H2 and CDR-H3) and light chain complementarity determining regions 1 to 3 (CDR-L1, CDR-L2 and CDR-L3):
  • amino acid sequences are respectively CDR-H1, CDR-H2 and CDR-H3 shown in SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:25; and, the amino acid sequences are respectively shown in SEQ ID NO:32 , CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NO: 30 and SEQ ID NO: 33;
  • the heavy chain of the anti-CLDN18.2 antibody or fragment thereof comprises a heavy chain variable region (VH), the light chain comprises a light chain variable region (VL), the heavy chain variable region (VH) and The light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof and the amino acid sequence shown in SEQ ID NO: 12 or a variant thereof; or, the heavy chain variable region ( The VH) and light chain variable region (VL) comprise the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof and the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, respectively.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the present invention or the anti-CLDN18.2 antibody or fragment thereof of the present invention, and optionally pharmaceutically acceptable Accepted excipients.
  • the adjuvant can be a carrier or an excipient and the like.
  • the pharmaceutical composition provided by the present invention may further comprise other drugs that can be used in combination with the antibody-drug conjugate of the present invention or the anti-CLDN18.2 antibody or its fragment, such as chemotherapeutic drugs EGFR inhibitors (Gefitinib, Erlotinib, Vandetanib, Sunitinib, etc.), PARP inhibitors (Olaparib, etc.), DNA alkylating agents (Bendamustine, etc.), HDAC inhibitors (Vorinostat, etc.), BTK inhibitors (Ibrutinib, etc.), C-met inhibitors (Tepotinib, etc.), Paclitaxel etc., or the pharmaceutical composition provided by the present invention can also be used in combination with other treatment methods such as chemotherapy regimen EOX.
  • chemotherapeutic drugs EGFR inhibitors Garfitinib, Erlotinib, Vandetanib, Sunitinib, etc.
  • PARP inhibitors Oparib, etc.
  • DNA alkylating agents
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises an antibody-drug conjugate mixture and a pharmaceutically acceptable carrier, the antibody-drug conjugate mixture comprising a plurality of the aforementioned antibody-drug conjugates.
  • the antibody drug conjugate mixture has an average n value of 2 to 8.
  • said antibody drug conjugate mixture comprises a plurality of said antibody drug conjugates, each of said antibody drug conjugates having an n value of 1 to 12, preferably 1 to 8, more preferably 3 to 8 , such as 4 to 8, 5 to 8, 6 to 8, or even 7 to 8.
  • the present invention also provides the use of the antibody drug conjugate, anti-CLDN18.2 antibody or fragment thereof or any pharmaceutical composition comprising them in the preparation of a medicament for the treatment of CLDN18.2 and CLDN18.2
  • the associated disease is preferably a tumor, more preferably a cancer.
  • the cancer is selected from pancreatic cancer, gastric cancer, colon cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer, gallbladder cancer and head and neck cancer.
  • the present invention also provides a method of treating a disease, the method comprising administering to a subject in need thereof the antibody drug conjugate, anti-CLDN18.2 antibody or fragment thereof provided by the present invention or comprising them any pharmaceutical composition.
  • the subject is a mammal, preferably a human.
  • the disease is a disease associated with CLDN18.2, preferably a tumor, more preferably a cancer.
  • the cancer is selected from pancreatic cancer, gastric cancer, colon cancer, esophageal cancer, liver cancer, ovarian cancer, lung cancer, gallbladder cancer and head and neck cancer.
  • high-CHOK1 cells stably expressing human CLDN18.2 on the cell surface are used to immunize mice to obtain hybridoma cells secreting anti-human CLDN18.2 antibodies, and then a chimeric antibody is constructed from mouse antibodies secreted by hybridoma cells, After proving that the chimeric antibody retains the specific binding of the mouse antibody to the antigen, the humanization of the heavy chain and light chain variable regions was carried out, and a number of different humanized antibodies against human CLDN18.2 were obtained.
  • the anti-CLDN18.2 antibody provided by the present invention has a strong affinity for the antigen CLDN18.2, and has significant complement-dependent cytotoxicity (CDC) activity and antibody-dependent cytotoxicity (ADCC) activity on target expressing cells, and The activity is stronger than or comparable to IMAB362 with the same target.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cytotoxicity
  • the anti-CLDN18.2 antibody provided by the present invention minimizes murine amino acids, thereby reducing the possibility of causing a rejection immune response in humans.
  • the anti-CLDN18.2 antibody of the present invention has better endocytosis activity, and the antibody-drug conjugate prepared with small molecule drugs is more stable.
  • the ADC prepared by using the anti-CLDN18.2 antibody of the present invention enters into the cell after phagocytosis of immune cells mediated by the targeting effect and/or Fc effect of the antibody, and can effectively achieve the release of the coupled drug.
  • the anti-CLDN18.2 antibody provided by the present invention is more suitable for preparing an antibody-drug conjugate with drugs, etc., taking into account both safety and effectiveness.
  • Fig. 1 Epitope research results of the antibodies of the present invention and Isotype antibodies
  • Figure 2 Binding activity of the antibody of the present invention to cell surface CLDN18.2;
  • FIG. 3 Internalization activity of the antibody of the present invention binding to cell surface CLDN18.2;
  • Figure 8 HIC analysis results of antibody drug conjugate 8k13-hz35-M-20200318-1;
  • Figure 10 RPLC analysis results of antibody drug conjugate 8k13-hz35-EX-DAR4-20200922;
  • FIG. 11 SEC analysis results of antibody drug conjugate 8k13-hz35-EX-DAR4-20200922;
  • Figure 12 HIC analysis results of antibody drug conjugate 8k13-hz35-EX-DAR8-20200421;
  • Figure 13 SEC analysis results of antibody drug conjugate 8k13-hz35-EX-DAR8-20200421;
  • Figure 14 RPLC analysis results of antibody drug conjugate 8k13-hz35-EX2-DAR4-20200922;
  • Figure 15 SEC analysis results of antibody drug conjugate 8k13-hz35-EX2-DAR4-20200922;
  • Figure 16 RPLC analysis results of antibody drug conjugate 18k13-hz35-EX2-DAR8-20200922;
  • Figure 17 SEC analysis results of antibody drug conjugate 18k13-hz35-EX2-DAR8-20200922;
  • Figure 18 HIC analysis results of antibody drug conjugate 16k15-hz35-M-20200318-1;
  • Figure 20 HIC analysis results of antibody drug conjugate 16k15-hz35-EX-20200421;
  • Figure 21 SEC analysis results of antibody drug conjugate 16k15-hz35-EX-20200421;
  • Figure 26 The killing effect of the antibody-drug conjugates of the present invention on cells without CLDN18.2 expression;
  • Figure 27 The killing effect of the antibody-drug conjugates of the present invention on cells with high CLDN18.2 expression
  • Figure 28 The stimulating effect of the antibody-drug conjugates of the present invention on the secretion of pro-inflammatory factor TNF ⁇ by PBMC;
  • Figure 29 Stimulatory effect of the antibody-drug conjugates of the present invention on pIRF3 expression
  • Figure 30 The therapeutic effect of the antibody-drug conjugate of the present invention on mouse subcutaneously transplanted tumor
  • Figure 31 The effect of the antibody-drug conjugates of the present invention on the body weight of tumor-bearing mice;
  • Figure 32 The effect of the antibody-drug conjugates of the present invention on the feeding of tumor-bearing mice;
  • Figure 33 The therapeutic effect of the antibody-drug conjugate of the present invention on mouse subcutaneously transplanted tumor
  • Figure 34 The therapeutic effect (individual tumor weight) of the antibody-drug conjugates of the present invention on human gastric cancer KATO III/18.2 mice subcutaneously transplanted; **P ⁇ 0.001, ***P ⁇ 0.001, vs solvent.
  • Antigen human CLDN18.2 see NP_001002026.1
  • antigen human CLDN18.1 see NP_057453.1.
  • the heavy and light chain sequences of Sacituzumab are shown in SEQ ID NO:46 and SEQ ID NO:47.
  • the heavy chain constant region sequences of the human monoclonal antibody IgG1 subclass and the light chain constant region sequences of the kappa subclass are shown in SEQ ID NO:48 and SEQ ID NO:49, respectively.
  • mice were immunized with CHOK1 cells stably expressing human CLDN18.2 protein on the cell surface. After 1 month, the mouse serum was analyzed by flow cytometry (FACS), and the spleen of the mice with high serum antibody titers was taken. , spleen cells isolated by standard methods and myeloma cells P3X63Ag8.653 were fused using PEG or electrofusion.
  • the fused hybridoma cells were inoculated in a 384-well plate, and after culturing for 10-14 days, the supernatant was taken to analyze the antibodies secreted by the hybridoma cells by FACS, and several clones were obtained by screening, and the clones were able to bind to the cell surface and stably express human CLDN18.
  • 2 protein CHOK1 cells but not able to bind to the cell surface of CHOK1 cells stably expressing human CLDN18.1 protein.
  • the screened clones were single-celled by limiting dilution, and each hybridoma clone obtained after 3 rounds secreted only one antibody.
  • VH mouse anti Heavy chain variable region
  • VL Light chain variable region 8k13 SEQ ID NO: 1 SEQ ID NO: 2 16k15 SEQ ID NO: 3 SEQ ID NO: 4
  • the heavy chain variable region sequence of the murine anti-human CLDN18.2 monoclonal antibody and the heavy chain constant region sequence of the published human monoclonal antibody IgG1 subclass were spliced together, and constructed into mammalian In an animal cell expression vector; the light chain variable region sequence of the murine anti-human CLDN18.2 monoclonal antibody and the light chain constant region sequence of the published human monoclonal antibody ⁇ subclass (see SEQ ID NO:49) are spliced together together, constructed into mammalian cell expression vectors.
  • the heavy chain vector and light chain vector of the constructed anti-human CLDN18.2 chimeric antibody were paired and mixed, and polyethyleneimine (PEI) was used to transfect HEK293 cells. After about 7 days, the cell supernatant was collected and purified using ProteinA to obtain anti-human CLDN18 .2 Chimeric Antibody Proteins.
  • PEI polyethyleneimine
  • the chimeric antibody was named "mouse anti-abbreviation-xiIgG".
  • the anti-human CLDN18.2 chimeric antibody was diluted with a gradient of 2 times starting from the initial concentration of 100 nM, with a total of 16 concentration points. 10ul of the antibody at each concentration point was added to the 384-well plate. Collect the CHOK1 cells expressing CLDN18.2 on the cell surface by centrifugation at 100g for 5 minutes at room temperature, wash the cells once with PBS containing 0.5% BSA, centrifuge at 100g for 5 minutes at room temperature, resuspend the cells to a density of about 2x106 cells/ml, and add 10ul to Antibodies have been added to the wells of a 384-well plate. After 1 hour incubation at 4°C, fluorescently labeled goat anti-human IgG secondary antibody was added. Following an additional 1 hour incubation at 4°C, the cell population was analyzed for mean fluorescence readings by flow cytometry.
  • the amino acid sequence regions of the six antigenic complementarity determinants (CDRs) in the heavy and light chains of the mouse antibody and the framework regions supporting the conservative three-dimensional conformation of the antibody were determined. Then, the known human antibody sequences were searched by analysis, and the human antibody heavy chain variable region sequence that was most similar to the mouse anti-antibody was selected, and its antibody framework region sequence was selected as the template, and the mouse anti-heavy chain CDRs were combined with the human antibody framework region. , and finally generate a humanized antibody heavy chain variable region sequence. In the same process, a humanized antibody light chain variable region sequence was generated.
  • Antibodies in which mouse anti-CDRs are directly grafted into human framework regions often show a sharp drop in binding activity, so individual amino acids in the framework regions need to be changed from human to murine.
  • To determine the reverse mutation site one is to compare the designed humanized antibody sequence with the original mouse antibody sequence to check which amino acids are different; the other is to check whether these amino acids play an important role in supporting the structure of the antibody or binding to the antigen. important role.
  • PTM post-translational modification sites
  • the humanized antibody of the present invention is named "mouse anti-abbreviation-hz".
  • the mouse anti-CDR antibody directly grafted to the human framework region was named “mouse anti-abbreviation-hz00”.
  • Antibodies obtained by further modification are numbered according to different sequences. For example, the numbering of antibody 8k13-hz11 is derived from the numbers 8K13_vh_hz1 and 8K13_vl_hz1 of its heavy chain and light chain variable regions.
  • Antibody-antigen interaction force was measured by GE BIAcore instrument S200.
  • GE Human antibody capture kit (Cat. No. BR-1008-39, Lot10261753), first saturate and couple the maximum amount of anti-human Fc antibody in the CM5 analysis channel and control sample channel of the sensor chip, and then flow through the analysis channel containing the anti-human Fc antibody. 7.5 ⁇ g/ml anti-human CLDN18.2 chimeric antibody, humanized antibody or IMAB362 buffer to make it evenly distributed, and finally flow through the serially diluted antigen sample (initial concentration 20nM, 1 : 3 diluted 8 concentration points, and set the 0.741nm concentration point to repeat), and measure the light reaction value after antibody-antigen binding. After fitting and analysis by the instrument software, the binding constant Kon, the dissociation constant Koff and the affinity constant KD of the antibody were finally obtained.
  • the humanized antibody of the present invention was analyzed using the commercial human serum whole complement of Quidel company, and its ability to induce complement-dependent cytotoxicity (CDC) to CHOK1, BxPC3 and NCI-N87 cells stably expressing human CLDN18.2 was analyzed.
  • CDC complement-dependent cytotoxicity
  • Cells were mixed with the antibody to be tested at a final concentration of 250 ⁇ g/ml to 3.8 ng/ml; 6.25% human serum complete complement dissolved in cell culture medium RPMI-1640 was added and incubated at 37°C for 3 hours; then passed through CCK -8 kit was used for cytotoxicity detection; finally, the absorbance at 450nm was detected by MD microplate reader.
  • the 4-parameter fitting curve was performed by using softmax pro7 software through the absorbance value, and the EC50 value of the sample was calculated.
  • ADCC Antibody-Dependent Cytotoxicity
  • effector cells engineered Jurkat cells stably expressing the Fc ⁇ RIIIa-Fc ⁇ RIa ⁇ hybrid receptor and firefly luciferase driven by the NFAT response element were used.
  • the biological activity of antibodies in the ADCC mechanism of action was quantified by luciferase produced by NFAT pathway activation.
  • Epitopes of the antibodies of the present invention and control antibodies were investigated using an epitope competition method.
  • Rituximab irrelevant antibody, i.e. control antibody
  • 8k13-hz35 SEQ ID NO:8+SEQ ID NO:12
  • 16k15-hz35 SEQ ID NO:16+SEQ ID NO:22
  • Fab forms antibodies Isotype- Fab, 8k13-hz35-Fab and 16k15-hz35-Fab.
  • the competitor antibody IMAB362 was first diluted to 0.05ug/ml.
  • the 8k13-hz35-Fab, 16k15-hz35-Fab and Isotype-Fab to be analyzed were diluted to a concentration of 200 nM with a solution containing a competing antibody, and a 2-fold gradient dilution was performed, with a total of 8 concentration points. 10ul of the antibody at each concentration point was added to the 384-well plate.
  • CLDN18.2 CHOK1 cells were collected by centrifugation at 100 g at room temperature for 5 minutes, washed once with PBS containing 0.5% BSA, and centrifuged at 100 g at room temperature for 5 minutes.
  • Example 8 FACS detection of the binding activity of anti-human CLDN18.2 humanized antibody to human CLDN18.2 recombinant protein on the surface of CHO cells
  • the suspensions of CHO cells (CHO/CLDN18.2) recombinantly expressing human CLDN18.2 were mixed with humanized antibodies (8k13-hz35, 16k15-hz35, 8k13-hz24, 16k15-hz22) (the initial concentration was 5 ⁇ g/mL) 3-fold serial dilution in 5 gradients) was incubated at 4°C for 60 min, and a negative control (NC) IgG1 isotype control antibody NC-huIgG1 was set. After washing the cells 3 times with PBS, 1:200 diluted goat anti-human IgG-FITC (Cat: F9512, Sigma) was added and incubated at 4°C for 45 min.
  • MFI mean fluorescence intensity
  • Antibodies 8k13-hz35 and 16k15-hz35 can effectively bind to the recombinant human CLDN18.2 protein on the surface of CHO cells, and the binding ability is comparable to that of antibodies 8k13-hz24 and 16k15-hz22.
  • NCI-N87 CLDN18.2 cells recombinantly expressing human CLDN18.2 (Cat.: KC-1222, Kangyuan Bochuang) were seeded in a 96-well cell culture plate at a density of 2 ⁇ 10 3 cells/well, and cultured for 24 Hour. Discard the supernatant, block with RPMI 1640 (containing 10% FBS), block at 37°C for 60 min, centrifuge, and wash twice with RPMI 1640 (containing 10% FBS); Mix-n-Stain TM CF TM 488A (Cat: MX488AS100, Sigma) will be used.
  • the drug linkers employed in the following examples include:
  • CL2A-SN38 a camptothecin-like topoisomerase I inhibitor-ADC linker, abbreviated "EX2" in the context of the present invention.
  • MC-VC-MMAE, MMAE microtubule inhibitor-ADC linker abbreviated "M” in the context of the present invention.
  • TLR8 benzoazepine TLR8 agonist-ADC linker
  • STING cyclic dinucleotide STING agonist-ADC linker
  • An antibody solution (10 mg/ml antibody concentration) was prepared in phosphate buffer (50 mM, pH 6.5; abbreviated as PBS6.5/EDTA) containing 50 mM sodium chloride and 2 mM EDTA, to which was added TCEP (Sigma-Aldrich) Aqueous solution to make TCEP 2-10 times the molar equivalent of the antibody, and then incubated at 25°C for 2h.
  • phosphate buffer 50 mM, pH 6.5; abbreviated as PBS6.5/EDTA
  • TCEP Sigma-Aldrich
  • the antibody concentration after reduction was adjusted to 5 mg/ml using PBS6.5/EDTA.
  • Add N,N-dimethylacetamide (DMA) then add the drug linker in dimethyl sulfoxide (drug linker concentration is 10 mg/ml) to make the drug linker 6-12 molar equivalents of the antibody, then add the drug
  • the dimethyl sulfoxide solution of the linker (the concentration of the drug linker is 10 mg/ml), the drug linker is 5-12 times the molar equivalent of the antibody, and the volume ratio of DMA in the whole reaction system is controlled to be 10-20%.
  • the solution was continued to stir at room temperature for 30-60 minutes to couple the drug linker to the antibody.
  • the antibody drug conjugate concentration in the conjugated sample was determined using the following antibody drug conjugate concentration assay method.
  • the concentration of the linked drug in the sample can be obtained by measuring the UV absorbance at a wavelength of 280 nm, followed by the following calculation.
  • the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (addition of absorbance), it is assumed that the molar absorbance of the antibody and the drug linker before and after the antibody is conjugated with the drug linker
  • the antibody concentration and the drug concentration in the antibody drug conjugate are represented by the following relational expressions.
  • the DAR value is the measured value.
  • the antibody concentration and the drug concentration in the antibody drug conjugate are represented by the following relational expressions.
  • the DAR value is the measured value.
  • the antibody concentration and drug concentration in the above antibody drug conjugate are shown in the following relationship.
  • the DAR value is the measured value.
  • the antibody concentration and the drug concentration in the antibody drug conjugate are represented by the following relational expressions.
  • the DAR value is the measured value.
  • the antibody concentration and the drug concentration in the antibody drug conjugate are represented by the following relational expressions.
  • the DAR value is the measured value.
  • Sample preparation for HPLC analysis Add antibody drug conjugate (about 1 mg/mL, 60 ⁇ L) to dithiothreitol aqueous solution (100 mM, 15 ⁇ L), mix well, and incubate at 37°C for 30 minutes for HPLC analysis.
  • Analytical column Agilent PLRP-S, 2.1*50mm, 8 ⁇ M, PN PL1912-1802
  • Sample preparation for HPLC analysis using mobile phase as a diluent, dilute the antibody drug conjugate to 2 mg/ml, filter with a 0.22 ⁇ M filter, and use for HPLC analysis.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 2.0eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 2.1 eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of antibody-drug conjugate in the solution was measured to be 7.7 mg/mL.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 2.2eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was measured to be 7.7 mg/mL.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 2.1 eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was measured to be 7.7 mg/mL.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 10eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was measured to be 7.7 mg/mL.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 2.2eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of antibody-drug conjugate in the solution was measured to be 4.5 mg/mL.
  • the antibody 8k13-hz35 was reduced (the amount of TCEP was 10.1 eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of antibody-drug conjugate in the solution was measured to be 4.4 mg/mL.
  • the antibody 16k15-hz35 was reduced (the amount of TCEP was 2.3eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was measured to be 7.7 mg/mL.
  • the antibody 16k15-hz35 was reduced (the amount of TCEP was 9.9eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was measured to be 7.7 mg/mL.
  • the antibody IMAB362 was reduced (the amount of TCEP was 2.2eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was determined to be 6.9 mg/mL.
  • the antibody IMAB362 was reduced (the amount of TCEP is 8eq of the antibody) using the operation described in "1. Reduction of the antibody" in the experimental method section above to obtain a reduced antibody solution.
  • the concentration of the antibody-drug conjugate in the solution was determined to be 6.1 mg/mL.
  • Example Group 2 of the present invention According to the analysis results of size exclusion chromatography in Examples 10 and 11 in the preparation of antibody drug conjugates in Example Group 2 of the present invention, compared with 8k13-hz35 and 16k15-hz35, two ADC samples IMAB362 obtained by coupling the control antibody IMAB362 -M-1 and IMAB362-M-2, with high aggregate content (the detected monomer content was 90.13% and 92.16%, respectively); that is, after the control antibody IMAB362 was coupled to obtain ADC, the physicochemical properties were unstable and aggregates were easily generated , which may raise various safety and efficacy concerns.
  • control antibody IMAB362 conjugated two ADC samples regardless of adding a low equivalent reducing agent (IMAB362-1, 2.2 times the molar equivalent of the antibody) or a high equivalent reducing agent (IMAB362-M-2, antibody 8 times the molar equivalent), the DAR of the prepared ADC samples is all around 3.3, and the DAR2 and DAR4 components are mainly prepared, and the DAR6 and DAR8 components are very few, and the drug load is not normally distributed, which may bring various drug effects. aspects of the problem. Based on the above two points, the control antibody IMAB362 is not suitable for the preparation of ADC.
  • the antibody drug conjugates prepared in Examples 4 and 5 in Example Group 2 were used as test drugs.
  • the above cells in logarithmic growth phase were seeded into 96-well cell culture plates at a density of 5,000 cells per well, and then different concentrations of antibody-drug conjugates were added, and 2-4 replicates were set for each drug concentration.
  • Wells, and blank control (only cells), acted for 120h (according to the cell growth rate, ensuring sufficient number of cell divisions), and determined EC 50 .
  • CLDN18.2-highly expressing cell line NCI-N87 (NCI-N87 CLDN18.2) and CLDN18.2-non-expressing cell line NCI-N87 (NCI-N87) as in vitro cell activity experimental models to study the different provided by the present invention Killing effect of antibody-drug conjugates on tumor cells with high and low expression levels of CLDN18.2.
  • test drugs are shown in Table 7, and each ADC is prepared with reference to the respective examples, wherein SAC is Sacituzumab, and the sequence is shown above; IL6 is TocilizuMab, purchased from Target Molecule Corp. The test process is the same as that of Example 1.
  • the cell killing effect on NCI-N87 cell line without CLDN18.2 expression is shown in Table 7 and FIG. 26 .
  • ADC-2 of the present invention 8k13-hz35-M - - 4 Naked Anti-2 of the present invention 8k13-hz35 - - N/A Positive control ADC SAC-M + 134.8 4.5 Negative control ADC IL-6-M - - N/A small molecule drug MMAE + 1200 N/A
  • the cell killing effect on the NCI-N87 cell line with high CLDN18.2 expression is shown in Table 8 and FIG. 27 .
  • Sample type sample name killing effect EC50(pM) DAR ADC-1 of the present invention 16k15-hz35-M + 63.55 4.1 Naked Anti-1 of the present invention 16k15-hz35 - - N/A
  • ADC-2 of the present invention 8k13-hz35 + 61.58 4 Naked Anti-2 of the present invention 8k13-hz35 - - N/A
  • Positive control ADC SAC-M + 64.25 4.5
  • Negative control ADC IL-6-M - - N/A small molecule drug MMAE + 209.7 N/A
  • the ADC obtained by using the antibody-conjugated TLR8 agonist of the present invention cannot directly kill tumor cells, but after phagocytosis of tumor cells by immune cells (mainly myeloid antigen-presenting cells) in the tumor microenvironment mediated by antibodies, immune cells TLR8 agonists are released from lysosomes. TLR8 agonists can activate immune cells after binding to TLR8 in immune cells, increase the secretion of pro-inflammatory cytokines, recruit immune cells, enhance tumor neoantigen presentation, and stimulate tumor-specific T cell generation. Ultimately achieve the purpose of killing tumor cells.
  • immune cells mainly myeloid antigen-presenting cells
  • NCI-N87 cell line expressing CLDN18.2 as the target cell, co-culture with human peripheral blood mononuclear cells (PBMC), adding a certain amount of the antibody-drug conjugate provided by the present invention, and measuring the pro-inflammatory factors in the cell supernatant expression.
  • PBMC peripheral blood mononuclear cells
  • the results are shown in Figure 28.
  • the ADC provided by the present invention can significantly stimulate the PBMC to secrete the pro-inflammatory factor TNF ⁇ , while the naked antibody and the negative control have no significant stimulating effect.
  • TLR8 agonist can mediate immune cell phagocytosis through the targeting effect and Fc effect of the antibody, deliver the TLR8 agonist to the target site, and activate immune cells.
  • the ADC obtained by using the antibody-conjugated STING agonist of the present invention is incubated with tumor cells (NCI-N87 CLDN18.2) expressing CLDN18.2 on the surface, the ADC can enter the cell through the endocytosis of the target, and protease in the cell Under the action of breaking the linker, the STING agonist is released. After the STING agonist is released, it can bind and activate the STING receptor, thereby inducing the phosphorylation of TBK1 (TANK binding kinase 1), activating the IRF3 (Interferon regulatory factor 3) signaling pathway and the NF- ⁇ B signaling pathway. Increase the expression of phosphorylated TRF3 (pIRF3).
  • TBK1 TANK binding kinase 1
  • IRF3 Interferon regulatory factor 3
  • the activation of the cells can be determined.
  • NCI-N87 CLDN18.2 was cultured in vitro to logarithmic growth phase, and seeded into 24-well cell plates at the number of 200,000/well. The medium was starved without serum, and cultured overnight at 37°C, 5% CO 2 . . The next day, a certain concentration of ADC (8k13-hz35-STING-D4-1203 prepared in Example 2 of Example Group 2), naked antibody (8k13-hz35), negative control (NC IgG-STING, NC IgG was an unrelated human antibody) or a STING agonist (compound STING), co-cultured for about 6 h.
  • ADC 8k13-hz35-STING-D4-1203 prepared in Example 2 of Example Group 2
  • naked antibody 8k13-hz35
  • NC IgG-STING NC IgG was an unrelated human antibody
  • compound STING compound STING
  • the ratio is the relative expression level of pIRF3.
  • the results are shown in Figure 29.
  • the ADC of the present invention can significantly stimulate the expression of pIRF3 in target cells, while the naked antibody and negative control have no obvious stimulation effect.
  • Small molecule drugs can also stimulate pIRF3, but the intensity is significantly lower than that of the ADC of the present invention. It is indicated that after the STING agonist is coupled to the antibody of the present invention, the STING agonist can be delivered to the target site through the targeting effect of the antibody to activate the STING signaling pathway.
  • Example group 4 ADC in vivo efficacy test
  • KATO III cells Human gastric cancer KATO III cells were purchased from the Cell Bank of the Chinese Academy of Sciences. KATO III was transfected with human CLDN18.2 gene and highly expressed CLDN18.2 protein, named KATO III/18.2. KATO III/18.2 cells were cultured in a 10 cm petri dish, and the culture conditions were RPMI 1640 medium with 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO 2 air. Passage 2 times a week, when cells are in exponential growth phase, harvest cells, count, and seed.
  • NOD-Scid mice 35 days, ⁇ , were purchased from Shanghai Lingchang Biotechnology Co., Ltd. License number: SCXK (Shanghai) 2018-0003. Certificate number 20180003012511. Breeding environment: SPF grade.
  • mice were subcutaneously inoculated with 1 ⁇ 10 7 KATO III/18.2 cells, and after tumors grew to 100-150 mm 3 , animals were grouped according to tumor volume (D0).
  • the specific dosage and dosage regimen are shown in Table 9. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific index is T/C (%) or tumor growth inhibition rate % (TGI%).
  • Partial tumor regression was defined if the tumor was smaller than the initial volume, i.e., T ⁇ T0 or C ⁇ C0; complete tumor regression (CR) was defined if the tumor disappeared completely.
  • PR Partial tumor regression
  • C complete tumor regression
  • P value refers to the comparison with the normal saline group.
  • 8k13-hz35-EX(DAR8) (3mg/kg, IV, D0) had a tumor inhibition rate of 98% on subcutaneously transplanted tumors in KATO III/18.2 mice, and 2/6 mice had partial tumor regression;
  • the tumor inhibition rate of 8k13-hz35-EX(DAR4) (6mg/kg, IV, D0) on subcutaneously transplanted tumors in KATO III/18.2 mice was 115%, and 3/6 mice had partial tumor regression;
  • 8k13-hz35-EX2(DAR4) (3, 6mg/kg, IV, D0) had tumor inhibition rates of 63% and 97% for subcutaneously transplanted tumors in KATO

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Abstract

本发明公开了包含抗CLDN18.2的抗体或其片段的抗体药物偶联物。此外，本发明还公开了包含该抗体药物偶联物的药物组合物，以及该抗体药物偶联物和该药物组合物在制备药物中的用途。

Description

包含抗CLDN18.2的抗体或其抗原结合片段的抗体药物偶联物及其用途
相关申请的交叉引用
本专利申请要求于2021年2月9日提交的申请号为CN202110181327.3的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明属于生物技术领域，具体地，本发明涉及靶向CLDN18.2的抗体药物偶联物及其应用。
背景技术
近年来全球恶性肿瘤发病率逐年升高，据世界卫生组织(World Health Organization)和美国癌症协会(American Cancer Society)统计数据显示，0-74岁患癌症风险已上升至20.2％，其中男性患癌症风险高达22.4％，女性患癌症风险达18.2％。
胃癌是全球第五大最常见癌症和第三大癌症死亡原因。据统计，2018年新增胃癌100多万病例，死亡78.3万例。目前临床上胃癌的治疗以手术、化疗和放疗为主，尽管已有多种治疗方案，但5年生存率仍然很低。
随着靶向治疗手段的出现，针对不同途径的不同分子被开发出来用于胃癌的治疗。但是，目前研制的靶向药物仍存在不足。例如曲妥珠单抗，在Her2阳性乳腺癌中取得了成功，但是对胃癌的治疗效果并不理想；同样，针对表皮生长因子受体(epidermal growth factor receptor，EGFR)和哺乳动物类雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)的抑制剂在胃肿瘤的III期临床试验中也被证实无效。仍需要寻求新靶点来进行胃癌的靶向治疗。
闭合蛋白(CLDN)为一个蛋白质家族，于1998年首次由Shorichiro Tsukita等人发现，该家族的成员具有重要的机体生理作用，如参与细胞旁通透性、电导调节，维持上皮和内皮细胞间分子交换的紧密连接等。CLDN18是CLDN家族包含的至少24个成员之一，具有CLDN18.1和CLDN18.2两种异构体，其中CLDN18.2是一种胃特异性亚型，通常埋藏在胃粘膜的分化上皮细胞中，正常组织中很少或不表达，而在癌细胞中广泛表达，是一种高度选择性的分子。当恶性肿瘤发生时，细胞间分子交换的紧密连接遭到破坏， 肿瘤细胞表面的CLDN18.2表位由此暴露出来，使得CLDN18.2可能成为靶向治疗的特异性位点。
已发现CLDN18.2在原发性胃癌及其转移癌中表达，在胃癌患者中，53％的患者具有CLDN18.2的高丰度表达、20％患者具有中丰度表达；而在胃癌脑转移患者的组织切片中，在转移灶中发现45.16％的患者具有CLDN18.2的高丰度表达。事实上，研究表明CLDN18.2在人类多种癌症中出现异位激活，高度选择性地、稳定地表达于特定肿瘤组织，比如在胰腺癌、食管癌、卵巢癌和肺肿瘤中。正是CLDN18.2在多个癌组织中的高度选择性表达，以及在特定癌种如胃癌、胰腺癌等患者中的高阳性率，使得CLDN18.2成为一个颇具潜力的靶向治疗与肿瘤免疫治疗新靶点，引起了国内外研究者的关注。
日本制药企业安斯泰来针对这一靶点开发了抗体药物Claudiximab(IMAB362)，其在临床I期/II期显示了较好的治疗效果，2018年7月已经启动了该药物的三期临床试验。目前临床上主推的方案为胃癌EOX+Claudiximab的组合疗法，而Claudiximab的单药使用效果结果不佳。
从目前的治疗情况来看，胃癌、尤其是晚期胃癌恶性程度高、预后差，常规化疗失败后，治疗手段有限；而免疫治疗仅能使部分患者获益，而且可用的免疫治疗抑制剂数量也极为有限，目前患者能够选择的治疗药物并不多，并且客观缓解率不高。因此，仍需要开发治疗效果更为显著、适用性更广的药物，以满足临床的迫切需求。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合人CLDN18.2的高亲和力抗体，其中通过人源化设计，使该抗体最大程度减少鼠源氨基酸的数目，以拥有更好的体内安全性和应用前景；在此基础上，进一步筛选具有更强细胞内吞效应的抗体，将其与小分子化学药物制备成抗体药物偶联物(Antibody-Drug Conjugate，ADC)，利用该抗体对CLDN18.2表达细胞的靶向性和经由靶点的强的内吞效应，结合该小分子化学药物的作用，来增强针对CLDN18.2的抗体药物的治疗效果。
因此，本发明的一个目的是提供特异性结合人CLDN18.2的抗体或其片段；其中，所述的抗体的片段涵盖抗体的各种功能性片段，例如其抗原结合部分，如Fab、F(ab’) 2或scFv片段。本发明的另一个目的是提供采用该抗体 或其片段制备的抗体药物偶联物。
本发明的技术方案如下。
一方面，本发明提供一种抗体药物偶联物，其包含与药物共价连接的抗CLDN18.2的抗体或其片段。
在本发明提供的抗体药物偶联物中，所述抗CLDN18.2的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
(i)氨基酸序列分别如SEQ ID NO:23、SEQ ID NO:26和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；
(ii)氨基酸序列分别如SEQ ID NO:27、SEQ ID NO:28和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；
(iii)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:40所示的CDR-L1、CDR-L2和CDR-L3；
(iv)氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:43所示的CDR-L1、CDR-L2和CDR-L3；或
(v)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:43所示的CDR-L1、CDR-L2和CDR-L3。
优选地，在本发明提供的抗体药物偶联物中，所述抗CLDN18.2的抗体或其片段的重链包含重链可变区(VH)，所述重链可变区(VH)包含SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列或其变体；和/或，所述抗CLDN18.2的抗体或其片段的轻链包含轻链可变区(VL)，所述轻链可变区(VL)包含：选自SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列或其变体。
或者，优选地，在本发明提供的抗体药物偶联物中，所述抗CLDN18.2的抗体或其片段的重链包含重链可变区(VH)，所述重链可变区(VH)包含SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列或其变体；和/或，所 述抗CLDN18.2的抗体或其片段的轻链包含轻链可变区(VL)，所述轻链可变区(VL)包含选自SEQ ID NO:19或SEQ ID NO:22所示的氨基酸序列或其变体。
更优选地，在本发明提供的抗体药物偶联物中，所述抗CLDN18.2的抗体或其片段的重链和轻链分别包含如下所示的重链可变区(VH)和轻链可变区(VL)：
(i)如SEQ ID NO:7所示的氨基酸序列或其变体；和，如SEQ ID NO:11所示的氨基酸序列或其变体；
(ii)如SEQ ID NO:8所示的氨基酸序列或其变体；和，如SEQ ID NO:12所示的氨基酸序列或其变体；
(iii)如SEQ ID NO:15所示的氨基酸序列或其变体；和，如SEQ ID NO:19所示的氨基酸序列或其变体；
(iv)如SEQ ID NO:16所示的氨基酸序列或其变体；和，如SEQ ID NO:22所示的氨基酸序列或其变体。
本发明的上下文中，“氨基酸序列的变体”是指与所述氨基酸序列具有至少75％序列同一性(例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性)的氨基酸序列。
在本发明提供的抗体药物偶联物中，所述抗CLDN18.2的抗体或其片段可以为针对CLDN18.2的单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式，或者，所述抗体或其片段为针对CLDN18.2的半抗体或半抗体的抗原结合片段，例如scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；关于本发明提供的抗体的片段，优选地，所述片段为抗体的能够特异性结合CLDN18.2的任何片段。并且，就抗CLDN18.2的抗体或其片段而言，优选地，所述CLDN18.2为人CLDN18.2。
特别地，在本发明提供的抗体药物偶联物中，所述抗CLDN18.2的抗体或其片段至少包含抗体的重链可变区和轻链可变区，而这两者均可分别包括上述CDR1至CDR13以及间隔的框架区FR1至FR4，各个区域的排列方式按照FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的顺序。在这一方面，上文所述的氨基酸序列的变体与氨基酸序列之间的“至少75％序列同一性”可能由重链可变区和轻链可变区中框架区FR1至FR4中任一个存在的差异导致，或 者，也可能由重链可变区和轻链可变区以外的任意结构域或序列中存在的差异导致。这种差异可以是任何位置的氨基酸缺失、添加或置换，其中置换可以是保守置换或非保守置换。
在本发明提供的抗体药物偶联物中，优选地，所述抗CLDN18.2的抗体或其片段还包含人或鼠的恒定区，优选包含人或鼠的轻链恒定区(CL)和/或重链恒定区(CH)；更优选地，所述抗体或其片段包含选自IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
优选地，所述抗体为单克隆抗体，优选为鼠源、嵌合或人源化的单克隆抗体；更优选地，所述单克隆抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型。
根据本发明的具体实施方式，所述单克隆抗体的重链恒定区包含如SEQ ID NO:48所示的氨基酸序列或其变体。或者，根据本发明的具体实施方式，所述单克隆抗体的轻链恒定区包含如SEQ ID NO:49所示氨基酸序列或其变体。
具体地，本发明提供的抗体药物偶联物具有分子式Ab-[L-D]n，其中Ab表示本发明提供的抗CLDN18.2的抗体或其片段，L表示接头，D表示药物，n表示相对于每一分子Ab的药物平均连接数(DAR)。
在本发明提供的抗体药物偶联物中，所述药物D为细胞毒性类小分子药物和/或免疫治疗剂类小分子药物。其中，所述细胞毒性类小分子药物可以为微管蛋白抑制剂和/或拓扑异构酶I抑制剂，微管蛋白抑制剂可以为澳瑞他汀类化合物，如MMAE、MMAF等；拓扑异构酶I抑制剂可以为喜树碱类化合物，如SN38、Exatecan以及Dxd。这些化合物的结构示例见下：
Figure PCTCN2022075689-appb-000001
所述免疫治疗剂类小分子药物可以为STING激动剂和/或TLR7/8激动 剂。其中，STING激动剂可以为环二核苷酸类化合物，例如如下所示的化合物10和11等；TLR7/8激动剂可以为咪唑并喹啉类化合物和/或苯并氮卓类化合物，例如如下所示的化合物1、2、3、4、5、6、7、8和9等。
Figure PCTCN2022075689-appb-000002
在本发明提供的抗体药物偶联物中，所述接头L可以为半胱氨酸偶联接头或赖氨酸偶联接头，其释放机制为可切割或不可切割的。可切割的接头如可选自：MC-Val-Cit-PAB、MC-Val-Ala和MC-Gly-Gly-Phe-Gly等；不可切割的接头如可选自MCC、PEG 4Mal和mc等。
具体地，在本发明提供的抗体药物偶联物中，细胞毒性类药物接头(即“[L-D]”部分，在本发明的上下文中“药物接头”、“[L-D]”和“含药接头”可互换使用)可以为：
1)喜树碱类拓扑异构酶I抑制剂-ADC接头，MC-GGFG-Dxd(简称为 EX)和CL2A-SN38(简称为EX2)：
Figure PCTCN2022075689-appb-000003
2)澳瑞他汀类微管蛋白抑制剂-ADC接头，MC-VC-MMAE(简称为M)：
Figure PCTCN2022075689-appb-000004
免疫治疗剂类药物接头(即“[L-D]”部分)可以为：
1)苯并氮卓类TLR8激动剂-ADC接头可以为
Figure PCTCN2022075689-appb-000005
Figure PCTCN2022075689-appb-000006
2)环二核苷酸类STING激动剂-ADC接头可以为：
Figure PCTCN2022075689-appb-000007
根据本发明的具体实施方式，在本发明提供的抗体药物偶联物中的“[L-D]”为实施例部分采用的特定化合物，其中MC-GGFG-Dxd缩写为“EX”、CL2A-SN38缩写为“EX2”、MC-VC-MMAE缩写为“M”、L-A缩写为“TLR8”和L-B缩写为“STING”。相应地，本发明提供的抗体药物偶联物可命名为“抗体简称-[L-D]缩写”。
优选地，在本发明提供的抗体药物偶联物中，n为1至12、优选1至8、更优选3至8，例如4至8、5至8、6至8、乃至7至8。
另一方面，本发明还提供所述抗体药物偶联物的制备方法，所述方法包括以下步骤：
(1)用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
(2)用药物接头(连接子-药物缀合物)与步骤(1)得到的经还原后的抗体在缓冲液与有机溶剂的混合液中进行交联，得到抗体药物偶联物。
优选地，所述方法包括以下步骤：
1)还原：将抗体原液用反应缓冲液稀释，加入抗体1.5-2.5倍摩尔比的还原剂如TCEP，反应液于室温搅拌1-4小时；
2)偶联：用反应缓冲液调整抗体还原液的浓度，再加入抗体4.0-6.0倍摩尔比的药物接头的有机溶剂如二甲基亚砜溶液，将该溶液在室温条件下继续搅拌30-60分钟；
3)纯化：将抗体偶联液浓缩，色谱分离收集主成分，超滤。
根据本发明的具体实施方式，本发明提供DAR为4的抗体药物偶联物的制备方法，包括：
1)还原：将抗体原液用反应缓冲液稀释至约10mg/mL，加入抗体1.5-2.5倍摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动2小时；
2)偶联：将抗体还原液用反应缓冲液调整浓度至5mg/mL，再加入抗体4.0-6.0倍摩尔比的药物接头的二甲基亚砜溶液，将该溶液在室温条件下继续搅拌30-60分钟；
3)纯化：将抗体偶联液浓缩至15mg/mL左右，添加缓冲液调整电导率为100ms/cm，上样至NAP-25色谱柱，分离并收集主成分；最后超滤离心管将最终样品置换至pH值7.4的50mM的磷酸氢二钠-磷酸二氢钠缓冲盐中，除菌过滤。
根据本发明的具体实施方式，本发明提供DAR为8的抗体药物偶联物的制备方法，包括：
1)还原：将抗体原液用反应缓冲液稀释至约10mg/mL，加入抗体9.0-11倍摩尔比的三(2-羧乙基)膦盐酸盐(TCEP)，反应液于25℃搅动2小时；
2)偶联：将抗体还原液用反应缓冲液调整浓度至5mg/mL，再加入抗体10-12倍摩尔比的药物接头的二甲基亚砜溶液，将该溶液在室温条件下继续搅拌30-60分钟；
3)纯化：将抗体偶联液浓缩至15mg/mL左右，添加缓冲液调整电导率为100ms/cm。上样至NAP-25色谱柱，分离并收集主成分；最后超滤离心管将最终样品置换至pH值7.4的50mM的磷酸氢二钠-磷酸二氢钠缓冲盐中，除菌过滤。
在本发明的另一方面，本发明提供一种抗CLDN18.2的抗体或其片段。上文就抗体药物偶联物中包含的抗CLDN18.2的抗体或其片段进行的描述与定义均适用于这一方面的抗CLDN18.2的抗体或其片段。
关于具体序列，优选地，本发明提供的抗CLDN18.2的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
(i)氨基酸序列分别如SEQ ID NO:23、SEQ ID NO:26和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；
(ii)氨基酸序列分别如SEQ ID NO:27、SEQ ID NO:28和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；
(iii)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:40所示的CDR-L1、CDR-L2和CDR-L3；
(iv)氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:43所示的CDR-L1、CDR-L2和CDR-L3；或
(v)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:43所示的CDR-L1、CDR-L2和CDR-L3。
优选地，所述抗CLDN18.2的抗体或其片段的重链包含重链可变区(VH)，轻链包含轻链可变区(VL)，所述重链可变区(VH)和轻链可变区(VL)分别包含SEQ ID NO:8所示的氨基酸序列或其变体和SEQ ID NO:12所示的氨基酸序列或其变体；或者，所述重链可变区(VH)和轻链可变区(VL)分别包含SEQ ID NO:16所示的氨基酸序列或其变体和SEQ ID NO:22所示的氨基酸序列或其变体。
又一方面，本发明还提供一种药物组合物，所述药物组合物包含本发明的抗体药物偶联物或者包含本发明的抗CLDN18.2的抗体或其片段，以及可选的药学上可接受的辅料。其中，所述辅料可以是载体或赋形剂等。
进一步地，本发明提供的药物组合物还可包含能够与本发明的抗体药物偶联物或者抗CLDN18.2的抗体或其片段联合使用的其他药物，例如化疗药物EGFR抑制剂(Gefitinib、Erlotinib、Vandetanib、Sunitinib等)、PARP抑 制剂(Olaparib等)、DNA烷化剂(Bendamustine等)、HDAC抑制剂(Vorinostat等)、BTK抑制剂(Ibrutinib等)、C-met抑制剂(Tepotinib等)、紫杉醇等，或者本发明提供的药物组合物还可与其他治疗方法如化疗方案EOX联合使用。
又一方面，本发明提供了一种药物组合物。根据本发明的实施例，该药物组合物包含抗体药物偶联物混合物和药学上可接受的载体，所述抗体药物偶联物混合物包含多种如前述的抗体药物偶联物。
任选地，所述抗体药物偶联物混合物的平均n值为2至8。
任选地，所述抗体药物偶联物混合物包含多种所述抗体药物偶联物，每种所述抗体药物偶联物的n值为1至12、优选1至8、更优选3至8，例如4至8、5至8、6至8、乃至7至8。
再一方面，本发明还提供所述抗体药物偶联物、抗CLDN18.2的抗体或其片段或包含它们任意的药物组合物在制备药物中的用途，所述药物用于治疗与CLDN18.2相关的疾病，优选为肿瘤，更优选为癌症。进一步优选地，所述癌症选自胰腺癌、胃癌、结肠癌、食管癌、肝癌、卵巢癌、肺癌、胆囊癌和头颈癌。
又一方面，本发明还提供一种治疗疾病的方法，所述方法包括向有此需要的受试者施用本发明提供的抗体药物偶联物、抗CLDN18.2的抗体或其片段或包含它们任意的药物组合物。其中所述受试者为哺乳类动物，优选地为人。所述疾病为与CLDN18.2相关的疾病，优选为肿瘤，更优选为癌症。进一步优选地，所述癌症选自胰腺癌、胃癌、结肠癌、食管癌、肝癌、卵巢癌、肺癌、胆囊癌和头颈癌。
相对于现有技术，本发明实现了以下有益的技术效果：
首先，在本发明中使用细胞表面稳定表达人CLDN18.2的高CHOK1细胞免疫小鼠，获得分泌抗人CLDN18.2抗体的杂交瘤细胞，然后由杂交瘤细胞分泌的鼠抗构建嵌合抗体，在证明该嵌合抗体保留了鼠抗与抗原的特异性结合，进行重链和轻链可变区的人源化改造，得到了多个针对人CLDN18.2的不同人源化抗体。实验证明，本发明提供的抗CLDN18.2抗体对抗原CLDN18.2具有强亲和力，对靶点表达细胞具有显著的补体依赖性细胞毒(CDC)活性和抗体依赖性细胞毒(ADCC)活性，且活性强于同靶点的IMAB362或与其相当。特别是，与本身作为嵌合抗体的IMAB362相比，本发明提供的抗CLDN18.2抗体最大程度地减少了鼠源氨基酸，由此能够降低 在人体内引起排斥免疫反应的可能性。
进一步地，实验证明，相比于IMAB362，本发明的抗CLDN18.2抗体具有更优的内吞活性，与小分子药物制备得到的抗体药物偶联物稳定性也更强。采用本发明的抗CLDN18.2抗体制备得到的ADC，在通过该抗体的靶向作用和/或Fc效应介导免疫细胞吞噬后进入细胞内部，能够有效实现所偶联的药物释放，在示例性的细胞毒性类小分子药物或免疫治疗剂类小分子药物的情况下，实现了细胞毒性类小分子药物对CLDN18.2表达肿瘤细胞的显著的杀伤作用，或实现免疫治疗剂类小分子药物对细胞内对应受体以及下游信号通路的显著的活化作用。因此，本发明提供的抗CLDN18.2抗体更适合与药物等制备成抗体药物偶联物，兼顾安全性和有效性。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1：本发明的抗体与Isotype抗体的表位研究结果；
图2：本发明的抗体与细胞表面CLDN18.2的结合活性；
图3：本发明的抗体结合细胞表面CLDN18.2的内化活性；
图4：抗体药物偶联物8k13-hz35-TLR8-1103的HIC分析结果；
图5：抗体药物偶联物8k13-hz35-TLR8-1103的SEC分析结果；
图6：抗体药物偶联物8k13-hz35-STING-D4-1203的RPLC分析结果；
图7：抗体药物偶联物8k13-hz35-STING-D4-1203的SEC分析结果；
图8：抗体药物偶联物8k13-hz35-M-20200318-1的HIC分析结果；
图9：抗体药物偶联物8k13-hz35-M-20200318-1的SEC分析结果；
图10：抗体药物偶联物8k13-hz35-EX-DAR4-20200922的RPLC分析结果；
图11：抗体药物偶联物8k13-hz35-EX-DAR4-20200922的SEC分析结果；
图12：抗体药物偶联物8k13-hz35-EX-DAR8-20200421的HIC分析结果；
图13：抗体药物偶联物8k13-hz35-EX-DAR8-20200421的SEC分析结果；
图14：抗体药物偶联物8k13-hz35-EX2-DAR4-20200922的RPLC分析结果；
图15：抗体药物偶联物8k13-hz35-EX2-DAR4-20200922的SEC分析结 果；
图16：抗体药物偶联物18k13-hz35-EX2-DAR8-20200922的RPLC分析结果；
图17：抗体药物偶联物18k13-hz35-EX2-DAR8-20200922的SEC分析结果；
图18：抗体药物偶联物16k15-hz35-M-20200318-1的HIC分析结果；
图19：抗体药物偶联物16k15-hz35-M-20200318-1的SEC分析结果；
图20：抗体药物偶联物16k15-hz35-EX-20200421的HIC分析结果；
图21：抗体药物偶联物16k15-hz35-EX-20200421的SEC分析结果；
图22：抗体药物偶联物IMAB362-M-1的HIC分析结果；
图23：抗体药物偶联物IMAB362-M-1的SEC分析结果；
图24：抗体药物偶联物IMAB362-M-2的HIC分析结果；
图25：抗体药物偶联物IMAB362-M-2的SEC分析结果；
图26：本发明的抗体药物偶联物对无CLDN18.2表达的细胞的杀伤效果；
图27：本发明的抗体药物偶联物对CLDN18.2高表达的细胞的杀伤效果；
图28：本发明的抗体药物偶联物对PBMC分泌促炎因子TNFα的刺激作用；
图29：本发明的抗体药物偶联物对pIRF3表达的刺激作用；
图30：本发明的抗体药物偶联物对小鼠皮下移植瘤的疗效；
图31：本发明的抗体药物偶联物对荷瘤小鼠体重的影响；
图32：本发明的抗体药物偶联物对荷瘤小鼠摄食的影响；
图33：本发明的抗体药物偶联物对小鼠皮下移植瘤的疗效；
图34：本发明的抗体药物偶联物对人胃癌KATO III/18.2小鼠皮下移植瘤的疗效(个体肿瘤重量)；**P<0.001,***P<0.001,vs溶剂。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂等，如无特殊说明，均为市售购买产品。
(一)用于制备抗体药物偶联物的抗体的制备与表征
以下实施例中：
抗原人CLDN18.2参见NP_001002026.1，抗原人CLDN18.1参见NP_057453.1。
IMAB362的重链和轻链序列参见SEQ ID NO:44和SEQ ID NO:45。
Sacituzumab的重链和轻链序列参见SEQ ID NO:46和SEQ ID NO:47。
人单克隆抗体IgG1亚类的重链恒定区序列和κ亚类的轻链恒定区序列分别示于SEQ ID NO:48和SEQ ID NO:49。
实施例1鼠源单克隆抗体的筛选
使用细胞表面稳定表达人CLDN18.2蛋白的CHOK1细胞免疫Balb/c小鼠，1个月后用流式细胞仪术(FACS)分析小鼠血清，取血清抗体滴度高的小鼠取其脾脏，标准方法分离得到的脾细胞与骨髓瘤细胞P3X63Ag8.653使用PEG或者电融合方法进行融合。将融合后的杂交瘤细胞接种于384孔板中，培养10-14天后，取上清用FACS分析杂交瘤细胞分泌的抗体，筛选得到若干克隆，所述克隆能够结合细胞表面稳定表达人CLDN18.2蛋白的CHOK1细胞而不能够结合细胞表面稳定表达人CLDN18.1蛋白的CHOK1细胞。通过有限稀释的方法将筛选到的克隆单细胞化，3轮之后得到的每个杂交瘤细胞克隆只分泌一个抗体。
将分泌抗人CLDN18.2抗体的杂交瘤细胞扩大培养后，按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA；利用5×PrimeScript RT Master Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(Anke Krebber.1997)和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列；利用PCR clean-up Gel extraction试剂盒(Macherey-Nagel公司)纯化PCR扩增产物；按照pClone007 Simple Vector Kit试剂盒(擎科生物科技有限公司)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。见表1。
表1.鼠抗的轻重链可变区
鼠抗 重链可变区(VH) 轻链可变区(VL)
8k13 SEQ ID NO:1 SEQ ID NO:2
16k15 SEQ ID NO:3 SEQ ID NO:4
鼠抗8k13
重链可变区
>8K13_vh(SEQ ID NO:1；CDR按顺序为：SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25)
Figure PCTCN2022075689-appb-000008
轻链可变区
>8K13_vl(SEQ ID NO:2；CDR按顺序为：SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31)
Figure PCTCN2022075689-appb-000009
鼠抗16k15
重链可变区
>16K15_vh(SEQ ID NO:3；CDR按顺序为：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36)
Figure PCTCN2022075689-appb-000010
轻链可变区
>16K15_vl(SEQ ID NO:4；CDR按顺序为：SEQ ID NO:39、SEQ ID NO:30、SEQ ID NO:40)
Figure PCTCN2022075689-appb-000011
实施例2抗人CLDN18.2嵌合抗体的制备
将鼠源抗人CLDN18.2单克隆抗体的重链可变区序列和公开发表的人单克隆抗体IgG1亚类的重链恒定区序列(参见SEQ ID NO:48)拼接在一起，构建到哺乳动物细胞表达载体中；将鼠源抗人CLDN18.2单克隆抗体的轻链可变区序列和公开发表的人单克隆抗体κ亚类的轻链恒定区序列(参见SEQ ID NO:49)拼接在一起，构建到哺乳动物细胞表达载体中。构建好的抗人CLDN18.2嵌合抗体的重链载体和轻链载体配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用ProteinA纯化得到抗人CLDN18.2嵌合抗体蛋白。
嵌合抗体命名为“鼠抗简称-xiIgG”。
实施例3抗人CLDN18.2嵌合抗体的体外细胞结合实验
将抗人CLDN18.2嵌合抗体从100nM的起始浓度开始做2倍的梯度倍比稀释，共16个浓度点，各个浓度点的抗体取10ul加入384孔板。100g室温离心5分钟收集细胞表面表达CLDN18.2的CHOK1细胞，用含0.5％BSA的PBS洗涤细胞一次，100g室温离心5分钟，重悬细胞为密度约2x10 6个细胞/ml，取10ul加入到已加抗体的384孔板的孔中。4℃孵育1小时后，加入荧光标记的羊抗人IgG二抗。继续于4℃孵育1小时后，用流式细胞仪分析细胞群的平均荧光读值。
结果显示嵌合抗体与表达人CLDN18.2细胞有nM级别的特异结合。
实施例4抗人CLDN18.2鼠抗的人源化
综合Kabat、Chothia的抗体编码方案，确定鼠抗的重链和轻链中6个抗原互补决定簇(CDR)的氨基酸序列区域及支撑抗体保守三维构象的框架区域(framework region)。随后通过分析搜索已知人源抗体序列，选择与鼠抗最为相似接近的人源抗体重链可变区序列，选择其抗体框架区序列作为模板，将鼠抗重链CDR与人源抗体框架区结合，最终生成人源化抗体重链可变区序列。同样过程，生成人源化抗体轻链可变区序列。
鼠抗CDR直接移植至人框架区的抗体常出现结合活性急剧下降，因此需要将框架区个别氨基酸从人源的改回鼠源的。确定回复突变位点，一是对照设计好的人源化抗体序列和原始的鼠抗序列，检查有哪些氨基酸不同；二是检查这些氨基酸是否对支持抗体结构起重要作用或者对与抗原的结合起重要作用。同时需要检查人源化设计后的序列是否有一些潜在的翻译后修饰位点(PTM)，如N(天冬酰胺)糖基化位点、N脱酰胺化位点、D(天冬氨酸)异构化位点等。
基于上述8K13_vh、8K13_vl、16K15_vh和16K15_vl的人源化版本示例如下：
鼠抗8k13的人源化
重链可变区
>8K13_vh_hz0(SEQ ID NO:5；CDR按顺序为：SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25)
Figure PCTCN2022075689-appb-000012
>8K13_vh_hz1(SEQ ID NO:6；CDR按顺序为：SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25)
Figure PCTCN2022075689-appb-000013
>8K13_vh_hz2(SEQ ID NO:7；CDR按顺序为：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:25)
Figure PCTCN2022075689-appb-000014
>8K13_vh_hz3(SEQ ID NO:8；CDR按顺序为：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:25)
Figure PCTCN2022075689-appb-000015
轻链可变区
>8K13_vl_hz0(SEQ ID NO:9；CDR按顺序为：SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31)
Figure PCTCN2022075689-appb-000016
>8K13_vl_hz1(SEQ ID NO:10；CDR按顺序为：SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31)
Figure PCTCN2022075689-appb-000017
>8K13_vl_hz4(SEQ ID NO:11；CDR按顺序为：SEQ ID NO:32、SEQ ID NO:30、SEQ ID NO:33)
Figure PCTCN2022075689-appb-000018
>8K13_vl_hz5(SEQ ID NO:12；CDR按顺序为：SEQ ID NO:32、SEQ ID NO:30、SEQ ID NO:33)
Figure PCTCN2022075689-appb-000019
鼠抗16k15的人源化
重链可变区
>16k15_vh_hz0(SEQ ID NO:13；CDR按顺序为：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36)
Figure PCTCN2022075689-appb-000020
Figure PCTCN2022075689-appb-000021
>16k15_vh_hz1(SEQ ID NO:14；CDR按顺序为：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36)
Figure PCTCN2022075689-appb-000022
>16k15_vh_hz2(SEQ ID NO:15；CDR按顺序为：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36)
Figure PCTCN2022075689-appb-000023
>16k15_vh_hz3(SEQ ID NO:16；CDR按顺序为：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36)
Figure PCTCN2022075689-appb-000024
轻链可变区
>16K15_vl_hz0(SEQ ID NO:17；CDR按顺序为：SEQ ID NO:39、SEQ ID NO:30、SEQ ID NO:40)
Figure PCTCN2022075689-appb-000025
>16K15_vl_hz1(SEQ ID NO:18；CDR按顺序为：SEQ ID NO:39、SEQ ID NO:30、SEQ ID NO:40)
Figure PCTCN2022075689-appb-000026
>16K15_vl_hz2(SEQ ID NO:19；CDR按顺序为：SEQ ID NO:41、SEQ ID NO:30、SEQ ID NO:40)
Figure PCTCN2022075689-appb-000027
>16K15_vl_hz3(SEQ ID NO:20；CDR按顺序为：SEQ ID NO:41、SEQ ID NO:30、SEQ ID NO:42)
Figure PCTCN2022075689-appb-000028
>16K15_vl_hz4(SEQ ID NO:21；CDR按顺序为：SEQ ID NO:41、SEQ ID NO:30、SEQ ID NO:43)
Figure PCTCN2022075689-appb-000029
>16k15_vl_hz5(SEQ ID NO:22；CDR按顺序为：SEQ ID NO:41、SEQ ID NO:30、SEQ ID NO:43)
Figure PCTCN2022075689-appb-000030
将人源化抗体重链可变区基因构建到含人单克隆抗体IgG1亚类的重链恒定区序列(参见SEQ ID NO:48)的哺乳动物细胞表达载体中；轻链可变区基因构建到含人单克隆抗体κ亚类的轻链恒定区序列(参见SEQ ID NO:49)的哺乳动物细胞表达载体中。构建好的抗人CLDN18.2人源化抗体的重链载体和轻链载体配对混合，使用聚乙烯亚胺(PEI)转染HEK293细胞，约7天后收集细胞上清，使用ProteinA纯化得到抗人CLDN18.2人源化抗体蛋白。
本发明的人源化抗体命名为“鼠抗简称-hz”。鼠抗CDR直接移植至人框架区的抗体命名为“鼠抗简称-hz00”。进一步改造得到的抗体根据序列不同进行编号，例如，抗体8k13-hz11的编号来自其重链和轻链可变区的编号8K13_vh_hz1和8K13_vl_hz1。
利用Fortebio(BLITZ pro1.1.0.28)仪器分析嵌合抗体及其人源化抗体与抗原人CLDN18.2的结合动力学参数。测定前先将NTA生物探针浸泡于PBS中10分钟；然后将该探针置于含100nM的抗原的PBS中300秒，捕获带His标签的抗原；进一步将探针与100nM抗体进行结合反应，结合时间400秒；之后将探针转移至PBS中，进行解离反应，时间为600秒。实验完毕，扣除空白对照响应值，用软件进行1：1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。结果见表2。
表2.鼠抗人源化后的结合动力学参数比较
Figure PCTCN2022075689-appb-000031
Figure PCTCN2022075689-appb-000032
实施例5抗人CLDN18.2人源化抗体的体外结合亲和力的动力学实验
采用GE公司BIAcore仪器S200测定抗体抗原相互作用力。
参考GE公司Human antibody capture kit(货号BR-1008-39，Lot10261753)操作说明，首先在传感芯片CM5分析通道和对照样品通道都饱和偶联最大量抗人Fc抗体，然后在分析通道流过含有7.5μg/ml抗人CLDN18.2嵌合抗体、人源化抗体或IMAB362的缓冲液使其均匀分布，最后在分析通道和对照样品通道一起流过梯度稀释的抗原样品(起始浓度20nM，1:3稀释8个浓度点，并且设定0.741nm浓度点重复)，测定抗体抗原结合后发生的光反应值。经仪器软件拟合分析，最终得到抗体的结合常数Kon和解离常数Koff，以及亲和力常数KD。
结果表明，抗人CLDN18.2人源化抗体的体外结合亲和力常数与原始鼠抗比没有显著的改变，见表3。
表3.抗体的结合动力学
抗体 ka(M-1s-1) kd(s-1) KD(M)
8k13-xiIgG 8.19E+04 5.73E-04 6.99E-09
8k13-hz24 7.09E+04 2.02E-03 2.85E-08
16k15-xiIgG 1.07E+05 1.44E-03 1.35E-08
16k15-hz22 5.71E+04 5.63E-04 9.85E-09
IMAB362 1.48E+06 0.201 1.36E-07
实施例6抗人CLDN18.2人源化抗体的体外细胞学试验
6.1补体依赖性细胞毒性(CDC)
使用Quidel公司的商品化人血清全补体对本发明人源化抗体进行分析，分析其对稳定表达人CLDN18.2的CHOK1、BxPC3和NCI-N87细胞诱导补体 依赖性细胞毒性(CDC)的能力。
将细胞与终浓度为250μg/ml至3.8ng/ml的待测抗体混匀；加入溶于细胞培养基RPMI-1640中的6.25％人血清全补体，在37℃下孵育3小时；然后通过CCK-8试剂盒进行细胞毒性检测；最终通过MD酶标仪检测450nm吸光度。通过吸光值采用softmax pro7软件进行4参数拟合曲线，计算样品的EC50值。
结果表明，抗人CLDN18.2人源化抗体针对靶点表达细胞有特异的补体依赖性细胞毒(CDC)活性，并且细胞杀伤活性显著优于IMAB362，见表4。
表4.抗体的CDC
靶点表达细胞 a.CHOK1 b.BxPC3 c.NCI-N87
抗体 EC50(nM) EC50(nM) EC50(nM)
8k13-hz24 21.27 28.53 57.7
16k15-hz22 5.2 12.22 17.36
IMAB362 30.45 52.23 474.2
6.2抗体依赖性细胞毒性(ADCC)
使用工程改造的Jurkat细胞作为效应细胞，该细胞稳定表达FcγRIIIa-FcεRIaγ杂合受体，由NFAT应答元件驱动表达萤火虫萤光素酶。抗体在ADCC作用机制中的生物活性通过NFAT通路活化产生的萤光素酶定量。将1.5E5个效应细胞与终浓度33μg/ml至85pg/ml的待测抗体混匀，然后将2.5E4个靶细胞加入其中(效应细胞与靶细胞E:T比例6:1)，在37℃下孵育16小时；然后通过Promega公司试剂盒Bio-Glo TM Luciferase Assay System进行检测；最终通过MD酶标仪检测LUM值。
数据处理方式如下，诱导倍数＝(受检孔读值–背景值)/(阴性对照孔读值–背景值)，用prism软件进行4参数拟合曲线，计算出样品的EC50值。
结果表明，抗人CLDN18.2人源化抗体针对靶点表达细胞有特异的抗体依赖性细胞毒(ADCC)活性，细胞杀伤活性与IMAB362相当，见表5。
表5.抗体的ADCC
靶点表达细胞 a.CHOK1 b.BxPC3 c.NCI-N87
抗体 EC50(nM) EC50(nM) EC50(nM)
8k13-hz24 0.5894 0.3045 0.1499
16k15-hz22 0.7122 0.1759 0.0677
IMAB362 0.5929 0.1056 0.0909
实施例7抗人CLDN18.2人源化抗体的表位研究
使用表位竞争法对本发明的抗体及对照抗体的表位进行研究。
制备Rituximab(无关抗体，即对照抗体)、8k13-hz35(SEQ ID NO:8+SEQ ID NO:12)和16k15-hz35(SEQ ID NO:16+SEQ ID NO:22)的Fab形式抗体Isotype-Fab、8k13-hz35-Fab和16k15-hz35-Fab。然后，首先将竞争抗体IMAB362稀释为0.05ug/ml。用含竞争抗体的溶液将待分析的8k13-hz35-Fab、16k15-hz35-Fab和Isotype-Fab稀释到200nM的浓度，做2倍的梯度倍比稀释，共8个浓度点。各个浓度点的抗体取10ul加入384孔板。100g室温离心5分钟收集细胞表面表达CLDN18.2 CHOK1细胞，用含0.5％BSA的PBS洗涤细胞一次，100g室温离心5分钟。重悬细胞为密度约2x106个细胞/毫升，取10ul加入到已加抗体的384孔板的孔中，4℃孵育过夜。第二天加入1:500稀释的荧光标记羊抗人IgG二抗10ul。于4℃孵育1小时后，用流式细胞仪分析细胞群的平均荧光读值。
结果见图1。可知，本发明的抗体8k13-hz35与IMAB362结合不同的抗原表位，而16k15-hz35与IMAB362存在抗原识别表位的竞争关系。
实施例8 FACS检测抗人CLDN18.2人源化抗体与CHO细胞表面人CLDN18.2重组蛋白的结合活性
将重组表达人CLDN18.2的CHO细胞(CHO/CLDN18.2)悬液分别与人源化抗体(8k13-hz35、16k15-hz35、8k13-hz24、16k15-hz22)(浓度为5μg/mL起始3倍连续稀释5个梯度)在4℃孵育60min，并设阴性对照(NC)IgG1同型对照抗体NC-huIgG1。以PBS洗涤细胞3次后，加入1:200稀释的羊抗人IgG-FITC(Cat：F9512，Sigma)并4℃孵育45min。PBS洗涤细胞3次后通过流式细胞仪(型号B49007AD，SNAW31211，BECKMAN COULTER)检测细胞的平均荧光强度(MFI)，以检测人源化抗体与CHO细胞表面的人CLDN18.2结合能力。
结果如图2所示，抗体8k13-hz35、16k15-hz35可以有效结合CHO细胞表面的人CLDN18.2重组蛋白，且结合能力与抗体8k13-hz24、16k15-hz22结合能力相当。
实施例9抗人CLDN18.2人源化抗体结合细胞表面CLDN18.2的内化活性
将重组表达人CLDN18.2的NCI-N87 CLDN18.2细胞(Cat.:KC-1222，康源博创)，以2×10 3个细胞/孔的密度接种于96孔细胞培养板，培养24小时。弃上清，用RPMI 1640(含10％FBS)，37℃封闭60min，离心，RPMI 1640(含10％FBS)洗涤2遍；将使用Mix-n-Stain TM CF TM 488A(Cat:MX488AS100，Sigma)标记的人源化抗体8k13-hz35、16k15-hz35、对照抗体IMAB362、内化阳性抗体Sacituzumab以及IgG同型对照抗体NC-huIgG1，用RPMI 1640(含10％FBS)稀释至1μg/mL，加入NCI-N87 CLDN18.2细胞中，100ul/孔。每个样品分为2组，一组在37℃下孵育作为内吞组，一组在4℃下孵育做为阴性对照；阴性对照孵育1小时后，4℃离心并PBS洗涤2次，用荧光显微镜观察并拍照，37℃内吞组孵育4小时后，室温离心并PBS洗涤2次，用荧光显微镜观察并拍照。
结果见图3，表明人源化抗体8k13-hz35和16k15-hz35在37℃条件下都能够被CLDN18.2介导内吞，在细胞质内呈点状分布。提示抗体人源化后仍然能够保持内化活性。
(二)抗体药物偶联物的制备与表征
以下实施例中采用的药物接头包括：
MC-GGFG-Dxd，喜树碱类拓扑异构酶I抑制剂-ADC接头，本发明上下文中缩写为“EX”。
Figure PCTCN2022075689-appb-000033
CL2A-SN38，喜树碱类拓扑异构酶I抑制剂-ADC接头，本发明上下文中缩写为“EX2”。
Figure PCTCN2022075689-appb-000034
MC-VC-MMAE，MMAE微管抑制剂-ADC接头，本发明上下文中缩写为“M”。
Figure PCTCN2022075689-appb-000035
L-A，苯并氮卓类TLR8激动剂-ADC接头，本发明上下文中缩写为“TLR8”。
Figure PCTCN2022075689-appb-000036
L-B，环二核苷酸类STING激动剂-ADC接头，本发明上下文中缩写为“STING”。
Figure PCTCN2022075689-appb-000037
示例性地，采用了以下实验方法：
1.抗体的还原
用含有50mM氯化钠和2mM EDTA的磷酸盐缓冲液(50mM，pH6.5；简称为PBS6.5/EDTA)制备抗体溶液(抗体浓度为10mg/ml)，向其中加入TCEP(Sigma-Aldrich)水溶液，使TCEP为抗体的2-10倍摩尔当量，然后在25℃孵育2h。
2.抗体与药物接头的偶联
使用PBS6.5/EDTA，将还原后抗体浓度调整为5mg/ml。加入N,N-二甲基乙酰胺(DMA)，然后加入药物接头的二甲基亚砜溶液(药物接头浓度为10mg/ml)，使药物接头为抗体的6-12摩尔当量，然后加入药物接头的二甲基亚砜溶液(药物接头浓度为10mg/ml)，使药物接头为抗体的5-12倍摩尔当量，且将DMA在整个反应体系中的体积比控制为10-20％。将该溶液在室温条件下继续搅拌30-60分钟，使药物接头与抗体偶联。
3.抗体药物偶联物的纯化
用磷酸缓冲液(pH7.4，浓度为50mM；简称为PB7.4)平衡NAP-25色谱柱，针对一根该NAP-25柱，装填一定量的抗体药物偶联反应液，然后分离获得用PB7.4洗脱的组分。使用Amicon Ultra-15(30000MWCO，Millipore Corporation)浓缩，除菌过滤。
使用如下抗体药物偶联物浓度测定方法测定偶联样品中抗体药物偶联物的浓度。
4.抗体药物偶联物浓度测定方法
4.1抗体药物偶联物浓度测定方法-EX
样品中的连接药物浓度可通过测定在280nm波长下的UV吸光度，然后进行下述计算而得到。
由于某一波长下的总吸光度等于存在于体系内的所有吸收化学物质种类的吸光度的和(吸光度的加成性)，所以，假设在抗体与药物接头偶联前后，抗体及药物接头的摩尔吸光系数不发生变化时，抗体药物偶联物中的抗体浓度及药物浓度如下述的关系式所示。
Figure PCTCN2022075689-appb-000038
因此，抗体偶联物浓度
Figure PCTCN2022075689-appb-000039
其中，
Figure PCTCN2022075689-appb-000040
DAR值为实测值。
4.2抗体-药物偶联物浓度测定方法-TLR8
如上，抗体药物偶联物中的抗体浓度及药物浓度如下述的关系式所示。
Figure PCTCN2022075689-appb-000041
因此，抗体偶联物浓度
Figure PCTCN2022075689-appb-000042
其中，
Figure PCTCN2022075689-appb-000043
DAR值为实测值。
4.3抗体-药物偶联物浓度测定方法-STING
如上抗体药物偶联物中的抗体浓度及药物浓度如下述的关系式所示。
Figure PCTCN2022075689-appb-000044
因此，抗体偶联物浓度
Figure PCTCN2022075689-appb-000045
其中，
Figure PCTCN2022075689-appb-000046
DAR值为实测值。
4.4抗体-药物偶联物浓度测定方法-M
如上，抗体药物偶联物中的抗体浓度及药物浓度如下述的关系式所示。
Figure PCTCN2022075689-appb-000047
因此，抗体偶联物浓度
Figure PCTCN2022075689-appb-000048
其中，
Figure PCTCN2022075689-appb-000049
DAR值为实测值。
4.5抗体-药物偶联物浓度测定方法-EX2
如上，抗体药物偶联物中的抗体浓度及药物浓度如下述的关系式所示。
Figure PCTCN2022075689-appb-000050
因此，抗体偶联物浓度
Figure PCTCN2022075689-appb-000051
其中，
Figure PCTCN2022075689-appb-000052
DAR值为实测值。
5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法
5.1疏水作用色谱法
HPLC分析用样品制备：以流动相B为稀释液，将抗体药物偶联物稀释至2mg/ml，0.22μM滤头过滤，用于HPLC分析。
DAR计算公式：
DAR＝∑(加权峰面积)/100，即DAR＝(D0峰面积比*0+D1峰面积比*1+D2峰面积比*2+D3峰面积比*3+D4峰面积比*4+D5峰面积比*5+D6峰面 积比*6+D7峰面积比*7+D8峰面积比*8)/100。
5.1.1在下述的测定条件下进行HIC分析
HPLC仪器：Waters Acquity Arc
流动相A：2.5M硫酸铵+125mM PB，pH 6.8
流动相B：125mM PB，pH 6.8
流动相C：IPA
流动相D：水
分析柱：Tosoh,TSKgel Butyl-NPR；4.6*100mm；2.5μm，PN 0042168
进样体积：20μL
流速：0.5mL/min
柱温：25℃
检测器：PDA检测器
检测波长：280nm
梯度：
时间(min) 流动相A 流动相B 流动相C 流动相D
0 60 0 5 35
15 0 50 5 45
20 0 50 5 45
20.1 60 0 5 35
35 60 0 5 35
5.1.2在下述的测定条件下进行HIC分析
HPLC仪器：Waters Acquity Arc
流动相A：2.5M硫酸铵+125mM PB，pH 6.8
流动相B：125mM PB，pH 6.8
流动相C：IPA
流动相D：水
分析柱：Tosoh,TSKgel Ether-5PW；7.5*75mm；2.5μm，PN 0008641
进样体积：50μL
流速：0.6mL/min
柱温：25℃
检测器：PDA检测器
检测波长：280nm
梯度：
时间(min) 流动相A 流动相B 流动相C 流动相D
0 60 0 5 35
30 0 50 5 45
33 0 50 5 45
33.1 60 0 5 35
45 60 0 5 35
5.2反相高效液相色谱法(RPLC)
HPLC分析用样品制备：将抗体药物偶联物(约1mg/mL，60μL)加入至二硫苏糖醇水溶液(100mM，15μL)中，混匀，37℃下孵育30分钟，用于HPLC分析。
DAR计算公式：
DAR＝2*(∑轻链加权峰面积+∑重链加权峰面积)/100.即：DAR＝2*(L0峰面积比*0+L1峰面积比*1+H0峰面积比*0+H1峰面积比*1+H2峰面积比*2+H3峰面积比*3)/100
5.2.1在下述的测定条件下进行RPLC分析
HPLC仪器：Waters Acquity Arc
流动相A：ACN+0.1％TFA
流动相B：H2O+0.1％TFA
分析柱：Agilent PLRP-S，2.1*50mm,8μM,
Figure PCTCN2022075689-appb-000053
PN PL1912-1802
进样体积：15μL
流速：0.25mL/min
柱温：80℃
检测器：PDA检测器
检测波长：280nm
梯度：
时间(min) ACN H2O
0 29 71
12.5 36 64
15 42 58
15.1 29 71
25 29 71
5.2.2在下述的测定条件下进行RPLC分析
HPLC仪器：Waters Acquity Arc
流动相A：ACN+0.1％TFA
流动相B：H2O+0.1％TFA
分析柱：Waters BioResolve RP mAb Polyphenyl，2.7μM，2.1*150mm，
Figure PCTCN2022075689-appb-000054
PN 186008946
进样体积：5μL
流速：0.25mL/min
柱温：80℃
检测器：PDA检测器
检测波长：280nm
梯度：
时间(min) ACN H2O
0 29 71
3 33 64
12.5 42 58
20 55 45
20.1 29 71
30 29 71
6.抗体药物偶联物分子大小异质性测定方法
在下述的测定条件下进行SEC分析：
HPLC分析用样品制备：以流动相为稀释液，将抗体药物偶联物稀释至2mg/ml，0.22μM滤头过滤，用于HPLC分析。
HPLC仪器：Waters Acquity Arc
流动相：100mM PB+200mM Arg·HCl+5％IPA，pH 6.8
分析柱：TOSOH TSKgel G3000 SWxl,7.8*300mm,5μM,
Figure PCTCN2022075689-appb-000055
PN0008541
进样体积：10μL
流速：0.6mL/min
柱温：30℃
检测器：PDA检测器
检测波长：280nm
梯度：等度洗脱
实施例组1药物接头的合成
实施例1苯并氮卓类TLR8激动剂-ADC接头的合成
Figure PCTCN2022075689-appb-000056
按照专利公开文件CN110612104A所述方法合成TLR8，其为白色固体，LC-MS(ESI):Pos(M+1)＝767。
实施例2环二核苷酸类STING激动剂-ADC接头的合成
Figure PCTCN2022075689-appb-000057
按照专利公开文件WO2018/100558A2所述方法合成STING，其为淡黄色固体，LC-MS(ESI):Pos(M+1)＝1164。
实施例组2抗体药物偶联物的制备
实施例1抗体药物偶联物1(8k13-hz35-TLR8-1103)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的2.0eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入TLR8的二甲基亚砜溶液(TLR8的用量为抗体的5.0eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
采用上文实验方法部分的“3.抗体药物偶联物的纯化”中所述操作，将 上述溶液纯化，得到含有标题抗体药物偶联物的溶液。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.2抗体-药物偶联物浓度测定方法-TLR8”中所述操作，测得溶液中抗体药物偶联物的浓度为6.3mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.2节中所述操作，测得药物平均连接数(DAR)为4.63(图4)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为98.72％(图5)。如图5所示，采用分子排阻色谱法分析分子大小异质性，除主峰外，高分子质量物质和抗体偶联药物中的碎片含量也能检测。
实施例2抗体药物偶联物2(8k13-hz35-STING-D4-1203)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的2.1eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入STING的二甲基亚砜溶液(STING用量为抗体的5.8eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.3抗体-药物偶联物浓度测定方法-STING”中所述操作，测得溶液中抗体药物偶联物的浓度为7.7mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.2.2节中所述操作，测得药物平均连接数(DAR)为4.02(图6)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为85.15％(图7)。
实施例3抗体药物偶联物3(8k13-hz35-M-20200318-1)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的2.2eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入M的二甲基亚砜溶液(M用量为抗体的5.5eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.4抗体-药物偶联物浓度测定方法-M”中所述操作，测得溶液中抗体药物偶联物的浓度为7.7mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.1节中所述操作，测得药物平均连接数(DAR)为4.04(图8)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为99.64％(图9)。
实施例4抗体药物偶联物4(8k13-hz35-EX-DAR4-20200922)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的2.1eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入EX的二甲基亚砜溶液(EX用量为抗体的5.2eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.1抗体-药物偶联物浓度测定方法-EX”中所述操作，测得溶液中抗体药物偶联物的浓度为7.7mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平 均连接数(DAR)的测定方法”第5.2.1节中所述操作，测得药物平均连接数(DAR)为3.53(图10)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为99.80％(图11)。
实施例5抗体药物偶联物5(8k13-hz35-EX-DAR8-20200421)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的10eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入EX的二甲基亚砜溶液(EX用量为抗体的11eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.1抗体-药物偶联物浓度测定方法-EX”中所述操作，测得溶液中抗体药物偶联物的浓度为7.7mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.1节中所述操作，测得药物平均连接数(DAR)为7.83(图12)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为98.81％(图13)。
实施例6抗体药物偶联物6(8k13-hz35-EX2-DAR4-20200922)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的2.2eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入EX2的二甲基亚砜溶液(EX用量为抗体的5.2eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.5抗体-药物偶联物浓度测定方法-EX2”中所述操作，测得溶液中抗体药物偶联物的浓度为4.5mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.2.2节中所述操作，测得药物平均连接数(DAR)为3.41(图14)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为99.85％(图15)。
实施例7抗体药物偶联物7(8k13-hz35-EX2-DAR8-20200922)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体8k13-hz35还原(TCEP用量为抗体的10.1eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入EX2的二甲基亚砜溶液(EX用量为抗体的12eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.5抗体-药物偶联物浓度测定方法-EX2”中所述操作，测得溶液中抗体药物偶联物的浓度为4.4mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.2.1节中所述操作，测得药物平均连接数(DAR)为7.12(图16)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为99.71％(图17)。
实施例8抗体药物偶联物8(16k15-hz35-M-20200318-1)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体16k15-hz35还原(TCEP用量为抗体的2.3eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入M的二甲基亚砜溶液(M用量为抗体的5.3eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.4抗体-药物偶联物浓度测定方法-M”中所述操作，测得溶液中抗体药物偶联物的浓度为7.7mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.1节中所述操作，测得药物平均连接数(DAR)为4.03(图18)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为99.18％(图19)。
实施例9抗体药物偶联物9(16k15-hz35-EX-20200421)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体16k15-hz35还原(TCEP用量为抗体的9.9eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入EX的二甲基亚砜溶液(EX用量为抗体的11.5eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.1抗体-药物偶联物浓度测定方法-EX”中所述操作，测得溶液中抗体药物偶联物的浓度为7.7mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.1节中所述操作，测得药物平均连接 数(DAR)为7.92(图20)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为98.39％(图21)。
实施例10抗体药物偶联物10(IMAB362-M-1)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体IMAB362还原(TCEP用量为抗体的2.2eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入M的二甲基亚砜溶液(M用量为抗体的5.2eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.4抗体-药物偶联物浓度测定方法-M”中所述操作，测得溶液中抗体药物偶联物的浓度为6.9mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.1节中所述操作，测得药物平均连接数(DAR)为3.28(图22)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为90.13％(图23)。
实施例11抗体药物偶联物11(IMAB362-M-2)
(1)抗体的还原
采用上文实验方法部分的“1.抗体的还原”中所述操作，将抗体IMAB362还原(TCEP用量为抗体的8eq)，得到还原后的抗体溶液。
(2)抗体与药物接头的偶联
采用上文实验方法部分的“2.抗体与药物接头的偶联”中所述操作，向所得还原液中加入M的二甲基亚砜溶液(M用量为抗体的10eq)，反应得到含有标题抗体药物偶联物的溶液。
(3)抗体药物偶联物的纯化
同实施例1。
(4)抗体药物偶联物分子特性评价
采用上文实验方法部分的“4.4抗体-药物偶联物浓度测定方法-M”中所述操作，测得溶液中抗体药物偶联物的浓度为6.1mg/mL。
采用上文实验方法部分的“5.抗体药物偶联物中每一分子抗体的药物平均连接数(DAR)的测定方法”第5.1.1节中所述操作，测得药物平均连接数(DAR)为3.31(图24)。
采用上文实验方法部分的“6.抗体药物偶联物分子大小异质性测定方法”中所述操作，测得主峰含量为92.16％(图25)。
根据本发明实施例组2抗体药物偶联物的制备中实施例10和11分子排阻色谱分析结果可知，相比8k13-hz35和16k15-hz35，对照抗体IMAB362偶联得到的两份ADC样品IMAB362-M-1和IMAB362-M-2，聚体含量高(检测到单体含量分别为90.13％和92.16％)；即，对照抗体IMAB362偶联得到ADC后，理化性质不稳定，易产生聚体，这可能带来各种安全性和有效性方面的问题。另外，疏水分析实验表明，对照抗体IMAB362偶联得到的两份ADC样品，不论加入低当量还原剂(IMAB362-1，抗体摩尔当量的2.2倍)或高当量还原剂(IMAB362-M-2，抗体摩尔当量的8倍)，制备得到ADC样品DAR均在3.3左右，且主要制备得到DAR2和DAR4组分，DAR6和DAR8组分很少，药物负载不成正态分布，这可能带来各种药效方面的问题。综合以上两点，对照抗体IMAB362不合适用于制备ADC。
实施例组3 ADC体外细胞活性测试
实施例1 ADC体外细胞活性测试1
使用CLDN18.2-高表达细胞系NCI-N87(NCI-N87 CLDN18.2)作为体外细胞活性实验模型，研究本发明提供的不同抗体药物偶联物对CLDN18.2高表达水平肿瘤细胞的杀伤作用。
实验步骤：根据如上实施例组2中实施例9样品制备方法，将抗体换成Trastuzumab制备得到Trastuzumab-EX(DAR＝8)样品。另外采用来自实施例组2中实施例4和5制备的抗体药物偶联物，作为测试药物。将处于对数生长期的上述细胞，分别以每孔5000个细胞的密度接种至96孔细胞培养板中，然后分别加入不同浓度的抗体药物偶联物，每个药物浓度设置2-4个复孔，及空白对照(仅细胞)，作用120h(根据细胞生长速度，保证细胞分裂足够 次数)，测定EC 50。
结果见表6。
表6.不同抗体药物偶联物对高表达CLDN18.2的NCI-N87细胞系的细胞杀伤效果
Figure PCTCN2022075689-appb-000058
实施例2 ADC体外细胞活性测试2
使用CLDN18.2-高表达细胞系NCI-N87(NCI-N87 CLDN18.2)和CLDN18.2-无表达细胞系NCI-N87(NCI-N87)作为体外细胞活性实验模型，研究本发明提供的不同抗体药物偶联物对CLDN18.2高表达及低表达水平肿瘤细胞的杀伤作用。
实验步骤：测试药物见表7，各个ADC均参照各自实施例制备，其中SAC为Sacituzumab，序列见上文；IL6为TocilizuMab，购自Target Molecule Corp。测试过程同实施例1。
对无CLDN18.2表达的NCI-N87细胞系的细胞杀伤效果见表7和图26。
表7.抗体药物偶联物对无CLDN18.2表达的NCI-N87细胞系的细胞杀伤效果
样品类型 样品名称 杀伤作用 EC50(pM) DAR
本发明的ADC-1 16k15-hz35-M - - 4.1
本发明的裸抗-1 16k15-hz35 - - N/A
本发明的ADC-2 8k13-hz35-M - - 4
本发明的裸抗-2 8k13-hz35 - - N/A
阳性对照ADC SAC-M + 134.8 4.5
阴性对照ADC IL-6-M - - N/A
小分子化药 MMAE + 1200 N/A
对CLDN18.2高表达的NCI-N87细胞系的细胞杀伤效果见表8和图27。
表8.抗体药物偶联物对CLDN18.2高表达的NCI-N87细胞系的细胞杀伤效果
样品类型 样品名称 杀伤作用 EC50(pM) DAR
本发明的ADC-1 16k15-hz35-M + 63.55 4.1
本发明的裸抗-1 16k15-hz35 - - N/A
本发明的ADC-2 8k13-hz35 + 61.58 4
本发明的裸抗-2 8k13-hz35 - - N/A
阳性对照ADC SAC-M + 64.25 4.5
阴性对照ADC IL-6-M - - N/A
小分子化药 MMAE + 209.7 N/A
如结果所示，两种细胞条件下，阳性对照ADC均有杀伤，阴性对照ADC均无杀伤，正负对照结果正常；小分子化药对两种细胞均有杀伤，但量效略有差异，半效杀伤浓度与文献报道一致。同时，两种裸抗分子(16k15-hz35和hz8k13-hz35)对两种细胞均无杀伤，而两种ADC分子(16k15-hz35-M和8k13-hz35-M)对NCI-N87均无杀伤，对NCI-N87 CLDN18.2均有杀伤，这说明NCI-N87 CLDN18.2细胞的杀伤依赖于抗原识别。
实施例3 ADC体外细胞活性测试3
采用本发明的抗体偶联TLR8激动剂得到的ADC并不能直接杀伤肿瘤细胞，而是通过抗体介导肿瘤微环境中的免疫细胞(主要是髓系抗原呈递细胞)吞噬肿瘤细胞后，在免疫细胞溶酶体处释放出TLR8激动剂，TLR8激动剂结合免疫细胞中的TLR8后可激活免疫细胞，提高促炎细胞因子分泌、招募免疫细胞、增强肿瘤新生抗原呈递、刺激肿瘤特异性T细胞生成，最终 达到杀灭肿瘤细胞的目的。
使用表达CLDN18.2的NCI-N87细胞系为靶细胞，与人外周血单核细胞(PBMC)共培养，加入一定量的本发明提供的抗体药物偶联物，测定细胞上清中促炎因子的表达情况。
实验步骤：使用新鲜分离的人PBMC或者冻存的PBMC，按照10～30万/孔的数量种入96孔细胞板，37℃，5％CO 2培养过夜。第二天按照1万/孔的数量种入NCI-N87 CLDN18.2细胞，然后再加入一定浓度的ADC(实施例组2的实施例1中制备的8k13-hz35-TLR8-1103)、裸抗(8k13-hz35)、阴性对照(NC IgG-TLR8，NC IgG是一种无关人源抗体)或TLR8激动剂(化合物TLR8)，共培养24-48h后，取细胞培养上清，ELISA测定上清中促炎细胞因子TNFα浓度。
结果见图28。如结果所示，本发明提供的ADC能明显刺激PBMC分泌促炎因子TNFα，裸抗和阴性对照无明显刺激效果，小分子药物也可以刺激PBMC分泌促炎因子，但强度明显低于本发明提供的ADC。说明将TLR8激动剂偶联在本发明的抗体上以后能通过抗体的靶向作用和Fc效应介导免疫细胞吞噬，将TLR8激动剂递送至靶标部位，激活免疫细胞。
实施例4 ADC体外细胞活性测试4
采用本发明的抗体偶联STING激动剂得到的ADC与表面表达CLDN18.2的肿瘤细胞(NCI-N87 CLDN18.2)孵育后，ADC可以通过靶点的内吞作用进入细胞内部，在细胞内部蛋白酶的作用下断裂linker，释放出STING激动剂。STING激动剂释放以后可以结合并激活STING受体，进而诱导TBK1(TANK binding kinase 1)磷酸化，激活IRF3(Interferon regulatory factor 3)信号通路和NF-κB信号通路，IRF3信号通路被激活后将会提高磷酸化TRF3(pIRF3)的表达量。
使用表达CLDN18.2的NCI-N87细胞系为靶细胞，加入一定量的本发明提供的抗体药物偶联物，通过测定pIRF3的表达量可以测定细胞的激活情况。
实验步骤：体外培养NCI-N87 CLDN18.2至对数生长期，按照20万/孔的数量种入24孔细胞板，培养基中不加血清做饥饿处理，37℃，5％CO 2培养过夜。第二天加入一定浓度的ADC(实施例组2的实施例2中制备的8k13-hz35-STING-D4-1203)、裸抗(8k13-hz35)、阴性对照(NC IgG-STING， NC IgG是一种无关人源抗体)或STING激动剂(化合物STING)，共培养约6h。培养结束后，收集细胞，加入含有蛋白酶抑制剂和磷酸酶抑制剂的细胞裂解液充分裂解细胞，离心收集细胞裂解后上清，通过Western-blotting分析总IRF3和pIRF3，pIRF3与总IRF3表达量的比值即为pIRF3的相对表达量。
结果见图29。如结果所示，本发明的ADC能明显刺激靶细胞中pIRF3表达，裸抗和阴性对照无明显刺激效果，小分子药物也可以刺激pIRF3，但强度明显低于本发明的ADC。说明将STING激动剂偶联在本发明的抗体上以后能通过抗体的靶向作用，将STING激动剂递送至靶标部位，激活STING信号通路。
实施例组4 ADC体内药效测试
实施例1 ADC体内药效测试
细胞：
人胃癌KATO III细胞购自中国科学院细胞库。使KATO III转染人CLDN18.2基因并高表达CLDN18.2蛋白，命名为KATO III/18.2。KATO III/18.2细胞用10cm培养皿培养，培养条件为RPMI 1640培养基中加10％胎牛血清以及青、链霉素，于37℃、含5％CO 2空气的培养箱中培养。一周2次传代，当细胞呈指数生长期时，收集细胞，计数，接种。
实验动物：
NOD-Scid小鼠，35日，♀，购自上海灵畅生物科技有限公司。许可证号：SCXK(沪)2018-0003。合格证编号20180003012511。饲养环境：SPF级。
实验步骤：
每只小鼠皮下接种1×10 7KATO III/18.2细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。具体给药剂量和给药方案见表9。每周测2次肿瘤体积，称小鼠体重，记录数据。
表9. 8k13-hz35-EX、8k13-hz35-EX2、对照抗体在人胃癌KATO III/18.2荷瘤小鼠的抗肿瘤作用试验设计
Figure PCTCN2022075689-appb-000059
Figure PCTCN2022075689-appb-000060
注：IV-静脉注射，给药开始于D0。
实验指标：实验指标为考察药物对肿瘤生长的影响，具体指标为T/C(％)或肿瘤生长抑制率％(TGI％)。
肿瘤体积(V)计算公式为V＝1/2×a×b 2(其中a、b分别表示长、宽)；T/C(％)＝(T-T 0)/(C-C 0)×100(其中T、C为实验结束时的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积)；肿瘤生长抑制率％(TGI％)＝100-T/C(％)；当肿瘤出现消退时，肿瘤生长抑制率％(TGI％)＝100-(T-T 0)/T 0×100；抑瘤率％＝(溶剂组肿瘤重量-治疗组肿瘤重量)/溶剂组肿瘤重量×100％。
如果肿瘤比起始体积缩小，即T<T0或C<C0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。实验结束、达到实验终点、或肿瘤体积达到1500mm 3，CO2麻醉处死动物，随后解剖取瘤并拍照。
结果见表10、表11以及图30至图34。
表10.CLAUDIXIMAB(IMAB362)、8k13-hz35-EX、8k13-hz35-EX2对人胃癌KATO III/18.2小鼠皮下移植瘤的疗效(根据肿瘤体积计算TGI％)
Figure PCTCN2022075689-appb-000061
Figure PCTCN2022075689-appb-000062
注：P值指与生理盐水组相比。
表11.CLAUDIXIMAB(IMAB362)、8k13-hz35-EX、8k13-hz35-EX2对人胃癌KATO III/18.2小鼠皮下移植瘤的疗效(根据肿瘤重量计算抑瘤率％)
Figure PCTCN2022075689-appb-000063
由实验结果可知，CLAUDIXIMAB(IMAB362)(6mg/kg,IV,D0)抑制人胃癌KATO III/18.2小鼠皮下移植瘤的生长，抑瘤率为63％。相比之下，8k13-hz35-EX(DAR8)(3mg/kg,IV,D0)对KATO III/18.2小鼠皮下移植瘤的抑瘤率为98％，有2/6小鼠肿瘤部分消退；8k13-hz35-EX(DAR4)(6mg/kg,IV,D0)对KATO III/18.2小鼠皮下移植瘤的抑瘤率为115％，有3/6小鼠肿瘤部分消退；8k13-hz35-EX2(DAR8)(3、6mg/kg,IV,D0)对KATO III/18.2小鼠皮下移植瘤的抑瘤率分别为74％和122％，分别有3/6和4/6小鼠肿瘤部分消退；8k13-hz35-EX2(DAR4)(3、6mg/kg,IV,D0)对KATO III/18.2小鼠皮下移植瘤的抑瘤率分别为63％和97％，6mg/kg剂量组1/6小鼠肿瘤部分消退(以上药物抑瘤率均根据肿瘤体积计算肿瘤生长抑制率％)。根据肿瘤重量计算抑瘤率得到相似的结果。荷瘤小鼠对以上药物均能很好耐受。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (12)

  1. 一种抗体药物偶联物，其包含与药物共价连接的抗CLDN18.2的抗体或其片段，其中所述抗CLDN18.2的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
    (i)氨基酸序列分别如SEQ ID NO:23、SEQ ID NO:26和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；
    (ii)氨基酸序列分别如SEQ ID NO:27、SEQ ID NO:28和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；
    (iii)氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:40所示的CDR-L1、CDR-L2和CDR-L3；或
    (iv)氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:43所示的CDR-L1、CDR-L2和CDR-L3。
  2. 根据权利要求1所述的抗体药物偶联物，其特征在于，所述抗CLDN18.2的抗体或其片段的重链包含重链可变区(VH)，所述重链可变区(VH)包含SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列或其变体；和/或，所述抗CLDN18.2的抗体或其片段的轻链包含轻链可变区(VL)，所述轻链可变区(VL)包含：选自SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列或其变体；
    或者，优选地，所述抗CLDN18.2的抗体或其片段的重链包含重链可变区(VH)，所述重链可变区(VH)包含SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列或其变体；和/或，所述抗CLDN18.2的抗体或其片段的轻链包含轻链可变区(VL)，所述轻链可变区(VL)包含选自SEQ ID NO:19或SEQ ID NO:22所示的氨基酸序列或其变体；
    更优选地，所述抗CLDN18.2的抗体或其片段的重链和轻链分别包含如 下所示的重链可变区(VH)和轻链可变区(VL)：
    (i)如SEQ ID NO:7所示的氨基酸序列或其变体；和，如SEQ ID NO:11所示的氨基酸序列或其变体；
    (ii)如SEQ ID NO:8所示的氨基酸序列或其变体；和，如SEQ ID NO:12所示的氨基酸序列或其变体；
    (iii)如SEQ ID NO:15所示的氨基酸序列或其变体；和，如SEQ ID NO:19所示的氨基酸序列或其变体；
    (iv)如SEQ ID NO:16所示的氨基酸序列或其变体；和，如SEQ ID NO:22所示的氨基酸序列或其变体。
  3. 根据权利要求1或2所述的抗体药物偶联物，其特征在于，所述抗CLDN18.2的抗体或其片段为针对CLDN18.2的单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式；或者，所述抗CLDN18.2的抗体或其片段为针对CLDN18.2的半抗体或半抗体的抗原结合片段；
    任选地，所述抗CLDN18.2的抗体或其片段为scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；
    优选地，所述抗体或其片段还包含人或鼠的恒定区，优选包含人或鼠的轻链恒定区(CL)和/或重链恒定区(CH)；
    更优选地，所述抗体或其抗原结合片段包含选自IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
  4. 根据权利要求1至3中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具有分子式Ab-[L-D]n，其中Ab表示抗CLDN18.2的抗体或其片段，L表示接头，D表示药物，n表示相对于每一分子Ab的药物平均连接数。
  5. 根据权利要求1至4中任一项所述的抗体药物偶联物，其特征在于，所述药物D为细胞毒性类小分子药物和/或免疫治疗剂类小分子药物；
    优选地，所述细胞毒性类小分子药物为微管蛋白抑制剂(如澳瑞他汀类化合物)和/或拓扑异构酶I抑制剂(如喜树碱类化合物)；
    优选地，所述免疫治疗剂类小分子药物为STING激动剂(如环二核苷酸类化合物)和/或TLR7/8激动剂(如咪唑并喹啉类化合物和/或苯并氮卓类化合物)；
    优选地，所述接头L为半胱氨酸偶联接头或赖氨酸偶联接头；
    优选地，所述接头L为可切割或不可切割的；
    优选地，n为1至12、优选1至8、更优选3至8。
  6. 权利要求1至5中任一项所述的抗体药物偶联物的制备方法，所述方法包括以下步骤：
    (1)用抗体与还原试剂在缓冲液中反应，得到经还原后的抗体；
    (2)用药物接头(连接子-药物缀合物)与步骤(1)得到的经还原后的抗体在缓冲液与有机溶剂的混合液中进行交联，得到抗体药物偶联物。
  7. 一种抗CLDN18.2的抗体或其片段，所述抗CLDN18.2的抗体或其片段包含重链和轻链，所述重链和轻链分别包含如下所示的重链互补决定区1至3(CDR-H1、CDR-H2和CDR-H3)和轻链互补决定区1至3(CDR-L1、CDR-L2和CDR-L3)：
    (A)氨基酸序列分别如SEQ ID NO:27、SEQ ID NO:28和SEQ ID NO:25所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:32、SEQ ID NO:30和SEQ ID NO:33所示的CDR-L1、CDR-L2和CDR-L3；或
    (B)氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:36所示的CDR-H1、CDR-H2和CDR-H3；和，氨基酸序列分别如SEQ ID NO:41、SEQ ID NO:30和SEQ ID NO:43所示的CDR-L1、CDR-L2和CDR-L3。
  8. 根据权利要求7所述的抗CLDN18.2的抗体或其片段，其特征在于，所述抗CLDN18.2的抗体或其片段的重链包含重链可变区(VH)，轻链包含轻链可变区(VL)，所述重链可变区(VH)和轻链可变区(VL)分别包含SEQ ID NO:8所示的氨基酸序列或其变体和SEQ ID NO:12所示的氨基酸序列或其变体；或者，所述重链可变区(VH)和轻链可变区(VL)分别包含SEQ ID NO:16所示的氨基酸序列或其变体和SEQ ID NO:22所示的氨基酸序列或其变体。
  9. 一种药物组合物，其包含权利要求1至5中任一项所述的抗体药物偶联物或者权利要求7至8中任一项所述的抗CLDN18.2的抗体或其片段。
  10. 一种药物组合物，其包含抗体药物偶联物混合物和药学上可接受的载体，所述抗体药物偶联物混合物包含多种如权利要求1至5中任一项所述的抗体药物偶联物；
    任选地，所述抗体药物偶联物混合物的平均n值为2至8；
    任选地，所述抗体药物偶联物混合物包含多种所述抗体药物偶联物，每 种所述抗体药物偶联物的n值为1至12、优选1至8、更优选3至8。
  11. 权利要求1至5中任一项所述的抗体药物偶联物、权利要求7至8中任一项所述的抗CLDN18.2的抗体或其片段或者权利要求9至10中任一项所述的药物组合物在制备药物中的用途，所述药物用于治疗与CLDN18.2相关的疾病，优选为肿瘤，更优选为癌症。
  12. 一种治疗疾病的方法，所述方法包括向有此需要的受试者施用权利要求1至5中任一项所述的抗体药物偶联物、权利要求7至8中任一项所述的抗CLDN18.2的抗体或其片段或者权利要求9至10中任一项所述的药物组合物，其中所述受试者为哺乳类动物，优选地为人；所述疾病为与CLDN18.2相关的疾病，优选为肿瘤，更优选为癌症。
PCT/CN2022/075689 2021-02-09 2022-02-09 包含抗cldn18.2的抗体或其抗原结合片段的抗体药物偶联物及其用途 WO2022171134A1 (zh)

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WO2024114667A1 (zh) * 2022-11-30 2024-06-06 正大天晴药业集团股份有限公司 抗cldn18.2抗体药物偶联物及其药物组合物和用途
CN117327182B (zh) * 2023-09-19 2024-06-04 上海交通大学医学院附属仁济医院 Cldn18.2单域抗体探针的制备方法及应用

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CN111110862A (zh) * 2018-11-01 2020-05-08 上海健信生物医药科技有限公司 抗cldn18．2抗体的药物偶联体及其制备方法和用途
CN112574307A (zh) * 2019-09-29 2021-03-30 迈威(上海)生物科技股份有限公司 抗人Claudin18.2抗体及其应用

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