WO2024017383A1 - 抗mct1抗体及其用途 - Google Patents

抗mct1抗体及其用途 Download PDF

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Publication number
WO2024017383A1
WO2024017383A1 PCT/CN2023/108737 CN2023108737W WO2024017383A1 WO 2024017383 A1 WO2024017383 A1 WO 2024017383A1 CN 2023108737 W CN2023108737 W CN 2023108737W WO 2024017383 A1 WO2024017383 A1 WO 2024017383A1
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antibody
variable region
seq
chain variable
heavy chain
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PCT/CN2023/108737
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English (en)
French (fr)
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蔡晓龙
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佰舟生物科技(苏州)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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of antibodies. Specifically, it relates to an anti-MCT1 antibody and its use.
  • MCTs Monocarboxylate transporters
  • SLC16A transporter family contains 14 family members in mammals.
  • MCT1-4 transport monocarboxylic acids, such as pyruvate, lactate and ketones, through hydrogen ion coupling.
  • MCT1 (alias SLC16A1) can bidirectionally transport monocarboxylic acids across the cell membrane. The transport direction depends on the relative concentration gradient of the substrate on both sides of the cell membrane.
  • MCT1 has high affinity for lactic acid and is expressed in almost all cell types.
  • MCT1 is considered to be the main pathway for lactate uptake by malignant cells that use lactate as metabolic fuel. High expression of MCT1 in cancer tissues is often associated with poor clinical prognosis in a variety of cancers, including gynecological, esophageal, gastrointestinal and other solid tumors.
  • MCT1 plays an important role in the transmembrane transport of lactic acid in tumor cells and in maintaining normal intracellular pH levels. In a series of in vitro and in vivo tumor models, blocking MCT1 has been shown to effectively inhibit tumor growth and induce tumor cell death.
  • the prior art has reported a new mechanism by which MCT1 blockers inhibit tumors by regulating tumor immune responses, which is related to regulating the function of Treg cells in the tumor microenvironment: Treg cells in the tumor microenvironment actively absorb lactic acid through MCT1 as a Metabolize substrates, thereby further maintaining the immunosuppressive function and high expression of PD-1; experiments show that the expression and function of MCT1 in Treg cells play an important role in blocking the therapeutic efficacy of PD-1 antibodies; MCT1 small molecule inhibitors can interact with PD- 1 Antibodies produce synergistic anti-tumor effects and demonstrate efficacy in multiple mouse tumor models in vivo.
  • MCT1-mediated lactate transport and thereby blocking glycolysis is also effective in treating autoimmune diseases.
  • Blocking MCT1 through small molecule inhibitors of MCT1 can inhibit the activity of effector T cells, thereby showing therapeutic efficacy in animal models of various autoimmune diseases, including collagen-induced arthritis, allograft rejection, etc. Model.
  • MCT1 inhibitors may have therapeutic effect on tumors and autoimmune diseases.
  • MCTs are widely expressed in various tissues, non-selective small molecule inhibitors of MCT1 will bring drug safety concerns.
  • Several existing small molecule inhibitors such as AZD3965 and AR-C117977 simultaneously inhibit MCT1, MCT2 and to a certain extent MCT3. Although they have all demonstrated efficacy in autoimmune disease models and tumor models, in preclinical and clinical studies of AZD3965, it was found that the compound's non-selectivity resulted in retinal and cardiac toxic effects, thus limiting the use of this compound. further development.
  • Neutralizing antibodies have a huge advantage in selectivity over small molecule inhibitors.
  • the performance and quantity of MCT1 antibodies that have been developed so far are still unsatisfactory. Therefore, there is a need in this field to develop a new anti-MCT1 antibody with high neutralizing activity and high selectivity to meet clinical needs.
  • the object of the present invention is to provide an anti-MCT1 antibody and its use.
  • an antibody heavy chain variable region includes the following three complementarity determining regions CDR (according to the Kabat numbering system):
  • the heavy chain variable region includes the following three complementarity determining regions CDR (according to the AbM numbering system):
  • the heavy chain variable region includes the following three complementarity determining regions CDR (according to the IMGT numbering system):
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining MCT1 binding affinity.
  • the heavy chain variable region includes the following complementarity determining regions: the heavy chain complementarity determining region HCDR1 of S15-C9 shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, HCDR2, HCDR3; or
  • the heavy chain variable region further includes a human FR region or a murine FR region.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 7.
  • the heavy chain variable region has the amino acid sequences shown in SEQ ID NO. 9, 10, 11, and 12.
  • amino acid sequence of the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • a heavy chain of an antibody said heavy chain having a heavy chain variable region as described in the first aspect of the present invention.
  • the heavy chain of the antibody further includes a heavy chain constant region.
  • the heavy chain constant region is of human, mouse or rabbit origin.
  • a light chain variable region of an antibody comprising the following three complementarity determining regions CDR (according to Kabat numbering system or AbM numbering system):
  • LCDR3 shown in SEQ ID NO.6, or the light chain variable region includes the following three complementarity determining regions CDR (according to the IMGT numbering system):
  • the sequence is LCDR2 as shown in STS,
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining MCT1 binding affinity.
  • the light chain variable region further includes a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO.: 8.
  • the light chain variable region has the amino acid sequences shown in SEQ ID NO.: 13, SEQ ID NO.: 14, and SEQ ID NO.: 15.
  • the amino acid sequence of the light chain variable region is at least 80%, 85%, 90%, 91%, 92% identical to the amino acid sequence shown in SEQ ID NO.: 8, 13, 14, and 15. %, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • a light chain of an antibody said light chain having a light chain variable region as described in the third aspect of the present invention.
  • the light chain of the antibody further includes a light chain constant region.
  • the light chain constant region is of human, mouse or rabbit origin.
  • an antibody having:
  • the antibody has: a heavy chain as described in the second aspect of the present invention; and/or a light chain as described in the fourth aspect of the present invention, wherein any one of the above amino acid sequences further includes any Derivative sequences that retain MCT1 binding affinity, optionally through the addition, deletion, modification and/or substitution of at least one amino acid.
  • the antibody is a monomer, a bivalent antibody, and/or a multivalent antibody.
  • the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor antibody (CAR).
  • CAR chimeric antigen receptor antibody
  • the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or combinations thereof.
  • the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes.
  • the animal is a non-human mammal, preferably a rat, a sheep, or a rabbit.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the number of added, deleted, modified and/or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.
  • the number of added, deleted, modified and/or substituted amino acids is 1-7, preferably 1-3, more preferably 1.
  • the at least one amino acid sequence that has been added, deleted, modified and/or substituted is an amino acid sequence with at least 80% homology.
  • the derived sequence with addition, deletion, modification and/or substitution of at least one amino acid has the catalytic function of inhibiting cell surface MCT1 or recombinant MCT1 protease.
  • the MCT1 is derived from mammals, preferably humans, mice, rats, cynomolgus monkeys, and rabbits.
  • the affinity EC 50 of the antibody for MCT1 is 0.05-10nM, preferably 0.1-6nM, more preferably 0.5-4nM; and, for MCT2, MCT3 or MCT4 (e.g. human MCT2, MCT3 or MCT4) each independently have an affinity EC50 >100 nM, preferably >1000 nM, more preferably >10000 nM.
  • the antibody binds to human MCT1 and blocks or inhibits the activity of MCT1, preferably with one or more properties selected from the following group:
  • T effector cells such as CD3+, CD4+ or CD8+ T effector cells
  • Treg regulatory T
  • Type 1 regulatory cells or Tr1 or TR1 type I regulatory T cells
  • a multispecific antibody comprising a first antigen-binding region, said first antigen-binding region comprising a heavy protein as described in the first aspect of the present invention. chain variable region; and a light chain variable region as described in the third aspect of the invention.
  • the multispecific antibody further includes a second antigen-binding region targeting a target selected from the following group: EGFR, TGF ⁇ , BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20 , CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7 , CCR8, CD47, CD39, CD73, CD147, Claudin 18.2, or combinations thereof.
  • a target selected from the following group: EGFR, TGF ⁇ , BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20 , CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP
  • the multispecific antibody further includes the Fc segment of the antibody.
  • the antigen-binding region is an antibody or an antibody fragment
  • the antibody fragment includes: (i) Fab fragment; (ii) F(ab') 2 fragment; (iii) Fd fragment; (iv) Fv fragment; (v) single chain Fv (scFv) molecule; (vi) dAb fragment.
  • a recombinant protein having:
  • Optional functional domains that improve protein physicochemical properties or druggability preferably polypeptides selected from the following group: antibody polypeptides or antibody domains (including Fc polypeptides or fragments thereof, for example, human IgG1, IgG2, IgG3 or IgG4 Fc region or fragment thereof), serum albumin, human or other primate serum albumin, monomer (also known as adnectin), affibody, designed ankyrin repeats protein (DARPin), anti-carrying protein anticalin, ethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), XTEN molecules, rPEG molecules, or fragments or variants of any of the foregoing.
  • antibody polypeptides or antibody domains including Fc polypeptides or fragments thereof, for example, human IgG1, IgG2, IgG3 or IgG4 Fc region or fragment thereof
  • serum albumin human or other primate serum albumin
  • monomer also known as adnect
  • the recombinant protein also includes
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, dimer, or multimer.
  • a CAR construct is provided, the scFv segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to MCT1, and the scFv has the characteristics of the present invention.
  • a recombinant immune cell is provided, the immune cell expressing an exogenous CAR construct as described in the eighth aspect of the present invention.
  • the immune cells are selected from the following group: NK cells and T cells.
  • the immune cells are from humans or non-human mammals (such as mice).
  • an antibody drug conjugate is provided, and the antibody drug conjugate contains:
  • an antibody portion selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a heavy chain as described in the third aspect of the invention
  • the light chain variable region described in the aspect, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, or a combination thereof and (b) coupled to the antibody portion
  • the coupling moiety is selected from the group consisting of a detectable label, a cytotoxic drug, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • subscript p is a value selected from 1-10, preferably 1-8.
  • the detectable label includes a radionuclide
  • the radionuclide includes:
  • Diagnostic isotope the said diagnostic isotope is selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and/or
  • Therapeutic isotope the therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or combination thereof.
  • the coupling part (D) is a cytotoxic drug
  • the cytotoxic drug is: a microtubule-targeting drug and/or a DNA-targeting drug and/or a topoisomerase inhibitor. agent. .
  • the microtubule targeting drug is selected from the following group: monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine derivatives DM1 and tubulysin .
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • maytansine derivatives DM1 and tubulysin maytansine derivatives DM1 and tubulysin .
  • an active ingredient selected from the group consisting of: a heavy chain variable region as described in the first aspect of the invention, a heavy chain variable region as described in the second aspect of the invention
  • Conjugates, or combinations thereof, the active ingredients are used for (a) preparing detection reagents, detection plates or kits; and/or (b) preparing drugs for preventing and/or treating MCT1-related diseases.
  • the detection reagent, detection plate or kit is used for:
  • the detection reagent, detection plate or kit is used to diagnose MCT1-related diseases.
  • the drug is used to treat or prevent tumors with high MCT1 expression.
  • the medicine is used for a purpose selected from the following group:
  • the MCT1-related disease is selected from the group consisting of cancer, autoimmune disease, metabolism-related disease, inflammation, graft-versus-host disease (GVHD), or a combination thereof.
  • the cancer includes solid tumors and blood cancers.
  • the cancer is a tumor with high expression of MCT1.
  • the tumor with high MCT1 expression is selected from the following group: breast cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, prostate cancer, rectal cancer, gastric cancer, liver cancer, lymphoma, renal cell carcinoma Carcinoma, glioma, melanoma, leukemia, lymphoma, head and neck squamous cell carcinoma, bladder cancer, or combinations thereof.
  • the tumor with high MCT1 expression refers to the ratio of MCT1 transcript and/or protein level L1 in tumor tissue to the transcript and/or protein level L0 in normal tissue, L1/L0 ⁇ 2 , preferably ⁇ 3.
  • the autoimmune diseases include: systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, ankylosing spondylitis, asthma, arteriosclerosis, Atherosclerosis, colitis, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter syndrome, psoriatic arthropathy, infectious arthritis, tuberculous arthritis, viral arthritis, fungal Arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, graft versus host disease (GvHD).
  • GvHD graft versus host disease
  • the metabolism-related diseases include: diabetes, food-borne obesity, fat inflammation, and gout.
  • the inflammation includes: chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, reperfusion injury, sarcoidosis, vasculitis, interstitial cystitis, norcomplementemic urticaria measles Vasculitis, pericarditis, myositis, gout, TNF receptor-associated periodic syndrome (TRAPSP), gingivitis, periodontitis, hepatitis, cirrhosis, pancreatitis, myocarditis, vasculitis, gastritis, gout, gouty joints inflammation, and inflammatory skin diseases such as psoriasis, atopic dermatitis, eczema, rosacea, urticaria, and acne.
  • TRAPSP TNF receptor-associated periodic syndrome
  • a pharmaceutical composition which pharmaceutical composition contains:
  • an active ingredient selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a heavy chain as described in the third aspect of the invention
  • the pharmaceutical composition also includes: other biologically active substances, such as drugs for treating tumors (such as PD-1 antibodies, TIGIT antibodies), mitochondrial inhibitors and/or biguanides and/or another A monocarboxylic acid transporter (MCT inhibitor).
  • drugs for treating tumors such as PD-1 antibodies, TIGIT antibodies
  • mitochondrial inhibitors and/or biguanides such as PD-1 antibodies, TIGIT antibodies
  • MCT inhibitor another A monocarboxylic acid transporter
  • the MCT inhibitor is selected from one or more inhibitors that inhibit MCT transporters (such as MCT1, MCT2, MCT3, MCT4, MCT5, MCT6, MCT7, MCT8, MCT9 or MCT10), so
  • the inhibitors optionally also include small molecules (eg, AZD3965 or AR-C117977), RNAi, antibodies, antibody fragments, or fusion proteins.
  • the pharmaceutical composition includes single drugs, compound drugs, or synergistic drugs.
  • the administration method of the pharmaceutical composition is selected from the following group: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spray and mist Inhaled.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • a polynucleotide encoding a polypeptide selected from the group consisting of:
  • the polynucleotide is DNA, linear or circular mRNA.
  • a vector is provided, said vector containing the polynucleotide according to the thirteenth aspect of the present invention.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, linear or circular mRNA, or other vectors.
  • a genetically engineered host cell contains the vector according to the fourteenth aspect of the present invention or the genome of the thirteenth aspect of the present invention is integrated therein. the polynucleotide.
  • a method for in vitro detection of MCT1 in (including diagnostic or non-diagnostic) samples comprising the steps:
  • a detection plate in the seventeenth aspect of the present invention, includes: a substrate (support plate) and a test strip.
  • the test strip contains the antibody according to the fifth aspect of the invention. Or the antibody drug conjugate according to the tenth aspect of the present invention.
  • a kit which includes:
  • a first container containing the antibody according to the fifth aspect of the present invention and/or
  • the kit contains the detection plate according to the seventeenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a method for treating MCT1-related diseases comprising: administering to a subject in need an antibody as described in the fifth aspect of the present invention, or as described in the tenth aspect of the present invention.
  • the method further includes: administering other drugs or treatment methods to the subject in need for combined treatment.
  • the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.
  • the anti-tumor immunotherapy drugs include PD-1 and PD-L1 monoclonal antibodies.
  • Figure 1 shows the identification of MCT1KO cell lines.
  • 293-wt Wild-type 293T cells.
  • 293-KO MCT1 knockout 293T cells.
  • Human IgG1 isotype Isotype control for MCT1 antibodies.
  • Figure 2 shows the binding ability of S15-C9 to human MCT1, human MCT2, human MCT3, human MCT4, mouse MCT1, rat MCT1 and cynomolgus monkey (Cyno) MCT1.
  • 293-wt is wild-type HEK293T cells expressing MCT1.
  • Figure 3 shows the binding ability of S15-C9 chimeric antibody S15-C9-hIgG1 and PTM mutant antibody S15-C9-hIgG1 PTM-1 to human MCT1.
  • Benchmark is the same anti-MCT1 control antibody.
  • Figure 4 shows the binding ability of S15-C9 chimeric antibody S15-C9-hIgG1 and PTM mutant antibody S15-C9-hIgG1 PTM-1 to cynomolgus monkey (Cyno) MCT1.
  • the Benchmark is the same anti-MCT1 control antibody. body.
  • Figure 5 shows the binding ability of S15-C9 chimeric antibody S15-C9-hIgG1 and PTM mutant antibody S15-C9-hIgG1 PTM-1 to human MCT2, 3, and 4.
  • Figure 6 shows that MCT1 knockdown significantly improves the tolerance of HEK293 cells to 3-BP cytotoxicity.
  • Figure 7 shows the treatment of mouse parental antibody S15-C9, human-mouse chimeric antibody S15-C9-hIgG1, and PTM mutant antibody S15-C9-PTM1 and Benchmark antibody to block the cytotoxicity caused by 3-BP.
  • the MCT1 antibody is effective against different subtypes of MCT. It has high selectivity. It binds to MCT1 with high affinity and hardly binds to other different subtypes of MCT (MCT2, MCT3, MCT4). The EC 50 difference is more than 100 times.
  • the MCT1 antibody of the present invention can efficiently block the function of MCT1.
  • the humanized antibodies of the present invention have high activity and selectivity similar to murine antibodies, and have lower immunogenicity. On this basis, the present invention was completed.
  • Monocarboxylate transporter also known as SLC16A transporter family (SLC16A, solution carrier family 16 member), contains 14 family members in mammals. Four of them (MCT1-4) transport monocarboxylic acids, such as pyruvate, lactate and ketones, through hydrogen ion coupling.
  • MCT1 is a proton-coupled monocarboxylate transporter.
  • MCT1 is a multiple-transmembrane protein responsible for the facilitated transport of key metabolites, including glycolysis products. It catalyzes many monocarboxylic acid compounds such as lactic acid, pyruvate, branched oxyacids derived from leucine, valine and isoleucine, and the ketone bodies acetoacetate, ⁇ -hydroxybutyrate, acetic acid, Rapid transport across the plasma membrane.
  • MCT1 mediates the import or export of lactate and ketone bodies.
  • MCT1 is one of the members of the largest family of surface membrane proteins called solute channel proteins (SLCs), whose functions are involved in the transmembrane transport of key cellular nutrients, metabolites, ions, hormones and lipids.
  • SLCs solute channel proteins
  • MCT1 belongs to the SLC16 transporter family, five of which have been shown to transport monocarboxylic acids, such as pyruvate, lactate, and ketones, in a pH-dependent and bidirectional facilitated manner.
  • MCT1 may also be referred to by any of the following names: monocarboxylate transporter 1, SLC16A1, HHF7, MCT, MCT1, MCT1D, solute carrier family 16 member 1.
  • SLC16A1 gene sequence: NM_001166496.2, protein sequence: NP_001159968.1.
  • High expression of MCT1 in cancer tissues is often associated with poor clinical prognosis in a variety of cancers, including gynecological, esophageal, gastrointestinal and other solid tumors.
  • MCT2 is a proton-coupled monocarboxylate transporter. It catalyzes many monocarboxylic acid compounds, such as lactic acid, pyruvate, branched-chain oxyacids derived from leucine, valine, and isoleucine, and the ketone bodies acetoacetate, beta-hydroxybutyrate, and acetic acid, Rapidly transported across the plasma membrane. It also functions as a high-affinity pyruvate transporter. MCT2 may also be referred to by any of the following names: monocarboxylate transporter 2, SLC16A7, MCT2, solute carrier family 16 member 7. In humans, it is encoded by the SLC16A7 gene (NM_001270622.2).
  • MCT3 is a proton-coupled monocarboxylate transporter. It catalyzes many monocarboxylic acid compounds, such as lactic acid, pyruvate, branched-chain oxyacids derived from leucine, valine, and isoleucine, and the ketone bodies acetoacetate, ⁇ -hydroxybutyrate, and acetate, across Rapid transport across the plasma membrane. It also functions as a high-affinity pyruvate transporter. MCT3 expression is restricted to the retinal pigment epithelium and choroid plexus epithelium, which are located on the basal membrane, whereas MCT1 is located on the apical membrane.
  • MCT3 may also be referred to by any of the following names: monocarboxylate transporter 3, SLC16A8, MCT3, REMP, solute carrier family 16 member 8. In humans, it is encoded by the SLC16A8 gene (NM_001394131.1).
  • MCT4 is a proton-coupled monocarboxylate transporter.
  • MCT4 may also be referred to by any of the following names: monocarboxylate transporter 4, SLC16A3, MCT 3, MCT 4, MCT-3, MCT-4, MCT3, MCT4, solute carrier family 16 member 3. In humans, it is encoded by the SLC16A3 gene (NM_001042422.3).
  • antibody or "immunoglobulin” is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains.
  • VH variable domain
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. .
  • Special amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not uniform distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial ⁇ -sheet structure can be formed.
  • the CDRs in each chain are held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be assigned to one of two distinct classes (termed kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant region. There are 5 main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • variable regions which are separated into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a cyclic structure, and the ⁇ -sheets formed by the FRs between them are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. You can determine which amino acids constitute the FR or CDR region by comparing the amino acid sequences of antibodies of the same type.
  • FR framework region
  • the light chain and heavy chain of immunoglobulins each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, and FR4-H respectively.
  • the light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain may thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof.
  • the derivative of the human antibody FR is essentially the same as the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, or 96%. , 97%, 98% or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to that of a naturally occurring human antibody. .
  • HCDR1 and CDR-H1 are used interchangeably and both refer to the CDR1 of the heavy chain variable region;
  • HCDR2 and “CDR-H2” are used interchangeably and both refer to the heavy chain variable region.
  • HCDR3 and “CDR-H3” are used interchangeably, both referring to CDR3 of the heavy chain variable region.
  • LCDR1 and CDR-L1 are used interchangeably, both referring to CDR1 of the light chain variable region;
  • LCDR2 and “CDR-L2” are used interchangeably, both referring to CDR2 of the light chain variable region;
  • LCDR3 and “CDR-L3” are used interchangeably, both referring to CDR3 of the light chain variable region.
  • Kabat numbering system As used herein, “Kabat numbering system”, “IMGT numbering system”, “AbM numbering system”, “Chothia numbering system” refer to the determination of complementary binding determining region CDRs under different assignment systems.
  • the assignment system can readily determine the precise amino acid sequence boundaries of a given CDR or FR using a number of numbering systems well known in the art, including: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed.
  • the boundaries of a given CDR or FR may differ depending on the protocol used for identification.
  • the Kabat scheme is based on structural alignment
  • the Chothia scheme is based on structural information.
  • the numbering in both the Kabat and Chothia schemes is based on the sequence length of the most common antibody regions, where insertions are provided by intervening letters (e.g. "30a") and deletions occur in some antibodies.
  • the two schemes place certain insertions and deletions ("indels") in different locations, resulting in different numbering.
  • Contact scheme is based on complex Analysis of the crystal structure and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used by Oxford Molecular's AbM antibody modeling software.
  • CDR of a given antibody or region thereof (such as a variable region thereof) encompasses CDRs defined by any of the above schemes or other known schemes.
  • CDR CDR3
  • FRs of a given antibody or region thereof encompass FRs defined by any of the above schemes or other known schemes.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • the antibody in the present invention can be a full-length protein (such as IgG1, IgG2a, IgG2b or IgG2c), or a protein fragment containing an antigen-antibody binding domain or an antigen-binding fragment (such as Fab, F(ab'), sdAb, scFv fragment ).
  • a full-length protein such as IgG1, IgG2a, IgG2b or IgG2c
  • a protein fragment containing an antigen-antibody binding domain or an antigen-binding fragment such as Fab, F(ab'), sdAb, scFv fragment.
  • antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv ( scFv) molecule; (vi) dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., independent complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3 -CDR3-FR4 peptide.
  • CDRs independent complementarity determining regions
  • the antibody or antigen-binding fragment thereof of the present invention is selected from the following group: camelid single domain antibody, scFv, scFv dimer, BsFv, dsFv, dsFv2, dsFv-dsFv', Fv fragment, Fab, Fab ', F(ab') 2 , ds bifunctional antibody, Nanobody, domain antibody or bivalent domain antibody.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard recombinant DNA techniques and are useful antibodies.
  • a chimeric antibody is a molecule in which different parts come from different animal species, for example a chimeric antibody with a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin.
  • Humanized antibodies refer to antibody molecules derived from non-human species, with one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules.
  • CDRs complementarity determining regions
  • antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes conservative variants thereof, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, optimally at most Three amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • Recombinant protein fusion protein
  • the invention also provides a recombinant protein, which includes one or more of the heavy chain CDR1, heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3) of the anti-MCT1 antibody (S15-C9), and/or, MCT1 One or more of the light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) of the antibody,
  • the sequence of the heavy chain HCDR1-3 is as follows:
  • the sequence of the light chain LCDR1-3 is as follows:
  • ASVFSRSWIN SEQ ID NO.16
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining MCT1 binding affinity.
  • DG ⁇ DA point mutation
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology or sequence identity of at least 80%, preferably at least 85%, and more preferably be at least 90%, preferably at least 95% of the amino acid sequence.
  • the recombinant protein of the present invention includes the heavy chain variable region of the MCT1 murine antibody and/or the light chain variable region of the MCT1 antibody, and the heavy chain variable region of the antibody contains SEQ ID NO.
  • the amino acid sequence shown in .7; the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.:8.
  • the recombinant protein of the present invention includes the heavy chain variable region of the MCT1 humanized antibody and the light chain variable region of the MCT1 antibody, and the heavy chain variable region of the antibody contains SEQ ID NO. 9, 10, 11, and 12; the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO.: 13, 14, or 15.
  • any one of the above amino acid sequences also includes a derivative sequence optionally added, deleted, modified and/or substituted at least one amino acid, and capable of retaining MCT1 binding affinity.
  • the recombinant protein also includes an antibody heavy chain constant region and/or an antibody light chain constant region.
  • the antibody heavy chain constant region is conventional in the art, and is preferably a murine antibody heavy chain constant region or A human antibody heavy chain constant region, more preferably a human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in this field, and is preferably a murine light chain antibody constant region or a human antibody light chain constant region, and is more preferably a human antibody light chain constant region.
  • the recombinant protein is a conventional protein in this field.
  • it is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv). ), one or more of single domain antibody (single domain antibody, sdAb) and single region antibody (Signle-domain antibody), as well as monoclonal antibodies or polyclonal antibodies prepared from the above antibodies.
  • the monoclonal antibodies can be developed through a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the full-length antibody protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region.
  • the heavy chain variable region and light chain variable region of the protein together with the human heavy chain constant region and the human light chain constant region constitute a fully human antibody full-length protein.
  • the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody is a conventional single-chain antibody in this field, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, and includes the light chain variable region, the light chain constant region and the Fd segment of the heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab').
  • the present invention also provides a nucleic acid (including DNA, linear or circular mRNA) encoding the above-mentioned antibody (such as an anti-MCT1 antibody) or a recombinant protein or the heavy chain variable region or the light chain variable region of an anti-MCT1 antibody. .
  • the preparation method of the nucleic acid is a conventional preparation method in the field, and preferably includes the following steps: obtaining the nucleic acid molecule encoding the above protein through gene cloning technology, or obtaining the nucleic acid molecule encoding the above protein through artificial full sequence synthesis. .
  • the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitutions, deletions, changes, insertions or additions to provide a polynucleotide homologue.
  • Homologs of the polynucleotides of the present invention can be prepared by substituting, deleting or adding one or more bases in the gene encoding the protein sequence within the range of maintaining antibody activity.
  • the invention also provides a recombinant expression vector containing the nucleic acid.
  • the recombinant expression vector can be obtained by conventional methods in the art, that is, it is constructed by connecting the nucleic acid molecule of the present invention to various expression vectors.
  • the expression vector is a variety of conventional vectors in the art, as long as it can accommodate the aforementioned nucleic acid molecules.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, etc.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a conventional preparation method in this field, preferably: it is prepared by transforming the above recombinant expression vector into a host cell.
  • the host cells are various conventional host cells in the art, as long as the recombinant expression vector can stably replicate itself and the nucleic acid carried can be effectively expressed.
  • the host cell is E.coli TG1 or E.coli BL21 cells (expressing single chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies).
  • the preferred recombinant expression transformant of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the transformation method is a conventional transformation method in this field, preferably a chemical transformation method, a heat shock method or an electroporation method.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genome library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form single-chain antibodies.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the DNA sequence encoding the antibody (or fragment thereof, or derivative thereof) of the present invention can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art.
  • the antibody of the present invention can be purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassays (RIA) or enzyme-linked immunosorbent assays. (ELISA)) to determine.
  • the binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the antibody of the present invention can be expressed within the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, infiltration sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • ADCs Antibody-drug conjugates
  • the invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the invention.
  • ADCs antibody-drug conjugates
  • the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule may be one or more of toxic proteins, chemotherapeutic drugs, small molecule drugs or radionuclides.
  • the antibody of the present invention and the effector molecule can be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds.
  • the non-selective coupling agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc.
  • the coupling agent utilizing carboxyl groups may be any one or more of aconitic anhydride coupling agents (such as aconitic anhydride) and acyl hydrazone coupling agents (the coupling site is an acyl hydrazone).
  • antibodies are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (e.g. glycol polymers) and therapeutic agents.
  • imaging reagents such as chromophores and fluorescent groups
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers e.g. glycol polymers
  • therapeutic agents e.g. glycol polymers
  • Antibodies can be coupled to functional agents to form antibody-functional agent conjugates.
  • Functional agents eg drugs, detection reagents, stabilizers
  • the functional agent can be linked to the antibody directly or indirectly through a linker.
  • Antibodies can be conjugated with drugs to form antibody drug conjugates (ADCs).
  • ADCs contain a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers.
  • Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g. at the target site, This releases the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases, such as lysosomal or endosomal proteases, or sugar linkers, such as those that can be degraded by glucuronides. Enzymatic degradation of glucuronide-containing linkers.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH-sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Nondegradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.
  • the linker Before being connected to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is achieved through the active reactive group.
  • Thiol-specific reactive groups are preferred and include, for example, maleimides, halogenated amides (e.g., iodine, bromo, or chlorinated); halogenated esters (e.g., iodine, bromo, or chlorinated). ); Halogenated methyl ketones (e.g. iodine, bromo or chlorinated), benzyl halides (e.g.
  • Linkers may include, for example, maleimides linked to the antibody via thiosuccinimide.
  • the drug can be any cytotoxic, cytostatic, or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can form a bond with the linker.
  • the drug is directly attached to the linker, the drug has reactive groups before being attached to the antibody.
  • Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Catharanthus roseus alkaloids, etc.
  • particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines and vinca alkaloids.
  • auristatins camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines and vinca alkaloids.
  • drug-linkers can be used to form ADCs in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive part of the linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug, forming an ADC.
  • linker typically, functional groups on the linker are selected to facilitate specific reaction with appropriate reactive groups on the drug moiety.
  • azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety.
  • the drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines) and activated esters, such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • ketones and aldehydes suitable for reaction with hydrazides and alkoxyamines
  • phosphines suitable for reaction with azides
  • isocyanates and isothiocyanates suitable for reaction with amines
  • activated esters such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols).
  • the present invention also provides a method for preparing an ADC, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • methods of the invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate.
  • the methods of the present invention further comprise: conjugating the antibody linker conjugate to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.
  • the antibody drug conjugate ADC is represented by the following molecular formula:
  • Ab is an antibody
  • the antibodies of the invention or their ADCs can be used in detection applications, for example in detecting samples to provide diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art. Biopsies used in the present invention may thus include, for example, resection samples of tumors, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the invention also provides a kit containing the antibody (or fragment thereof) of the invention.
  • the kit further includes a container, instructions for use, a buffer, etc.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
  • the pharmaceutical composition is in various conventional dosage forms in this field, preferably in the form of solid, semi-solid or liquid, and can be in the form of aqueous solution, non-aqueous solution or suspension, and more preferably in the form of tablets, capsules, granules , injections or infusions, etc.
  • the antibody of the present invention can also be expressed in cells from a nucleotide sequence and used for cell therapy.
  • the antibody can be used for chimeric antigen receptor T cell immunotherapy (CAR-T).
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and/or treating diseases related to abnormal expression or function of MCT1.
  • the pharmaceutical composition of the present invention can be directly used to bind MCT1 protein molecules, and thus can be used for prevention and treatment. Cancer and other diseases.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. Additionally, the polypeptides of the invention may be used with other therapeutic agents.
  • the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in this field, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in this field, preferably including pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of the pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.
  • the dosage of the pharmaceutical composition is an effective amount
  • the effective amount is an amount that can alleviate or delay the progression of diseases, degenerative or damaging conditions.
  • the effective amount can be determined on an individual basis and will be based in part on considerations of the condition to be treated and the results sought. One skilled in the art can determine an effective amount by using such factors as the above on an individual basis and using no more than routine experimentation.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the present invention provides the use of the above pharmaceutical composition in the preparation of drugs for preventing and/or treating diseases related to abnormal expression or function of MCT1.
  • the disease related to abnormal expression or function of MCT1 is tumor/cancer.
  • the pharmaceutical composition also includes: other biologically active substances.
  • the biologically active substances are selected from immunosuppressive drugs, chemotherapy agents, biguanide drugs or small molecule MCT1 inhibitors.
  • metformin or another antidiabetic agent, or an anti-inflammatory agent, or the other therapeutic agent is a mitochondrial inhibitor agent and/or biguanide, or the other therapeutic agent is selected from metformin, phenformin, alexidine, bisbiguanide, Buformim, chlorhexidine , Chlorproguanil, Phenylbiguanide, Polyaminopropyl biguanide, Polyhexanide, Moroxydine, Glipizide, Glide Glyburide, Repaglinide, Saxagliptin, Sitagliptin, PyrvinumPamoate, Proguanil, Doxycycline, Atovaquone (Atovaquone), Canagliflozin, Glitazones (such as Troglitazone, Pi
  • the MCT1 antibody of the present invention has high biological activity and high selectivity, and has high affinity for cells expressing MCT1 (EC 50 can be as high as about 2nM as determined by FACS); it has high affinity for different subtypes of MCT (MCT2, MCT3, MCT4 ) has high selectivity (barely binds MCT2, MCT3 and MCT4, More than 100-fold difference from the EC50 for binding to MCT1).
  • the MCT1 antibody of the present invention is a blocking antibody and can effectively inhibit the channel function of MCT1 (3-BP test Bmax>95%, IC50 is about 20nM), and inhibit the proliferation of immune cells (Raji cell proliferation test Bmax> 95%).
  • the humanized MCT1 antibody of the present invention has high activity and selectivity similar to mouse-derived antibodies, and has lower immunogenicity; the humanized antibody not only has good efficacy in multiple tumor/autoimmune disease models It has significant therapeutic effect and is also suitable for other diseases related to high expression/high activity of MCT1, such as metabolic diseases.
  • Wild-type HEK-293T cells mainly express MCT1, while the expression of MCT2, MCT3, and MCT4 is very low or not expressed.
  • MCT1 knockout cell lines (293-KO, MCT1KO cells) were constructed through gene targeting methods, and western blot and flow cytometry were used to prove that MCT1 was completely knocked out in the obtained cell lines (the results are shown in Figure 1) .
  • lentivirus containing hMCT1 (NM_001166496.2) expression element was used to transfect the CHO cell line. After puromycin screening, a CHO-hMCT1 cell line with stable and high expression of hMCT1 was obtained. BALB/c and C57BL/6 mice were immunized (each 5 mice), the dosage is 1 ⁇ 10 7 cells/mouse to prepare immune splenocytes; after multiple immunizations, the two mice with the highest antibody titers in the serum were selected for further hybridoma preparation.
  • Step 2 Preparation of hybridoma cells and screening of antibody supernatant
  • mice On the 4th day after the last immunization, the spleens of the mice were removed and splenocytes were isolated, electrofused with SP2/0 cells, resuspended in complete medium containing HAT and then inoculated into 96-well plates for culture. A total of 56 cells of 96 cells were obtained. orifice plate.
  • Example 3 Detection of the binding ability of the antibody of the present invention to human hMCT1/2/3/4
  • S15-C9 Use flow cytometry to evaluate the binding ability of S15-C9 to human MCT1 (wild-type HEK293T), human MCT2, human MCT3, human MCT4, mouse MCT1, rat MCT1 and cynomolgus monkey MCT1.
  • the method is as follows:
  • Antibody incubation concentration 0ng/ml, 1.524158ng/ml, 4.572474ng/ml, 13.71742ng/ml, 41.15226ng/ml, 123.4568ng/ml, 370.3704ng/ml, 1111.111ng/ml, 3333.333ng/ml, 10000ng/ ml.
  • the positive monoclonal antibody S15-C9 obtained through screening can specifically bind to human MCT1, but does not bind to human MCT2, human MCT3, or human MCT4.
  • S15-C9 can bind to cynomolgus MCT1, weakly bind to rat MCT, but not mouse MCT1.
  • the binding activity of S15-C9 to human MCT1, human MCT2, human MCT3, human MCT4, mouse MCT1, rat MCT1 and cynomolgus monkey MCT1 is shown in Table 1.
  • S15-C9 showed very high specificity, very high affinity and could significantly inhibit the activity of MCT1 (3-BP cytotoxicity experiment), it was selected for sequencing and subsequent research.
  • the CDR sequence of S15-C9 determined according to other rules is as follows:
  • ASVFSRSWIN SEQ ID NO.16
  • variable region sequences of the antibody heavy chain and light chain obtained above were cloned into the After the human IgG1 heavy chain constant region and Kappa chain constant region vectors are identified correctly, the constructed chimeric antibodies are expressed and purified using transfection technology and mammalian expression cells (CHO-S or HEK-293 cells). The obtained human-mouse chimeric antibody was named S15-C9 hIgG1.
  • the S15-C9 chimeric antibody S15-C9-hIgG1 and the PTM mutant antibody S15-C9-hIgG1 PTM-1 can bind to human MCT1 and cynomolgus monkey MCT1.
  • the affinity activity of S15-C9-IgG1 for human MCT1 is 3-4 times higher than that of Benchmark.
  • the S15-C9 chimeric antibody S15-C9-hIgG1 and the PTM mutant antibody S15-C9-hIgG1 PTM-1 do not bind to human MCT2, human MCT3, or human MCT4.
  • the calculation formula for the binding ability of each antibody to human MCT2, 3, and 4 is: MFI(hMCT2/3/4)/MFI(hMCT1)*100%.
  • Example 5 The antibody of the present invention inhibits MCT1 activity and thereby blocks the cytotoxicity of 3-bromopyruvate (3-BP)
  • 3-BP is a strong alkylating agent that mainly enters cells through MCT1 and causes cytotoxicity.
  • MCT1 gene knockout significantly improved the tolerance of 293T cells to the cytotoxicity caused by 3-BP, which was nearly 100 times higher than that of wildtype cells, further confirming that MCT1 mediates the entry of 3-BP.
  • Cells play a key role in initiating cell death (Figure 6).
  • AZD3965 (Selleck), a specific small molecule inhibitor of MCT1, was used as a positive control. It was found that AZD3965 can almost completely block the cytotoxicity of 3-BP at 100 nM.
  • HEK293T cells were seeded into a 96-well plate at 2.5x 10 4 /well and cultured overnight. Dilute the antibody solution at a ratio of 1:2 and add it to the cell culture plate. The final working concentrations are 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39ug/ml. After incubation for 3 hours, add 3-BP (working concentration: 85uM). Incubate in a 37°C incubator for 6 hours, remove the cells, use Promega's CellTiter-Glo detection solution (35ul/well), and read Luminescence on Cytation3 (Biotek). Test the cell viability of each well.
  • Well cell viability% (well reading-PBS well reading)/(positive control well reading-PBS well reading)*100%
  • the mouse parental antibody S15-C9, the human-mouse chimeric antibody S15-C9-hIgG1, and the PTM mutant antibody S15-C9-PTM1 can all inhibit MCT1 activity, thereby blocking the cells caused by 3-BP. toxicity.
  • the Human IgG1 isotype control (Isotype) has no effect.
  • Table 3 shows the IC50 of the half effective concentration of S15-C9 and its chimeric antibody S15-C9-hIgG1 and the PTM mutant antibody S15-C9-hIgG1.
  • PTM-1 blocks 3-BP and causes cytotoxicity.
  • the IC 50 of S15-C9 and its chimeric antibody S15-C9-hIgG1 in the 3-BP experiment is 2-3 times lower than that of Benchmark.
  • the package Buried residues, residues that directly interact with the CDR region, and residues that have an important impact on the conformation of VL and VH were back mutated to obtain four variable regions of humanized heavy chains (SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12) and the variable regions of three humanized light chains (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15).
  • the inventor also measured the activity of 12 humanized antibodies in the 3-BP cytotoxicity experiment.
  • the experimental method is as described in Example 5.
  • the growth inhibitory effect of 12 humanized antibodies on human Raji cells was also determined.
  • the experimental method was as described in Example 7. Experimental results showed that all humanized antibodies showed significant inhibition of Raji cell proliferation.
  • Example 7 MCT1 antibody inhibits human Raji cell proliferation
  • the mouse parental antibody S15-C9, the human-mouse chimeric antibody S15-C9-hIgG1, and the PTM mutant antibody S15-C9-PTM1 can all inhibit Raji cell proliferation, while the Human IgG1 isotype control has no effect.
  • the epitope of the MCT1 antibody of the present invention is the MCT1 amino acid sequence 282-300, sequence: VFLSSYGKSQHYSSEKSAF (SEQ ID NO. 38); further, it is the amino acid sequence 291-293, sequence: QHY.

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Abstract

提供了一种抗MCT1抗体及其用途。所述MCT1抗体及其人源化抗体具有高中和活性和高选择性，可以高效阻断MCT1的功能，并分别在肿瘤模型/自身免疫病模型中展示了良好的治疗活性。

Description

抗MCT1抗体及其用途 技术领域
本发明涉及抗体领域。具体地，涉及一种抗MCT1抗体及其用途。
背景技术
单羧酸转运蛋白(MCT)，也称为SLC16A转运蛋白家族(SLC16A)，在哺乳动物中包含14个家族成员。其中的4个成员(MCT1-4)通过氢离子偶联方式转运单羧酸，如丙酮酸，乳酸和酮类。MCT1(别名SLC16A1)可以双向跨细胞膜转运单羧酸，转运方向取决于底物在细胞膜两侧的相对浓度梯度，MCT1对于乳酸具有高亲和力，并在几乎所有细胞类型中表达。MCT1被认为是以乳酸为代谢燃料的恶性细胞摄取乳酸的主要途径。MCT1在癌症组织中的高表达通常与多种癌症临床预后结果不佳具有相关性，其中包括妇科、食道、胃肠道等实体肿瘤。
肿瘤细胞获取能量的方式高度依赖于有氧糖酵解而非氧化磷酸化，即便在有充足氧气的情况下，肿瘤细胞的糖酵解速率也远远高于正常细胞(Warburg Effect)；糖酵解可以为肿瘤细胞提供能量和其高度活跃合成代谢所需的营养物质如氨基酸等。糖酵解过程会产生大量的乳酸，从而导致细胞内酸化，MCT1在肿瘤细胞的乳酸跨膜转运并维持胞内正常PH水平中发挥重要作用。在一系列离体在体肿瘤模型中，阻断MCT1已经被证明能够有效的抑制肿瘤生长，诱导肿瘤细胞死亡。
现有技术中报道了MCT1阻断剂通过调节肿瘤免疫反应从而抑制肿瘤的新机制，其与调节肿瘤微环境中的Treg细胞功能有关：在肿瘤微环境中的Treg细胞会通过MCT1主动吸收乳酸作为代谢底物，从而进一步保持免疫抑制功能并高表达PD-1；实验表明Treg细胞中的MCT1的表达和功能对于阻断PD-1抗体治疗功效起重要作用；MCT1小分子抑制剂能够和PD-1抗体产生协同抗肿瘤效用，在多个小鼠肿瘤在体模型中展示疗效。
抑制MCT1介导的乳酸转运进而阻断糖酵解对于治疗自身免疫性疾病同样有效。通过MCT1小分子抑制剂阻断MCT1可以抑制效应T细胞(effector T cell)的活性，进而在多种自身免疫疾病的动物模型中显示治疗功效，包括胶原诱导的关节炎、同种异体移植排斥等模型。综上所述，MCT1抑制剂可能对于肿瘤和自身免疫性疾病有治疗效用。
由于MCTs在多种组织中广泛表达，非选择性的MCT1小分子抑制剂会带来药物安全性隐忧。现有的几个小分子抑制剂如AZD3965和AR-C117977都同时抑制MCT1，MCT2和一定程度的MCT3。尽管它们都在自身免疫疾病模型和肿瘤模型中展示了疗效，但在AZD3965的临床前研究和临床研究中，发现了化合物的非选择性导致了视网膜、心脏的毒性作用，从而限制了该化合物的进一步开发。
相对于小分子抑制剂，中和抗体在选择性上具有巨大优势。然而，目前已开发的MCT1抗体的性能、数量尚难以令人满意。因此本领域需要开发一种高中和活性、高选择性的新型抗MCT1抗体，以满足临床上的需求。
发明内容
本发明的目的在于提供一种抗MCT1抗体及其用途。
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR(根据Kabat编号系统)：
SEQ ID NO.1所示的HCDR1，
SEQ ID NO.2所示的HCDR2，和
SEQ ID NO.3所示的HCDR3；
或
SEQ ID NO.1所示的HCDR1，
SEQ ID NO.23所示的HCDR2，
SEQ ID NO.3所示的HCDR3；或者
所述的重链可变区包括以下三个互补决定区CDR(根据AbM编号系统)：
SEQ ID NO.16所示的HCDR1，
SEQ ID NO.17所示的HCDR2，和
SEQ ID NO.3所示的HCDR3
或
SEQ ID NO.16所示的HCDR1，
SEQ ID NO.24所示的HCDR2，
SEQ ID NO.3所示的HCDR3；或者
所述的重链可变区包括以下三个互补决定区CDR(根据IMGT编号系统)：
SEQ ID NO.18所示的HCDR1，
SEQ ID NO.19所示的HCDR2，
SEQ ID NO.20所示的HCDR3；
或
SEQ ID NO.18所示的HCDR1，
SEQ ID NO.25所示的HCDR2，
SEQ ID NO.20所示的HCDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
在另一优选例中，所述重链可变区包括以下互补决定区：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示S15-C9的重链互补决定区HCDR1、HCDR2、HCDR3；或
SEQ ID NO.1、SEQ ID NO.23、SEQ ID NO.3所示S15-C9-hIgG1 PTM-1的重链互补决定区HCDR1、HCDR2、HCDR3。
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述重链可变区具有SEQ ID NO.7所示的氨基酸序列。
在另一优选例中，所述重链可变区具有SEQ ID NO.9、10、11、12所示的氨基酸序列。
在另一优选例中，所述重链可变区的氨基酸序列与SEQ ID NO.7、9、10、11、12所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明的第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。
在本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区包括以下三个互补决定区CDR(根据Kabat编号系统或AbM编号系统)：
SEQ ID NO.4所示的LCDR1，
SEQ ID NO.5所示的LCDR2，
SEQ ID NO.6所示的LCDR3，或者，所述的轻链可变区包括以下三个互补决定区CDR(根据IMGT编号系统)：
SEQ ID NO.21所示的LCDR1，
序列如STS所示的LCDR2，
SEQ ID NO.22所示的LCDR3；
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:13、SEQ ID NO.:14、SEQ ID NO.:15所示的氨基酸序列。
在另一优选例中，所述轻链可变区的氨基酸序列与SEQ ID NO.:8、13、14、15所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性。
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明的第三方面所述的轻链可变区。
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。
在本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明的第一方面所述的重链可变区；和/或
(2)如本发明的第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明的第二方面所述的重链；和/或如本发明的第四方面所述的轻链，其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
在另一优选例中，所述抗体为单体、二价抗体、和/或多价抗体。
在另一优选例中，所述抗体为动物源抗体、人源化抗体、嵌合抗体或嵌合抗原受体抗体(CAR)。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述人源化抗体的CDR区包含1、2、或3个氨基酸的变化。
在另一优选例中，所述的动物为非人哺乳动物，较佳地为鼠、羊、兔。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量，不超过初始氨基酸序列总氨基酸数量的40％，较佳地为20％，更佳地为10％。
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-7个，较佳地为1-3个，更佳地为1个。
在另一优选例中，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸的衍生序列具有抑制细胞表面MCT1或重组MCT1蛋白酶催化功能。
在另一优选例中，所述MCT1来源于哺乳动物，较佳地人、小鼠、大鼠、食蟹猴、兔。
在另一优选例中，所述抗体对MCT1(例如人MCT1)的亲和力EC50为0.05-10nM，较佳地为0.1-6nM，更佳地为0.5-4nM；并且，对MCT2、MCT3或MCT4(例如人MCT2、MCT3或MCT4)的亲和力EC50各自独立地>100nM，较佳地>1000nM，更佳地>10000nM。
在另一优选例中，所述抗体结合人MCT1，并且阻断或抑制所述MCT1的活性，较佳地具有选自下组的一种或多种特性：
(1)抑制人MCT1介导的乳酸运输；
(2)抑制单羧酸、丙酮酸、衍生自亮氨酸、缬氨酸和异亮氨酸的支链含氧酸、酮体、乙酰乙酸、β-羟基丁酸、乙酸、乳酸、细胞营养物、代谢物、离子、激素、脂质和酮类物质中的一种或多种的转运；
(3)抑制CD3/CD28刺激的T细胞的增殖；
(4)抑制活化T细胞或B细胞的增殖；
(5)抑制一种或多种炎性细胞因子的产生；
(6)降低T效应细胞的活性和/或数量，例如CD3+、CD4+或CD8+T效应细胞；
(7)降低浆细胞(Plasma Cell)的活性和/或数量；
(7)增加调节性T(Treg)细胞，尤其是I型调节性T细胞(Type 1 regulatory cells or Tr1 or TR1)的比例或活性；
(8)抑制混合淋巴细胞反应中的同种异体活化。
在本发明的第六方面，提供了一种多特异性抗体，所述的多特异性抗体包含第一抗原结合区，所述第一抗原结合区包含如本发明的第一方面所述的重链可变区；和如本发明的第三方面所述的轻链可变区。
在另一优选例中，所述多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：EGFR、TGFβ、BCMA、B7H6、GUCY2C、DLL3、CD38、CD123、CD19、CD20、CD22、B7-H3、GPC3、HER2、PMSA、CD28、4-1BB、OX40、CD40、CD27、CD3、CTLA4、PD1、PDL1、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、TLR7、CCR8、CD47、CD39、CD73、CD147、Claudin 18.2，或其组合。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
在另一优选例中，所述抗原结合区为抗体或抗体片段，所述抗体片段包括：(i)Fab片段；(ii)F(ab')2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段。
在本发明的第七方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体；以及
(ii)任选的提升蛋白理化性质或成药性的功能域，较佳地选自下组的多肽：抗体多肽或抗体结构域(包含Fc多肽或其片段，例如，人IgG1、IgG2、IgG3或IgG4Fc区或其片段)、血清白蛋白、人或其他灵长类动物血清白蛋白、单体(也称为阿德乐汀(adnectin))、亲和体(affibody)、经设计的锚蛋白重复蛋白(DARPin)、抗运载蛋 白(anticalin)、乙二醇(PEG)、单甲氧基聚乙二醇(mPEG)、XTEN分子、rPEG分子，或任意前述的片段或变体。
在另一优选例中，所述的重组蛋白还包括
(iii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在本发明的第八方面，提供了一种CAR构建物，所述的CAR构建物的单克隆抗体抗原结合区域的scFv段为特异性结合于MCT1的结合区，并且所述scFv具有如本发明的第一方面所述的重链可变区；和如本发明的第三方面所述的轻链可变区。
在本发明的第九方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明的第八方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第十方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、或如本发明的第五方面所述的抗体、或其组合；和(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、细胞毒性药物、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述抗体药物偶联物ADC如下分子式所示：
其中：
Ab是抗MCT1的抗体，
LU是接头；
D是药物；
而且下标p是选自1-10，较佳地1-8的值。
在另一优选例中，所述的可检测标记物包括放射性核素，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述的偶联部分(D)为细胞毒性药物，并且所述的细胞毒性药物为：微管靶向药物和/或DNA靶向药物和/或拓扑异构酶抑制剂。。
在另一优选例中，所述的微管靶向药物选自下组：单甲基澳瑞他汀E(MMAE)、单甲基澳瑞他汀F(MMAF)、美登素衍生物DM1和tubulysin。
在本发明的第十一方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、如本发明的第五方面所述的抗体、如本发明的第六方面所述的多特异性抗体、如本发明的第七方面所述的重组蛋白、如本发明的第九方面所述的免疫细胞、如本发明的第十方面所述的抗体药物偶联物、或其组合，所述活性成分用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗MCT1相关疾病的药物。
在另一优选例中，所述检测试剂、检测板或试剂盒用于：
(1)检测样品中的MCT1蛋白；
(2)检测肿瘤细胞中内源性的MCT1蛋白；和/或
(3)检测表达MCT1蛋白的肿瘤细胞。
在另一优选例中，所述的检测试剂、检测板或试剂盒用于诊断MCT1相关疾病。
在另一优选例中，所述的药物用于治疗或预防MCT1高表达的肿瘤。
在另一优选例中，所述的药物用于选自下组的用途：
(a)特异结合肿瘤细胞，和/或肿瘤微环境中的免疫/基质细胞的MCT1；
(b)抑制肿瘤细胞，和/或肿瘤微环境中的免疫/基质细胞的MCT1活性和功能；
(c)抑制或降低肿瘤细胞增殖；
(d)增加肿瘤对免疫治疗药物的敏感性，提高抗肿瘤免疫治疗药物组合疗法的协同作用；
(e)抑制肿瘤生长，提高联合用药的抗肿瘤疗效。
在另一优选例中，所述MCT1相关疾病选自下组：癌症、自身免疫疾病、代谢相关疾病、炎症、移植物抗宿主病(GVHD)、或其组合。
在另一优选例中，所述的癌症包括实体瘤、血液癌。
在另一优选例中，所述的癌症为MCT1高表达的肿瘤。
在另一优选例中，所述的MCT1高表达的肿瘤选自下组：乳腺癌、肺癌、胰腺癌、宫颈癌、卵巢癌、前列腺癌癌、直肠癌、胃癌、肝癌、淋巴瘤、肾细胞癌、脑胶质瘤、黑色素瘤、白血病、淋巴瘤、头颈部鳞状细胞癌、膀胱癌、或其组合。
在另一优选例中，所述的MCT1高表达的肿瘤指肿瘤组织中MCT1转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。
在另一优选例中，所述自身免疫疾病包括：系统性红斑狼疮、类风湿关节炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症、强直性脊柱炎、哮喘、动脉粥样硬化、结肠炎、风湿性关节炎、骨关节炎、强直性脊柱炎、痛风、莱特尔综合征、牛皮癣性关节病、感染性关节炎、结核性关节炎、病毒性关节炎、真菌性关节炎、肾小球性肾炎、全身性红斑狼疮、克罗恩病、溃疡性结肠炎、急性肺损伤、慢性阻塞性肺疾病、特发性肺纤维化、移植物抗宿主病(GvHD)。
在另一优选例中，所述代谢相关疾病包括：糖尿病、食源性肥胖、脂肪炎症、痛风。
在另一优选例中，所述炎症包括：慢性前列腺炎、肾小球性肾炎、骨盆炎性疾病、再灌注损伤、类肉瘤病、血管炎、间质性膀胱炎、正常补体血症性荨麻疹性 血管炎、心包炎、肌炎、痛风、TNF受体相关的周期综合征(TRAPSP)、齿龈炎、牙周炎、肝炎、肝硬化、胰腺炎、心肌炎、血管炎、胃炎、痛风、痛风性关节炎、以及炎性皮肤病，例如牛皮癣、特应性皮炎、湿疹、红斑痤疮、荨麻疹和粉刺。
在本发明的第十二方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、如本发明的第五方面所述的抗体、如本发明的第六方面所述的多特异性抗体、本发明的第七方面所述的重组蛋白、如本发明的第九方面所述的免疫细胞、如本发明的第十方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述药物组合物中还包括：其他生物活性物质，如治疗肿瘤的药物(例如PD-1抗体、TIGIT抗体)、线粒体抑制剂和/或双胍类和/或另一种单羧酸转运蛋白(MCT抑制剂)。
在另一优选例中，所述MCT抑制剂选自一种或多种抑制MCT转运蛋白(例如MCT1、MCT2、MCT3、MCT4、MCT5、MCT6、MCT7、MCT8、MCT9或MCT10)的抑制剂，所述抑制剂还可选地包含小分子(例如AZD3965或AR-C117977)、RNAi、抗体、抗体片段或融合蛋白。
在另一优选例中，所述的药物组合物包括单方药物、复方药物、或协同药物。
在另一优选例中，所述的药物组合物的施用方式选自下组：皮下注射、皮内注射、肌肉注射、静脉注射、腹腔注射、微针注射、口服、或口鼻腔喷入和雾化吸入。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在本发明的第十三方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明的第一方面所述的重链可变区、如本发明的第二方面所述的重链、 如本发明的第三方面所述的轻链可变区、如本发明的第四方面所述的轻链、如本发明的第五方面所述的抗体；或
(2)如本发明的第六方面所述的多特异性抗体或本发明的第七方面所述的重组蛋白；
(3)如本发明的第八方面所述的CAR构建物。
在另一优选例中，所述多核苷酸为DNA，线性或环状mRNA。
在本发明的第十四方面，提供了一种载体，所述的载体含有如本发明的第十三方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、线性或环状mRNA,或其他载体。
在本发明的第十五方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明的第十四方面所述的载体或基因组中整合有如本发明的第十三方面所述的多核苷酸。
在本发明的第十六方面，提供了一种体外检测(包括诊断性或非诊断性)样品中MCT1的方法，所述方法包括步骤：
(1)在体外，将所述样品与本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在MCT1。
在本发明的第十七方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明的第五方面所述的抗体或如本发明的第十方面所述的抗体药物偶联物。
在本发明的第十八方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明的第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明的第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明的第十七方面所述的检测板。
在本发明的第十八方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养如本发明的第十五方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明的第五方面所述的抗体、如本发明的第六方面所述的多特异性抗体、或如本发明的第七方面所述的重组蛋白。
在本发明的第十九方面，提供了一种治疗MCT1相关疾病的方法，所述方法包括：给需要的对象施用如本发明的第五方面所述的抗体、如本发明的第十方面所述的抗体药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在另一优选例中，所述的方法还包括：给需要的对象施用其他药物或治疗方法进行联合治疗。
在另一优选例中，所述的其他药物或治疗方法包括：抗肿瘤免疫治疗药物、肿瘤靶向药物、肿瘤化疗药物、肿瘤放射治疗。
在另一优选例中，所述的抗肿瘤免疫治疗药物包括PD-1、PD-L1单抗。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了MCT1KO细胞系的鉴定。293-wt：野生型293T细胞。293-KO：MCT1敲除的293T细胞。Human IgG1 isotype：MCT1抗体的同型对照。
图2显示了S15-C9对人MCT1、人MCT2、人MCT3、人MCT4、mouse MCT1、rat MCT1及食蟹猴(Cyno)MCT1的结合能力。其中293-wt为表达MCT1的野生型HEK293T细胞。
图3显示了S15-C9嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1对人MCT1的结合能力,Benchmark为同类anti-MCT1对照抗体。
图4显示了S15-C9嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1对食蟹猴(Cyno)MCT1的结合能力，Benchmark为同类anti-MCT1对照抗 体。
图5显示了S15-C9嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1对人MCT2、3、4的结合能力。
图6显示了MCT1敲除显著提高HEK293细胞对于3-BP细胞毒性的耐受。
图7显示了鼠源parental抗体S15-C9，人-鼠嵌合抗体S15-C9-hIgG1，和PTM突变抗体S15-C9-PTM1及Benchmark抗体处理HEK293细胞，阻断3-BP引起的细胞毒性。
具体实施方式
本发明人经过广泛而深入地研究，通过大量筛选，从5000多个克隆孔中筛选获得了一株具有高亲和力、高选择性的阻断型MCT1抗体，所述MCT1抗体对不同亚型的MCT具有高选择性，其以高亲和力结合MCT1、几乎不结合其他不同亚型的MCT(MCT2，MCT3，MCT4)，EC50差异超过100倍。本发明的MCT1抗体能够高效阻断MCT1的功能。此外，本发明的人源化抗体具有与鼠源抗体相似的高活性和高选择性，而且具有更低的免疫原性。在此基础上完成了本发明。
单羧酸转运蛋白(MCT)
单羧酸转运蛋白(MCT)，也称为SLC16A转运蛋白家族(SLC16A，solute carrier family 16 member)，在哺乳动物中包含14个家族成员。其中的4个成员(MCT1-4)通过氢离子偶联方式转运单羧酸，如丙酮酸，乳酸和酮类。
“MCT1”是一种质子偶联的单羧酸转运蛋白。MCT1是一种多次跨膜蛋白，负责关键代谢物包括糖酵解产物的易化转运。它催化许多单羧酸化合物，诸如乳酸、丙酮酸、衍生自亮氨酸、缬氨酸和异亮氨酸的支链含氧酸、和酮体乙酰乙酸、β-羟基丁酸盐、乙酸，跨质膜的快速转运。根据组织和环境，MCT1介导乳酸和酮体的输入或输出。MCT1是称为溶质通道蛋白(SLC)的最大表面膜蛋白家族的成员之一，其功能涉及关键细胞营养物质，代谢物，离子，激素和脂质的跨膜转运。MCT1属于SLC16转运蛋白家族，其中5种已被证明以pH依赖性和双向的易化方式转运单羧酸，诸如丙酮酸，乳酸和酮。MCT1也可以用以下任何一个名称来提及：单羧酸转运蛋白1，SLC16A1，HHF7，MCT，MCT1，MCT1D，溶质载体家族16成员1。在人类中，它由SLC16A1基因编码(基因序列： NM_001166496.2，蛋白序列：NP_001159968.1)。MCT1在癌症组织中的高表达通常与多种癌症临床预后结果不佳具有相关性，其中包括妇科、食道、胃肠道等实体肿瘤。
“MCT2”是一种质子偶联的单羧酸转运蛋白。它催化许多单羧酸化合物，诸如乳酸、丙酮酸、衍生自亮氨酸、缬氨酸和异亮氨酸的支链含氧酸、和酮体乙酰乙酸、β-羟基丁酸、和乙酸，跨质膜快速转运。它还具有高亲和力丙酮酸转运蛋白的功能。MCT2也可以用以下任何一个名称来提及：单羧酸转运蛋白2，SLC16A7，MCT2，溶质载体家族16成员7。在人类中，它由SLC16A7基因编码(NM_001270622.2)。
“MCT3”是一种质子偶联的单羧酸转运蛋白。它催化许多单羧酸化合物，诸如乳酸、丙酮酸、衍生自亮氨酸、缬氨酸和异亮氨酸的支链含氧酸、和酮体乙酰乙酸、β-羟基丁酸和乙酸，跨质膜快速转运。它还具有高亲和力丙酮酸转运蛋白的功能。MCT3的表达限于视网膜色素上皮和脉络丛上皮，其位于基底膜上，相对地MCT1位于顶端膜上。MCT3也可以通过以下任何一个名称来提及：单羧酸转运蛋白3，SLC16A8，MCT3，REMP，溶质载体家族16成员8。在人类中，它由SLC16A8基因编码(NM_001394131.1)。
“MCT4”是一种质子偶联的单羧酸转运蛋白。MCT4也可以用以下任何名称来提及：单羧酸转运蛋白4，SLC16A3，MCT 3，MCT 4，MCT-3，MCT-4，MCT3，MCT4，溶质载体家族16成员3。在人类中，它由SLC16A3基因编码(NM_001042422.3)。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀 地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
如本文所用，术语“框架区”(FR)指插入CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，“HCDR1”、与“CDR-H1”可互换使用，均指重链可变区的CDR1；“HCDR2”与“CDR-H2”可互换使用，均指重链可变区的CDR2；“HCDR3”与“CDR-H3”可互换使用，均指重链可变区的CDR3。“LCDR1”与“CDR-L1”可互换使用，均指轻链可变区的CDR1；“LCDR2”与“CDR-L2”可互换使用，均指轻链可变区的CDR2；“LCDR3”与“CDR-L3”可互换使用，均指轻链可变区的CDR3。
如本文所用，“Kabat编号系统”、“IMGT编号系统”、“AbM编号系统”、“Chothia编号系统”是指，在不同指派系统下确定互补结合决定区CDR。所述指派系统可以使用许多本领域熟知的编号系统容易地确定给定CDR或FR的精确氨基酸序列边界，这些方案包括：Kabat等人(1991),“Sequences of Proteins of ImmunologicalInterest,”第5版Public Health Service,National Institutes of Health,贝塞斯达,马里兰州(“Kabat”编号系统)；Al-Lazikani等人,(1997)JMB 273,927-948(“Chothia”编号系统)；Lefranc MP等人,“IMGT unique numbering forimmunoglobulin and T cell receptor variable domains andIg superfamily V-likedomains,”Dev Comp Immunol,2003年1月；27(1):55-77(“IMGT”编号系统)；和Martin等人,“Modelingantibody hypervariable loops:a combinedalgorithm,”PNAS,1989,86(23):9268-9272(“AbM”编号系统)；MacCallum等人,J.Mol.Biol.262:732-745(1996),“Antibody-antigeninteractions:Contact analysis and binding sitetopography,”J.Mol.Biol.262,732-745”(“Contact”编号系统)。
给定CDR或FR的边界可能取决于用于鉴定的方案而不同。例如，Kabat方案是基于结构比对，而Chothia方案是基于结构信息。Kabat和Chothia方案的编号都是基于最常见的抗体区域序列长度，其中通过插入字母提供插入(例如“30a”)并且在一些抗体中出现缺失。这两种方案将某些插入和缺失(“插入缺失(indel)”)放置在不同的位置，从而产生不同的编号。Contact方案是基于对复杂 晶体结构的分析，并且在许多方面与Chothia编号方案相似。AbM方案是介于Kabat与Chothia定义之间的折衷，其基于Oxford Molecular的AbM抗体建模软件所使用的方案。
因此，除非另有规定，否则应当理解，给定抗体或其区域(如其可变区)的“CDR”涵盖由任何上述方案或其他已知方案所定义的CDR。例如，在指定特定的CDR(例如CDR3)含有给定氨基酸序列的情况下，应理解，这样的CDR还可以具有由任何上述方案或其他已知方案所定义的相应CDR(例如CDR3)的序列。同样，除非另有规定，否则应当理解给定抗体或其区域(如其可变区)的FR涵盖由任何上述方案或其他已知方案所定义的FR。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
本发明中的抗体可以是全长蛋白(如IgG1,IgG2a,IgG2b或者IgG2c)，也可以是包含抗原抗体结合域或抗原结合片段的蛋白片段(例如Fab，F(ab'),sdAb,scFv片段)。
如本文所用，抗原结合片段的非限制性例子包括：(i)Fab片段；(ii)F(ab')2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)由模拟抗体高变区的氨基酸残基组成的最小识别单位(例如，独立的互补性决定区(CDR)如CDR3肽)或受约束的FR3-CDR3-FR4肽。在另一优选例，本发明的抗体或其抗原结合片段选自下组：驼源单域抗体、scFv、scFv二聚体、BsFv、dsFv、dsFv2、dsFv-dsFv'、Fv片段、Fab、Fab'、F(ab')2、ds双功能抗体、纳米抗体、域抗体或双价域抗体。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域。这些嵌合和人源化的 单克隆抗体可以采用本领域熟知的DNA重组技术制备。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
重组蛋白(融合蛋白)
本发明还提供一种重组蛋白，其包括抗MCT1抗体(S15-C9)的重链CDR1、重链CDR2(HCDR2)和重链CDR3(HCDR3)中的一种或多种，和/或，MCT1抗体的轻链CDR1(LCDR1)、轻链CDR2(LCDR2)和轻链CDR3(LCDR3)中的一种或多种，
所述重链HCDR1-3的序列如下：
重链可变区的CDR1(Kabat)RSWIN(SEQ ID NO.1)
重链可变区的CDR2(Kabat)RIYPGDGDINHNEKFKD(SEQ ID NO.2)
重链可变区的CDR3(Kabat)SGEGYYAAMDY(SEQ ID NO.3)
所述轻链LCDR1-3的序列如下：
轻链可变区的CDR1(Kabat或AbM)TASSSVRSNYLH(SEQ ID NO.4)
轻链可变区的CDR2(Kabat或AbM)STSNLAS(SEQ ID NO.5)
轻链可变区的CDR3(Kaba或AbM)HQYHRSPYT(SEQ ID NO.6)
或使用其他的编号系统的确定的重链CDR1-3序列如下：
重链可变区的CDR1(AbM规则)ASVFSRSWIN(SEQ ID NO.16)
重链可变区的CDR2(AbM规则)RIYPGDGDIN(SEQ ID NO.17)
重链可变区的CDR3(AbM规则)SGEGYYAAMDY(SEQ ID NO.3)或
重链可变区的CDR1(IMGT规则)ASVFSRSW(SEQ ID NO.18)
重链可变区的CDR2(IMGT规则)IYPGDGDI(SEQ ID NO.19)
重链可变区的CDR3(IMGT规则)VRSGEGYYAAMDY(SEQ ID NO.20)
或使用其他的编号系统的确定的轻链CDR1-3序列如下：
轻链可变区的CDR1(IMGT规则)SSVRSNY(SEQ ID NO.21)
轻链可变区的CDR2(IMGT规则)STS
轻链可变区的CDR3(IMGT规则)HQYHRSPYT(SEQ ID NO.22)
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
例如，为了避免翻译后修饰(PTM)的风险，对于重链可变区HCDR2的潜在的PTM位点进行了点突变(DG→DA)，所获得的抗体命名为S15-C9 hIgG1PTM1，使用不同的编号系统的确定的重链HCDR2序列如下：
S15-C9-hIgG1 PTM-1的HCDR2(Kabat规则)RIYPGDADINHNEKFKD(SEQ ID NO.23)
S15-C9-hIgG1 PTM-1的HCDR2(AbM规则)RIYPGDADIN(SEQ ID NO.24)
S15-C9-hIgG1 PTM-1的HCDR2(IMGT规则)IYPGDADI(SEQ ID NO.25)。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括MCT1鼠源抗体的重链可变区和/或MCT1抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.7所示的氨基酸序列；所述抗体的轻链可变区含有SEQ ID NO.:8所示的氨基酸序列。
在另一优选例中，本发明所述的重组蛋白包括MCT1人源化抗体的重链可变区和MCT1抗体的轻链可变区，所述抗体的重链可变区含有SEQ ID NO.9、10、11、12所示的氨基酸序列；所述抗体的轻链可变区含有SEQ ID NO.:13、14或15所示的氨基酸序列。
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
较佳地，所述的重组蛋白还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
所述的重组蛋白为本领域常规的蛋白质，较佳地，其为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab')。
核酸
本发明还提供一种核酸(包括DNA，线性或环状mRNA)，其编码上述的抗体(例如抗MCT1的抗体)或重组蛋白或抗MCT1的抗体的重链可变区或轻链可变区。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
载体
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli  BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
抗体的制备
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定 (ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
抗体-药物偶联物(ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解， 从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
检测用途和试剂盒
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明所述的药物组合物是用于预防和/或治疗与MCT1表达或功能异常相关的疾病的药物组合物。
本发明的药物组合物可直接用于结合MCT1蛋白分子，因而可用于预防和治疗 肿瘤等疾病。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提供上述药物组合物在制备预防和/或治疗与MCT1表达或功能异常相关的疾病的药物中的应用。较佳地，所述与MCT1表达或功能异常相关的疾病为肿瘤/癌症。
在另一优选例中，所述药物组合物中还包括：其他生物活性物质，例如，所述生物活性物质选自免疫抑制药物、化疗剂、双胍类药物或小分子MCT1抑制剂。例如，二甲双胍或另一种抗糖尿病药，或抗炎药，或所述其他治疗剂是线粒体抑制 剂和/或双胍类药物，或所述其他治疗剂选自二甲双胍(Metformin)、苯乙双胍(Phenformin)、阿来西定(Alexidine)、二双胍(Bisbiguanide)、Buformim、氯己定(Chlorohexidine)、氯丙胍(Chlorproguanil)、苯基双胍(Phenylbiguanide)、聚氨基丙基双胍(Polyaminopropyl biguanide)、聚己缩胍(Polyhexanide)、吗啉胍(Moroxydine)、格列吡嗪(Glipizide)、格列本脲(Glyburide)、瑞格列奈(Repaglinide)、沙格列汀(Saxagliptin)、西格列汀(Sitagliptin)、PyrvinumPamoate、氯胍(Proguanil)、多西环素(Doxycycline)、阿托伐醌(Atovaquone)、卡格列净(Canagliflozin)、格列酮类(Glitazones)(例如曲格列酮(Troglitazone),吡格列酮(Pioglitazone),罗格列酮(Rosiglitazone))，替加环素(Tigecycline)、Thiazolides(例如，硝唑尼特(Nitazoxanide))、水杨酰苯胺类(Salicylanilides)(如氯生太尔(Closantel)、氯羟柳胺(Oxyclozanide)、氯硝柳胺(Niclosamide))、派克昔林(Perhexiline)、普萘洛尔(Propronolol)、非诺贝特(Fenofibrate)、咪康唑(Miconazole)、奈法唑酮(Nefazodone)、喷他脒(Pentamidine)、氢化可的松(Hydrocortisone)、间位碘代苄胍(Metaiodobenzylguanidine)、氯尼达明(Lonidamine)、α-生育酚琥珀酸酯(维生素E的主要形式)、碳酸酐酶(Carbonic anhydrase)、ME344(MEI Pharma)、HIF1a抑制剂(例如，白杨素(Chrysin)、Chetomin、二甲双酚A(Dimethy-bisphenol A)、BAY84-2243)、SR13800、二甲基草酰甘氨酸(Dimethyloxaloylglycine,DMOG)、羰基氰化物对三氟甲氧基苯腙(FCCP)、羰基氰化物间氯苯腙(CCCP)、抗霉素A(Antimycin A)、寡霉素(Oligomycin)、沙利霉素(Salinomycin)、二硝基苯酚(Dinitrophenol)、鱼藤酮(Rotenone)、苯乙双胍(Phenformin)、Tyrphostin 9、Atpenin A5、小檗碱(Berberine)、叠氮化物(Azide)、氰化物(Cyanide)、氧化亚氮(Nitrous oxide)、三氧化二砷(Arsenic trioxide)、pyrvinium、卡格列净(Canagliflozin)、罗格列酮(Rosiglitazone)、异戊巴比妥(Amobarbital)、和厚朴酚(Honokiol)、牛蒡子苷元(Arctigenin)、咖啡酸苯基酯(Caffeic acid phenyl ester)、Perhenazine、Triflouroperazine、丙酮醛(Methylglyoxal)、以及包含任意前述的组合。
本发明的主要优点包括
(1)本发明的MCT1抗体具有高生物活性和高选择性，对于表达MCT1的细胞具有很高的亲和力(FACS测定EC50可高达约2nM)；对不同亚型的MCT(MCT2，MCT3，MCT4)具有高选择性(几乎不结合MCT2、MCT3和MCT4， 与结合MCT1的EC50差异超过100倍)。
(2)本发明的MCT1抗体为阻断型抗体，并且能高效抑制MCT1的通道功能(3-BP实验Bmax>95％，IC50大约20nM)，并抑制免疫细胞的增殖(Raji cell增殖实验Bmax>95％)。
(3)本发明的人源化MCT1抗体具有与鼠源抗体相似的高活性和高选择性，而且具有更低的免疫原性；人源化抗体不仅在多个肿瘤/自身免疫疾病模型中有显著的治疗效用，而且还适用于其他与MCT1高表达/高活性有关的疾病，如代谢类疾病等。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1用于筛选的工具细胞系的构建
在HEK-293T细胞基础上，构建用于筛选的工具细胞。野生型HEK-293T细胞主要表达MCT1，而MCT2，MCT3，MCT4的表达很低或不表达。
首先，通过基因打靶方法构建MCT1敲除细胞系(293-KO，即MCT1KO细胞)，并利用western blot、流式细胞术证明所获细胞系中MCT1被完全敲除(结果如图1所示)。
在所述MCT1KO细胞基础上，进一步构建多种工具细胞系：先构建Human MCT2(NM_001270622.2)、Human MCT3(NM_001394131.1)、Human MCT4(NM_001042422.3)、mouse MCT1(NM_009196.4)、rat MCT1(NM_012716.2)及食蟹猴(Cyno)MCT1(NM_001257704.1)的慢病毒，并分别转染293-KO细胞，获得表达Human MCT2、Human MCT3、Human MCT4、mouse MCT1、rat MCT1及Cyno MCT1，且不表达人MCT1的细胞系。
实施例2靶向人MCT1(hMCT1)单克隆抗体的发现和制备
步骤一，动物免疫
首先利用含有hMCT1(NM_001166496.2)表达元件的慢病毒转染CHO细胞系，经过嘌呤霉素筛选后获得稳定高表达hMCT1的CHO-hMCT1细胞系，免疫BALB/c和C57BL/6小鼠(各5只小鼠)，用量为1×107细胞数/只，以制备免疫脾细胞；经过多次免疫后取血清中抗体滴度最高的两只小鼠进行进一步的杂交瘤制备。
步骤二，杂交瘤细胞的制备与抗体上清筛选
在最后一次免疫后第4天，取小鼠脾脏并分离脾细胞，与SP2/0细胞进行电融合，用含HAT的完全培养基重悬后接种至96孔板中培养，共获得56块96孔板。
通过高通量流式进行阳性孔筛选，选择对于MCT1高表达细胞特异性结合并且不结合MCT1KO细胞的阳性孔，从中选取高结合力的孔共48个做亚克隆；通过有限稀释法进行克隆化培养，取其中存在克隆的孔的上清利用高通量流式细胞术进行进一步确认，共获得有结合活性的克隆孔210个，将这些孔进一步扩大培养至24孔，利用分别过表达hMCT2/3/4亚型的细胞进行负筛选，排除结合其他亚型MCT的克隆，选出只结合hMCT1的单克隆(共4个)进一步扩大化培养。
我们发现，只有极少数克隆才具有亚型选择性。扩增后进一步确认，经过三步筛选(hMCT1细胞，MCT1KO细胞，hMCT2/3/4亚型的细胞)后，对确认只结合hMCT1，不结合hMCT2/3/4的克隆进行扩大和建库。流式筛选具体步骤如下：
将2×104 293T细胞铺于U底96孔板，离心后去上清，加入50ul杂交瘤细胞培养基，室温孵育1小时，然后离心去除上清，每孔加入含2％FBS的PBS溶液100ul，混匀后离心去上清，然后加入50ul Alexa Fluor-647标记的Anti-mouse二抗，室温孵育1小时，孵育完成后利用高通量流式筛选阳性杂交瘤。
负向筛选时采用细胞为分别表达人hMCT2，hMCT3，hMCT4的细胞，其他操作与上述相同，筛选对hMCT2/3/4没有结合能力的杂交瘤。
实施例3:本发明抗体与人hMCT1/2/3/4的结合能力检测
将经过三轮筛选所获的优选单克隆进一步扩大培养，并纯化小鼠单克隆抗 体，命名为S15-C9。利用流式细胞术评价S15-C9对人MCT1(野生型HEK293T)、人MCT2、人MCT3、人MCT4、mouse MCT1、rat MCT1及食蟹猴MCT1的结合能力，方法如下：
将2×104细胞铺于U底96孔板，离心后去上清，按照如下抗体浓度加入50ul抗体溶液孵育，室温孵育1小时，然后离心去除上清，每孔加入含2％FBS的PBS溶液100ul，混匀后离心去上清，然后加入50ul Alexa Fluor-647标记的Anti-mouse二抗，室温孵育1小时，孵育完成后利用高通量流式进行鉴定。
抗体孵育浓度：0ng/ml、1.524158ng/ml、4.572474ng/ml、13.71742ng/ml、41.15226ng/ml、123.4568ng/ml、370.3704ng/ml、1111.111ng/ml、3333.333ng/ml、10000ng/ml。
如图2所示，筛选获得的阳性单抗S15-C9可以特异性的结合人MCT1，不结合人MCT2，人MCT3，或人MCT4。同时，S15-C9可以结合食蟹猴MCT1，弱结合rat MCT，但不结合mouse MCT1。S15-C9对人MCT1、人MCT2、人MCT3、人MCT4、mouse MCT1、rat MCT1及食蟹猴MCT1的结合活性如表1所示。
表1 S15-C9对人MCT1(wt)、人MCT2、人MCT3、人MCT4、mouse MCT1、rat MCT1及食蟹猴MCT1的结合活性
由于S15-C9表现出非常高的特异性、非常高的亲和力并能够对MCT1的活性有显著抑制作用(3-BP细胞毒性实验)，故被选定用于测序以及后续研究。
实施例4人-鼠嵌合抗体的制备
在已获得的高活性高选择性的鼠源单抗S15-C9的基础上，构建人-鼠嵌合抗体。对S15-C9克隆进行基因测序，通过Kabat数据库分析，确定S15-C9的重链可变区、轻链可变区的CDR氨基酸序列如下所示：
重链可变区的CDR1(Kabat规则)RSWIN(SEQ ID NO.1)
重链可变区的CDR2(Kabat规则)RIYPGDGDINHNEKFKD(SEQ ID NO.2)
重链可变区的CDR3(Kabat规则)SGEGYYAAMDY(SEQ ID NO.3)
轻链可变区的CDR1(Kabat规则)TASSSVRSNYLH(SEQ ID NO.4)
轻链可变区的CDR2(Kabat规则)STSNLAS(SEQ ID NO.5)
轻链可变区的CDR3(Kabat规则)HQYHRSPYT(SEQ ID NO.6)
VH氨基酸序列
VL氨基酸序列
根据其他规则确定的S15-C9的CDR序列如下：
重链可变区的CDR1(AbM规则)ASVFSRSWIN(SEQ ID NO.16)
重链可变区的CDR2(AbM规则)RIYPGDGDIN(SEQ ID NO.17)
重链可变区的CDR3(AbM规则)SGEGYYAAMDY(SEQ ID NO.3)
重链可变区的CDR1(IMGT规则)ASVFSRSW(SEQ ID NO.18)
重链可变区的CDR2(IMGT规则)IYPGDGDI(SEQ ID NO.19)
重链可变区的CDR3(IMGT规则)VRSGEGYYAAMDY(SEQ ID NO.20)
轻链可变区的CDR1(AbM规则)TASSSVRSNYLH(SEQ ID NO.4)
轻链可变区的CDR2(AbM规则)STSNLAS(SEQ ID NO.5)
轻链可变区的CDR3(AbM规则)HQYHRSPYT(SEQ ID NO.6)
轻链可变区的CDR1(IMGT规则)SSVRSNY(SEQ ID NO.21)
轻链可变区的CDR2(IMGT规则)STS
轻链可变区的CDR3(IMGT规则)HQYHRSPYT(SEQ ID NO.22)
通过设计引物将上述所获得抗体重链和轻链的可变区序列分别克隆入含 有人IgG1重链恒定区和Kappa链恒定区的载体，经鉴定无误后，利用转染技术和哺乳动物表达细胞(CHO-S或HEK-293细胞)将构建的嵌合型抗体表达，纯化，所获得的人-鼠嵌合型抗体，命名为S15-C9 hIgG1。为了避免PTM的风险，对于重链可变区的潜在的PTM位点进行了点突变(DG→DA)，所获得的抗体命名为S15-C9 hIgG1 PTM1(S15-C9 hIgG1 PTM1的重链CDR2(Kabat规则)：RIYPGDADINHNEKFKD，SEQ ID NO.23)。
如图3和图4所示，S15-C9嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1可以结合人MCT1和食蟹猴MCT1，发明人表达了现有技术(WO 2019/136300)的代表性人源化抗体序列作为阳性对照(Benchmark)，结合活性如表2所示。S15-C9-IgG1对人MCT1的亲和活性相较于Benchmark高3-4倍。
如图5所示，S15-C9嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1对人MCT2，人MCT3，或人MCT4都不结合。各抗体对人MCT2、3、4的结合能力计算公式为：MFI(hMCT2/3/4)/MFI(hMCT1)*100％。
表2 S15-C9嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1以及Benchmark抗体对人MCT1与猴MCT1结合的半数有效浓度。
实施例5：本发明抗体抑制MCT1活性从而阻断3-溴丙酮酸(3-Bromopyruvate，3-BP)的细胞毒性
3-BP是一种强烷化剂，主要通过MCT1进入细胞内引起细胞毒性。前期实验也显示，MCT1基因敲除显著提高了293T细胞对于3-BP引起的细胞毒性的耐受性，相较于wildtype细胞提高了近100倍，从而进一步证实了MCT1在介导3-BP进入细胞引发细胞死亡过程中起关键作用(图6)。
根据该实验，选择了85uM作为3-BP的工作浓度。本实验以MCT1的特异性小分子抑制剂AZD3965(Selleck)为阳性对照，发现AZD3965在100nM就可以几乎完全阻断3-BP的细胞毒性。
HEK293T细胞以2.5x 104/孔接种到96孔板，培养过夜。将抗体溶液倍比(1：2)稀释然后加入细胞培养板中，最终工作浓度为50，25，12.5，6.25，3.13，1.56，0.78，0.39ug/ml。孵育3小时后，加入3-BP(工作浓度为85uM)。37度培养箱中培养6小时，将细胞取出，使用Promega公司的CellTiter-Glo检测液(35ul/well)，在Cytation3(Biotek)读取Luminescence。检测每个孔的细胞活力(Cell viability)。
孔细胞活力％＝(孔读数-PBS孔读数)/(阳性对照孔读数-PBS孔读数)*100％
如图7所示，鼠源parental抗体S15-C9，人-鼠嵌合抗体S15-C9-hIgG1，和PTM突变抗体S15-C9-PTM1都可以抑制MCT1活性，从而阻断3-BP引起的细胞毒性。而Human IgG1同型对照(Isotype)没有作用。
表3为S15-C9及其嵌合抗体S15-C9-hIgG1及PTM突变抗体S15-C9-hIgG1 PTM-1阻断3-BP引起细胞毒性半数有效浓度IC50。S15-C9及其嵌合抗体S15-C9-hIgG1在3-BP实验中IC50相较于Benchmark低2-3倍。
表3
实施例6 S15-C9的人源化及活性测定
参照S15-C9的抗体重链可变区序列(SEQ ID NO:7)和轻链可变区序列(SEQ ID NO:8)，在Germline数据库中选取与其非CDR区匹配最好的人源化模板。然后将鼠源抗体S15-C9的CDR区移植到所选择的人源化模板上，替换人源模板的CDR区，再与lgG1恒定区重组，同时以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，得到4个人源化重链的可变区(SEQ ID NO:9,SEQ ID NO:10,SEQ ID NO:11,SEQ ID NO:12)及3个人源化轻链的可变区(SEQ ID NO:13,SEQ ID NO:14,SEQ ID NO:15)。
表4

基于所述改造的VH和VL，分别组合表达这些人源化的重链及轻链，最终共得到的12个人源化抗体，即S15-C9-H1至S15-C9-H12。各抗体相应的重链和轻链组合如下表所示：
表5
首先通过流式细胞仪测定这12个人源化抗体对表达人MCT1的HEK293的亲和活性(实验方法参照实施例3)，结果如表6所示。
表6人源化抗体对对表达人MCT1的HEK293的结合亲和力

发明人还测定了12个人源化抗体在3-BP细胞毒性实验中的活性，实验方法参照实施例5。
实验结果如表7所示，各人源化抗体几乎都可有效阻断3-BP引起的细胞毒性，并呈浓度依赖性。
表7人源化抗体阻断3-BP引起的细胞毒性
还测定12个人源化抗体在对人Raji细胞的生长抑制作用，实验方法参照实施例7。实验结果表明，所有人源化抗体均表现出显著抑制Raji细胞增殖。
实施例7 MCT1抗体抑制人Raji细胞增殖
取对数生长期Raji细胞，用1640完全培养基重悬细胞，以1*104细胞/孔接种到96孔板中，培养过夜。将抗体溶液倍比(1：3)稀释然后加入细胞培养板中，最终工作浓度为450、150、50、16.67、5.56、1.85、0.62、0.21、0.07ug/mL共九个浓度，human IgG1作为阴性对照。继续培养72小时后加入XTT测定细胞活力，酶标仪读数(OD450)，测定抑制百分率(％)
抑制百分率(％)＝100-(OD抗体处理孔-ODblank孔)/(ODPBS对照孔-ODblank孔)*100％
鼠源parental抗体S15-C9，人-鼠嵌合抗体S15-C9-hIgG1，和PTM突变抗体S15-C9-PTM1都可以抑制Raji细胞增殖，而Human IgG1同型对照没有作用。
实施例8 MCT1抗体表位测定
经表位实验测定，本发明的MCT1抗体的epitope为MCT1氨基酸序列282-300，序列：VFLSSYGKSQHYSSEKSAF(SEQ ID NO.38)；更进一步，为氨基酸序列291-293位点，序列：QHY。
本发明的抗体序列的对应不同的编号系统如下表所示：
表8.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR(根据Kabat编号系统)：
    SEQ ID NO.1所示的HCDR1，
    SEQ ID NO.2所示的HCDR2，和
    SEQ ID NO.3所示的HCDR3；
    或
    SEQ ID NO.1所示的HCDR1，
    SEQ ID NO.23所示的HCDR2，
    SEQ ID NO.3所示的HCDR3；或者
    所述的重链可变区包括以下三个互补决定区CDR(根据AbM编号系统)：
    SEQ ID NO.16所示的HCDR1，
    SEQ ID NO.17所示的HCDR2，和
    SEQ ID NO.3所示的HCDR3
    或
    SEQ ID NO.16所示的HCDR1，
    SEQ ID NO.24所示的HCDR2，
    SEQ ID NO.3所示的HCDR3；或者
    所述的重链可变区包括以下三个互补决定区CDR(根据IMGT编号系统)：
    SEQ ID NO.18所示的HCDR1，
    SEQ ID NO.19所示的HCDR2，
    SEQ ID NO.20所示的HCDR3；
    或
    SEQ ID NO.18所示的HCDR1，
    SEQ ID NO.25所示的HCDR2，
    SEQ ID NO.20所示的HCDR3；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述轻链可变区包括以下三个互补决定区CDR(根据Kabat编号系统或AbM编号系统)：
    SEQ ID NO.4所示的LCDR1，
    SEQ ID NO.5所示的LCDR2，
    SEQ ID NO.6所示的LCDR3，或者，所述的轻链可变区包括以下三个互补决定区CDR(根据IMGT编号系统)：
    SEQ ID NO.21所示的LCDR1，
    序列如STS所示的LCDR2，
    SEQ ID NO.22所示的LCDR3；
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链，其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留MCT1结合亲和力的衍生序列。
  6. 一种多特异性抗体，其特征在于，所述的多特异性抗体包含第一抗原结合区，所述第一抗原结合区包含如权利要求1所述的重链可变区；和如权利要求3所述的轻链可变区；其中，所述多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：EGFR、TGFβ、BCMA、B7H6、GUCY2C、DLL3、CD38、CD123、CD19、CD20、CD22、B7-H3、GPC3、HER2、PMSA、CD28、4-1BB、OX40、CD40、CD27、CD3、CTLA4、PD1、PDL1、BCMA、GLP-1、Trop2、TIGIT、LAG-3、FGL1、TLR7、CCR8、CD47、CD39、CD73、CD147、Claudin 18.2，或其组合。
  7. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或权利要求5所述的抗体；以及
    (ii)任选的提升蛋白理化性质或成药性的功能域，较佳地选自下组的多肽：抗体多肽或抗体结构域(包含Fc多肽或其片段，例如，人IgG1、IgG2、IgG3或IgG4Fc区或其片段)、血清白蛋白、人或其他灵长类动物血清白蛋白、单体(也称为阿德乐汀(adnectin))、亲和体(affibody)、经设计的锚蛋白重复蛋白(DARPin)、抗运载蛋白(anticalin)、乙二醇(PEG)、单甲氧基聚乙二醇(mPEG)、XTEN分子、rPEG分子，或任意前述的片段或变体。
  8. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFv段为特异性结合于MCT1的结合区，并且所述scFv具有如如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区。
  9. 一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如权利要求8所述的CAR构建物。
  10. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、细胞毒性药物、细胞因子、放射性核素、酶、或其组合。
  11. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体、如权利要求6所述的多特异性抗体、权利要求7所述的重组蛋白、权利要求9所述重组的免疫细胞、如权利要求10所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
  12. 一种多核苷酸，其特征在于，所述的多核苷酸编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体；或
    (2)如权利要求6所述的多特异性抗体或权利要求7所述的重组蛋白；
    (3)如权利要求8所述的CAR构建物。
  13. 一种载体，其特征在于，所述的载体含有如权利要求12所述的多核苷酸。
  14. 一种遗传工程化的宿主细胞，其特征在于，所述的宿主细胞含有如权利要求13所述的载体或基因组中整合有如权利要求13所述的多核苷酸。
  15. 一种治疗MCT1相关疾病的方法，其特征在于，所述方法包括：给需要的对象施用如权利要求5所述的抗体、或其抗体药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
PCT/CN2023/108737 2022-07-21 2023-07-21 抗mct1抗体及其用途 WO2024017383A1 (zh)

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