WO2023051621A1 - 抗lag3抗体、药物组合物及用途 - Google Patents

抗lag3抗体、药物组合物及用途 Download PDF

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WO2023051621A1
WO2023051621A1 PCT/CN2022/122185 CN2022122185W WO2023051621A1 WO 2023051621 A1 WO2023051621 A1 WO 2023051621A1 CN 2022122185 W CN2022122185 W CN 2022122185W WO 2023051621 A1 WO2023051621 A1 WO 2023051621A1
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antibody
seq
antigen
cancer
variable region
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French (fr)
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夏瑜
***
张鹏
李百勇
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中山康方生物医药有限公司
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Priority to AU2022355381A priority patent/AU2022355381A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention belongs to the field of biomedicine, and relates to an anti-LAG3 antibody, a pharmaceutical composition containing the anti-LAG3 antibody and its use.
  • Tumors are diseases that seriously endanger human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend.
  • the curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor.
  • conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve.
  • Lymphocyte-activation gene 3 (LAG3), or CD223, is a type I transmembrane protein composed of 498 amino acids, which belongs to the immunoglobulin superfamily (IgSF) member.
  • LAG3 is mainly expressed in activated CD4 + T cells and CD8 + T cells, in addition to natural killer (natural killer, NK) cells, B cells, regulatory T cells (regulatory T cells, Treg) and plasmacytoid dendritic cells ( Plasmacytoid dendritic cells, pDC) and other cells also express LAG3.
  • NK natural killer
  • Treg regulatory T cells
  • Plasmacytoid dendritic cells Plasmacytoid dendritic cells, pDC
  • the LAG3 molecular gene is located on human chromosome 12 (20p13.3), adjacent to the CD4 molecular gene, and both have the same exons and introns. Although the amino acid sequence homology between the two is only about 20%, the LAG3 molecule and the CD4 molecule have a high similarity in structure.
  • Major histocompatibility complex class II molecules MHC II
  • LSECtin liver sinusoidal endothelial cell agglutinin
  • galectin-3 galectin-3
  • MHC class II molecules are the main ligands of LAG3, and their affinity with MHC class II molecules (Kd: 60 nmol L-1) is 100 times that of CD4 molecules, indicating that LAG3 molecules can effectively compete for the binding of CD4 to MHC class II molecules and inhibit T cells. activation.
  • the expression of the immunosuppressive molecule LAG3 can be detected 24 hours after T cell activation, leading to T cell incapacity or apoptosis.
  • the LAG3 molecule forms a dimer molecule through its D1 domain (containing a proline-rich ring structure) and specifically binds to the MHCII molecule in the first signaling axis "CD3-TCR-MHCII" of CD4 + T cell activation, On the one hand, it blocks the signal transduction pathway of T cell activation, and on the other hand, the intracellular segment of LAG3 molecule (KIEELE motif) produces an immunosuppressive signal to down-regulate the activity of CD4 + T cells.
  • LAG3 molecules can promote the differentiation of Treg cells, participate in signal transduction and transcription activator 5 downstream signaling, thereby enhancing the inhibitory effect of Treg cells, which is one of the mechanisms for tumors to escape the killing of the immune system (Andrews Lawrence P, Marciscano Ariel E, Drake Charles G et al.LAG3(CD223) as a cancer immunotherapy target.[J].Immunol Rev,2017,276:80-96.).
  • LAG3 is overexpressed in tumor-infiltrating CD8 + T cells in various malignancies.
  • tumor-infiltrating New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific CD8 + T cells express high levels of PD-1 and LAG3 and have reduced ability to produce IFN- ⁇ and TNF- ⁇ , resulting in inactivation of lymphocytes.
  • Galectin-3 and LSECtin mainly interact with LAG3 to regulate the activation and function of CD8 + T cells.
  • melanoma antigen-specific T cells isolated from patients with melanoma metastasis were significantly upregulated in the expression of LAG3 and other immune checkpoint molecules CTLA-4 and TIM-3.
  • LAG3 antibody drugs have entered the clinical research stage.
  • Bristol-Myers Squibb's Relatlimab has made the fastest progress.
  • 10 clinical studies have been carried out, and most of them are combined drugs of Relatlimab and Nivolumab. It is used to treat tumors such as hematoma, melanoma, glioblastoma, renal cell carcinoma and non-small cell lung cancer.
  • the present inventor obtained an anti-LAG3 antibody through intensive research and creative work.
  • the inventors have surprisingly found that the anti-LAG3 antibody of the present invention (also referred to as antibody or antibody of the present invention) has superior affinity and/or specificity, and is even superior to a positive control antibody (such as Relatlimab) in one or more aspects .
  • a positive control antibody such as Relatlimab
  • One aspect of the present invention relates to an anti-LAG3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises: amino acid sequences shown in SEQ ID NOs: 9-11 respectively HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences shown in SEQ ID NOs: 12-14 respectively LCDR1-LCDR3;
  • the heavy chain variable region comprises: amino acid sequences such as HCDR1-HCDR3 shown in SEQ ID NOs: 9-11; and the light chain variable region comprises: amino acid sequences such as SEQ ID NO: 12, SEQ ID NO LCDR1-LCDR3 shown in :15 and SEQ ID NO:16;
  • the heavy chain variable region comprises: amino acid sequences such as HCDR1-HCDR3 shown in SEQ ID NOs: 9-11; and the light chain variable region comprises: amino acid sequences such as SEQ ID NO: 17, SEQ ID NO :15 and LCDR1-LCDR3 shown in SEQ ID NO:14.
  • the antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:4;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 6;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementary Determining region fragments, single chain antibodies, humanized antibodies or chimeric antibodies.
  • the antibody or antigen-binding fragment thereof wherein, the antibody is less than 0.2nM, such as less than 0.15nM, less than 0.1nM, less than 0.08nM, 0.06nM or less than 0.05nM or A smaller EC50 binding to human LAG3-mFc; preferably, said EC50 is determined by an indirect ELISA method.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, eg, from a human antibody.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody whose constant region is derived from a human antibody
  • the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment thereof wherein,
  • the heavy chain constant region of the anti-LAG3 antibody is Ig gamma-1 chain C region (for example, as shown in SEQ ID NO: 18) or Ig gamma-4 chain C region (for example, as shown in SEQ ID NO: 20) and the light chain constant region is an Ig kappa chain C region (eg, as shown in SEQ ID NO: 19).
  • the anti-LAG3 antibody is a monoclonal antibody.
  • the anti-LAG3 antibody is in the form of immunoglobulin.
  • the anti-LAG3 antibody is a single chain antibody.
  • an antibody-drug conjugate which includes an antibody or its antigen-binding fragment and a small molecule drug, wherein the antibody or its antigen-binding fragment is any one of the present invention
  • ADC antibody-drug conjugate
  • the small-molecule drug is a small-molecule cytotoxic drug; more preferably, the small-molecule drug is a tumor chemotherapy drug.
  • the chemotherapeutic drugs can be conventional tumor chemotherapeutic drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune preparations and the like.
  • the antibody-drug conjugate wherein the antibody or its antigen-binding fragment is connected to the small molecule drug through a linker;
  • the linker can be a person skilled in the art Known linkers are, for example, hydrazone bonds, disulfide bonds or peptide bonds.
  • the antibody-drug conjugate wherein the molar ratio of the antibody or its antigen-binding fragment to the small-molecule drug is 1: (2-4), for example, 1: 2, 1:3 or 1:4.
  • Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-LAG3 antibody of any one of the present invention.
  • Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a recombinant vector of the invention.
  • Another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the present invention, which comprises culturing the host cell of the present invention under suitable conditions, and recovering the obtained antibody from the cell culture. The steps of the antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof according to any one of the present invention, and the antibody-drug conjugate according to any one of the present invention; optionally, It also includes pharmaceutically acceptable excipients.
  • Another aspect of the present invention relates to the antibody or its antigen-binding fragment described in any one of the present invention, and the antibody-drug conjugate described in any one of the present invention in the preparation of a drug for treating and/or preventing tumor or anemia use in
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • Another aspect of the present invention relates to a method for treating and/or preventing tumor or anemia, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in any one of the present invention, the present invention The steps of the antibody drug conjugate described in any one of the inventions;
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously, A population of identical antibody molecules.
  • mAbs are highly specific for a single epitope on an antigen.
  • polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens.
  • Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but it can also be obtained by recombinant DNA technology (see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody).
  • Antibodies or antibody fragments, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated or “isolated” do not exclude the admixture of artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substance.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 5 M, such as less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • Binds antigen eg, PD-1 protein). KD can be determined using methods known to those skilled in the art, for example using a Fortebio Molecular Interaction Instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH regulators include but not limited to phosphate buffer
  • surfactants include but not limited to cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include but not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (such as a tumor);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the occurrence of a disease in a patient Amount of disease and its complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • lymphocyte-activation gene 3 when referring to the amino acid sequence of lymphocyte activation gene 3 (lymphocyte-activation gene 3, LAG3), it includes the full length of the LAG3 protein, or the extracellular fragment LAG3 ECD of LAG3 or a protein comprising LAG3 ECD Fragments; also include full-length fusion proteins of LAG3 protein or fusion proteins of LAG3 ECD, for example, fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • Fc protein fragments mFc or hFc
  • the anti-LAG3 antibody of the present invention has superior affinity and specificity
  • the anti-LAG3 antibody of the present invention can effectively block the interaction between LAG3 and MHC-II, and specifically relieve the immune suppression of the body by LAG3.
  • Figure 1 The detection results of the binding activity between H7L8(hG1WT) and antigen LAG3-mFc determined by indirect ELISA method.
  • Figure 2 ELISA method to determine the binding activity of H7L8(hG4WT), H7L9(hG4WT), H7L10(hG4WT) to antigen human LAG3-mFc.
  • FIG. 3 FACS detection of the binding activity of H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) to the surface antigen LAG3 of 293T-LAG3 cells.
  • Figure 4 The results of competitive flow cytometry determination of H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) and LAG3-mFc for binding to 293T-LAG3 cell membrane surface antigen MHC II.
  • Figure 5 The results of the mixed lymphocyte reaction MLR detection of the biological activity of anti-LAG3 antibody to promote IFN- ⁇ secretion.
  • Figure 6 The results of the mixed lymphocyte reaction MLR detection of the biological activity of the anti-LAG3 antibody in promoting IL-2 secretion.
  • Figure 7 Detection results of biological activity of anti-LAG antibody blocking the interaction between LAG-3 and MHC-II.
  • Relatlimab refers to the US Patent Publication: US20160326248A1 for its sequence.
  • amino acid sequence of the heavy chain refers to SEQ ID NO:1 in the patent disclosure
  • amino acid sequence of the light chain refers to SEQ ID NO:2 in the patent disclosure.
  • Relatlimab is an anti-LAG-3 antibody.
  • control antibody 14C12H1L1 is an anti-PD-1 antibody, prepared by Akeso Bio, batch number B105Y2080601.
  • the 293T-LAG3 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the 293T-LAG3 cell line was obtained by virus infection of HEK293T cells.
  • the virus was prepared using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-huLAG3FL-BSD (where LAG3, Genebank ID: NM_002277.4; vector plenti6.3/V5-BSD, purchased from Invitrogen, product number: K5315-20).
  • the Raji-PDL1 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the Raji-PDL1 cell line was obtained by virus infection of Raji cells using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-PDL1 (wherein PDL1, Genebank ID: NP_054862.1; vector plenti6. 3/V5, purchased from Invitrogen, catalog number: K5315-20).
  • the Jurkat-NFAT-PD1-LAG3 cell line was constructed by Zhongshan Akeso Biopharmaceutical Co., Ltd.
  • the Jurkat-NFAT-PD1-LAG3 cell line is obtained from PD-1 Effector cells (CPM), manufacturer: Promega, product number: J112A) cells infected by virus, the virus is prepared using 3rd Generation Lentiviral Systems, See, for example A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector is pCDH-huLAG3FL-RFP-NEO (wherein LAG3, Genebank ID: NM_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from Youbao Biology, product number: VT9005).
  • the nucleotide sequence of the heavy chain variable region H7v of H7L9 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:1.
  • the amino acid sequence of the heavy chain variable region H7v of H7L9 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:2.
  • the nucleotide sequence of the heavy chain variable region H7v of H7L10 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:1.
  • the amino acid sequence of the heavy chain variable region H7v of H7L10 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:2.
  • amino acid sequence of the CDR of antibody H7L8 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 9);
  • HCDR2 INHRGTT (SEQ ID NO: 10);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 11);
  • LCDR1 QTISSY (SEQ ID NO: 12);
  • LCDR2 DAS (SEQ ID NO: 13);
  • LCDR3 QQRSNWPIT (SEQ ID NO: 14).
  • amino acid sequence of the CDR of antibody H7L9 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 9);
  • HCDR2 INHRGTT (SEQ ID NO: 10);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 11);
  • LCDR1 QTISSY (SEQ ID NO: 12);
  • LCDR2 DGS (SEQ ID NO: 15);
  • LCDR3 QQRSNWPLT (SEQ ID NO: 16).
  • amino acid sequence of the CDR of antibody H7L10 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 9);
  • HCDR2 INHRGTT (SEQ ID NO: 10);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 11);
  • LCDR1 QSISSY (SEQ ID NO: 17);
  • LCDR2 DGS (SEQ ID NO: 15);
  • LCDR3 QQRSNWPIT (SEQ ID NO: 14).
  • the heavy chain cDNA sequence of H7L8 (hG1WT) (the variable region coding sequence is shown in SEQ ID NO: 1; the constant region is the Ig gamma-1 chain C region) and the cDNA sequence of the light chain (the variable region coding sequence is shown in SEQ ID NO: ID NO: 3; the constant region is human Ig kappa chain C region) were cloned into pUC57simple (provided by GenScript) vectors to obtain pUC57simple-H7 and pUC57simple-L8 plasmids, respectively.
  • pUC57simple provided by GenScript
  • Plasmids pUC57simple-H7 and pUC57simple-L8 were digested (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cells. After the cells were cultured for 7 days, the culture medium was concentrated by high-speed centrifugation and the supernatant was concentrated and loaded onto the HiTrap MabSelect SuRe column. The protein was eluted with Elution Buffer in one step, and the target sample was recovered and replaced with PBS.
  • the heavy chain cDNA sequences of H7L8 (hG4WT), H7L9 (hG4WT) and H7L10 (hG4WT) (the variable region coding sequence is shown in SEQ ID NO: 1; the constant region is Ig gamma-4 chain C region) and H7L8 (hG4WT ) light chain cDNA sequence (the variable region coding sequence is shown in SEQ ID NO: 3; the constant region is human Ig kappa chain C region), the cDNA sequence of the H7L9 (hG4WT) light chain (the variable region coding sequence is shown in SEQ ID Shown in NO: 5; the constant region is human Ig kappa chain C region), the cDNA sequence of H7L10 (hG4WT) light chain (the variable region coding sequence is shown in SEQ ID NO: 7; the constant region is human Ig kappa chain C region ) were respectively cloned into pUC57simple (provided by GenScript
  • the plasmids pUC57simple-H7 and pUC57simple-L8, pUC57simple-L9, pUC57simple-L10 were digested with restriction enzymes (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were respectively subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cell. After the cells were cultured for 7 days, the culture medium was concentrated by high-speed centrifugation and the supernatant was concentrated and loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer, and the target sample was recovered and replaced with PBS.
  • restriction enzymes HindIII & EcoRI
  • H7L8 H7L9
  • H7L10 H7L10
  • Human anti-Hen Egg Lysozyme IgG human anti-Hen Egg Lysozyme IgG, anti-HEL, namely human IgG, referred to as hIgG
  • its sequence comes from Affinity maturation increases the stability and plasticity of the Fv domain of published by Acierno et al.
  • Variable region sequence of Fab F10.6.6 sequence in anti-protein antibodies study (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The preparation method is as follows:
  • Experimental example 1 Determination of the binding activity of anti-LAG3 antibody and antigen by ELISA method
  • Relatlimab and H7L8 can effectively bind to the antigen human LAG3-mFc, and the binding efficiency is dose-dependent.
  • the absorbance intensity of each dose is shown in Table 1.
  • Embodiment 2 ELISA method measures the binding activity of anti-LAG3 antibody and antigen
  • Example 3 Detection of binding activity of anti-LAG3 antibody to cell surface antigen LAG3 by flow cytometry
  • the host cell 293T-LAG3 expressing LAG3 antigen obtained in the above steps was digested with conventional trypsin, and the number of cells in each collection tube was 3 ⁇ 10 5 , and 1% PBSA (containing 1 %BSA in PBS) to prepare LAG3 antibody dilutions with final concentrations of 0.0123nM, 0.123nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM, 100nM, and 300nM, and incubate with 293T cells expressing LAG3 on ice for 1 hour.
  • PBSA containing 1 %BSA in PBS
  • FITC goat anti-human IgG purchased from Jackson, catalog number: 109-095-098
  • 100 ⁇ L FITC goat anti-human IgG purchased from Jackson, catalog number: 109-095-098
  • 200 ⁇ L of 1% PBSA was added to resuspend the cells, and the fluorescent signal was detected with the FITC channel on the flow cytometer.
  • Table 3 shows the EC 50 of the binding efficiency of each anti-LAG3 antibody to the 293T-LAG3 surface antigen.
  • Table 3 The binding activity of anti-LAG3 antibody to 293T-LAG3 surface antigen detected by flow cytometry
  • the anti-LAG3 antibody can effectively bind the target LAG3 protein on the surface of host cell 293T-LAG3, and the binding activity of anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) to the surface antigen of 293T-LAG3 Comparable to the positive control antibody Relatlimab.
  • hIgG1 manufactured by Akeso, batch number: 20190410
  • a final concentration of 300 nM added to each tube according to the experimental design, and incubate 100 ⁇ L of each tube on ice for 1 h; add 200 ⁇ L of 1% PBSA to the incubated Raji cells, centrifuge at 600 ⁇ g for 5 min, and remove supernatant.
  • Raji-PDL1 cells were conventionally subcultured; revived PBMCs were cultured with 10 mL of 1640 complete medium, and stimulated with 0.5 ⁇ g/mL SEB (Staphylococcal enterotoxin B) (Denotec, product number: S010201) for two days.
  • Raji-PDL1 cells were treated with 25 ⁇ g/mL MMC (Stressmarq, product number: SIH-246-10MG), and placed in a 37°C, 5% CO 2 carbon dioxide incubator for 1 hour; PBMCs were collected after being stimulated by SEB for 2 days and treated with MMC.
  • the 1-hour Raji-PDL1 cells were washed twice with PBS and resuspended in complete medium (ie RPMI1640+10% FBS) for counting.
  • PBMC and Raji-PDL1 were each added to a U-shaped 96-well plate (Corning, model: 3799) at 10 ⁇ 10 4 cells/well for co-cultivation.
  • antibodies according to the experimental design the final concentration of each antibody is 300nM, 30nM, 3nM when used alone or in combination
  • co-cultivate in the incubator for 3 days; after 3 days, centrifuge at 1200rpm for 5min, collect the cell culture supernatant, and perform ELISA.
  • IFN- ⁇ detection the final concentration of each antibody is 300nM, 30nM, 3nM when used alone or in combination
  • the mixed culture of human PBMC and Raji-PDL1 cells can promote the secretion of IFN- ⁇ in PBMC, and the addition of antibodies in the mixed culture system can significantly induce PBMC to further secrete IFN- ⁇ .
  • anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) combined with 14C12H1L1 (hG1TM) and the positive control antibody Relatlimab combined with 14C12H1L1 (hG1TM) could promote IFN- ⁇ secretion. And its activity is quite.
  • Raji-PDL1 cells were conventionally subcultured; revived PBMCs were cultured with 10 mL of 1640 complete medium, and 0.5 ⁇ g/mL of SEB (Staphylococcal enterotoxin B, purchased from Denotec, Cat. No.: S010201) was added to stimulate for two days.
  • Raji-PDL1 cells were treated with 25 ⁇ g/mL MMC (Stressmarq, product number: SIH-246-10MG), and placed in a 37° C., 5% CO 2 carbon dioxide incubator for 1 hour.
  • PBMC and Raji-PDL1 were each added to a U-shaped 96-well plate (Corning, model: 3799) at 10 ⁇ 10 4 cells/well for co-culture.
  • add antibodies according to the experimental design the final concentration of each antibody is 300nM, 30nM, 3nM when used alone or in combination), and co-culture for 3 days; after 3 days, centrifuge at 1200rpm for 5min, collect the cell culture supernatant, and use ELISA method to detect IL-2 detection.
  • the mixed culture of human PBMCs (from healthy donors) and Raji-PDL1 cells can promote the secretion of IL-2 in PBMCs, and adding antibodies to the mixed culture system can significantly induce PBMCs to further increase
  • the secretion of IL-2 has a significant dose-dependent relationship.
  • anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) combined with 14C12H1L1 (hG1TM), positive control Relatlimab Both 14C12H1L1 (hG1TM) can promote IL-2 secretion, and their activities are equivalent.
  • Jurkat-NFAT-PD1-LAG3 cells and Raji cells were used as reporter gene system. After adding SEE superantigen, the TCR-NFAT signaling pathway was activated to induce the expression of luciferase.
  • LAG-3 on Jurkat cells binds to MHC-II on Raji cells, inhibits the NFAT signaling pathway, and down-regulates the expression of luciferase. The antibody releases inhibition and upregulates luciferase expression by specifically binding to LAG-3.
  • Jurkat-NFAT-PD1-LAG3 cells were inoculated into black-bottom 96-well plates (Corning, model: 3916) at 105 cells/well, 30 ⁇ L/well; antibodies were added according to the experimental design (final concentrations were 0.3nM, 3nM, 300nM) , 10 ⁇ L/well, and placed in a 37°C, 5% CO 2 incubator for pre-incubation for 30 min.
  • SEE Staphylococcal Enterotoxins E, purchased from Toxin Technology, product number: ET404
  • the SEE-treated Raji cells at 2 ⁇ 104 cells/well (40 ⁇ L/well) to the above-mentioned 96-well plate containing Jurkat-NFAT-PD1-LAG3 cells, the final volume of the system is 80 ⁇ L, mix well Afterwards, they were placed in a 37°C, 5% CO 2 incubator and incubated for 6h. After the incubation, the culture plate was taken out, equilibrated to room temperature, 80 ⁇ L/well of Bright-Glo TM Luciferase Assay System (purchased from Promega, product number: E2650) was added, and the RLU value was read after incubation in the dark for 2 minutes.
  • the isotype control hIgG1DM is made by Akeso Bio, batch number: 20181107
  • the isotype control hG4WT is made by Akeso Bio, batch number: 20190910.
  • anti-LAG antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT), and the positive control antibody Relatlimab can block the interaction between LAG-3 and MHC-II, and up-regulate the expression of luciferase , and the activities of anti-LAG antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) were better than the control antibody Relatlimab.

Abstract

属于生物医药领域，涉及一种抗LAG3抗体、包含该抗LAG3抗体的药物组合物及用途。具体地，涉及一种抗LAG3抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中，所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；和所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:12-14所示的LCDR1-LCDR3。抗LAG3抗体具有优越的亲和力和特异性，具有良好的应用前景。

Description

抗LAG3抗体、药物组合物及用途 技术领域
本发明属于生物医药领域，涉及一种抗LAG3抗体、包含该抗LAG3抗体的药物组合物及用途。
背景技术
肿瘤尤其是恶性肿瘤是当今世界严重危害人类健康的疾病，在各种疾病所致死亡中高居第二位。而且近年来，其发病率呈明显上升趋势。恶性肿瘤治疗效果差，晚期转移率高，预后多不佳。目前临床上所采用的常规治疗方法如放、化疗和手术治疗虽然在很大程度上缓解了病痛，延长了生存时间，但这些方法均存在很大的局限性，其疗效难以进一步提高。
淋巴细胞活化基因3(lymphocyte-activation gene 3，LAG3)，即CD223，是一种由498个氨基酸组成的Ⅰ型跨膜蛋白，属于免疫球蛋白超家族(immunoglobulin superfamily，IgSF)成员。LAG3主要表达于活化的CD4 +T细胞和CD8 +T细胞，另外自然杀伤(natural killer，NK)细胞、B细胞、调节性T细胞(regulatory T cell，Treg)和浆细胞样树突状细胞(plasmacytoid dendritic cells，pDC)等细胞也表达LAG3。(Ruffo Elisa,Wu Richard C,Bruno Tullia C et al.Lymphocyte-activation gene 3(LAG3):The next immune checkpoint receptor.[J].Semin Immunol,2019,42:101305.)。
LAG3分子基因定位于人12号染色体(20p13.3)上，与CD4分子基因相邻，二者具有相同的外显子和内含子。虽然二者的氨基酸序列同源性只有约20％，但LAG3分子与CD4分子在结构上具有较高的相似性。主要组织相容性复合体Ⅱ类分子(MHCⅡ)、肝窦内皮细胞凝集素(LSECtin)分子和半乳糖凝集素3(galectin-3)分子是LAG3分子的相关配体。MHCⅡ类分子是LAG3的主要配体，其与MHCⅡ类分子的亲和力(Kd：60nmol·L-1)是CD4分子的100倍，表明LAG3分子能够有效竞争CD4与MHCⅡ类分子的结合，抑制T细胞活化。
肿瘤微环境中，T细胞活化24小时后便能检测到免疫抑制分子LAG3分子的表达，进而导致T细胞失能或者凋亡。LAG3分子通过其D1结构域(含有1个富含脯氨酸的环状结构)形成二聚体分子与CD4 +T细胞活化第一信号轴“CD3-TCR-MHCⅡ”中MHCⅡ 分子特异性结合，一方面阻断T细胞活化的信号转导通路，另一方面LAG3分子胞内段(KIEELE基序)产生免疫抑制信号下调CD4 +T细胞活性。LAG3分子能促进Treg细胞分化，参与信号转导及转录激活因子5下游信号，从而增强Treg细胞的抑制作用，这是肿瘤逃逸免疫***杀伤的机制之一(Andrews Lawrence P,Marciscano Ariel E,Drake Charles G et al.LAG3(CD223)as a cancer immunotherapy target.[J].Immunol Rev,2017,276:80-96.)。
多项研究表明，LAG3在各种恶性肿瘤的肿瘤浸润性CD8 +T细胞中过表达。例如卵巢癌中，肿瘤浸润的纽约食管鳞状细胞癌1抗原(NY-ESO-1)特异性CD8 +T细胞表达高水平的PD-1和LAG3，产生IFN-γ和TNF-α的能力降低，进而导致淋巴细胞失活。Galectin-3和LSECtin主要与LAG3相互作用来调节CD8 +T细胞的活化与功能。另外从黑色素瘤转移患者中分离的黑色素瘤抗原特异性T细胞在LAG3及其他免疫检查点分子CTLA-4和TIM-3表达上调显著。(刘昊,李新颖,罗龙龙,等.淋巴细胞活化基因3分子生物学功能及其抗体药物临床应用研究进展[J].中国药理学与毒理学杂志,2019,33(01):70-78.)。
目前已有多种LAG3抗体药物进入临床研究阶段，其中，百时美施贵宝的Relatlimab进展最快，目前已开展了10个临床研究，绝大多数是以Relatlimab与纳武单抗进行联合用药，用于治疗血液瘤、黑色素瘤、神经胶质母细胞瘤、肾细胞癌和非小细胞肺癌等肿瘤。
目前，尚需要开发新的抗LAG3抗体药物。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种抗LAG3抗体。本发明人惊奇地发现，本发明的抗LAG3抗体(也简称为抗体或本发明的抗体)具有优越的亲和力和/或特异性，甚至在一个或多个方面优于阳性对照抗体(例如Relatlimab)。由此提供了下述发明：
本发明的一个方面涉及一种抗LAG3抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中，
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:12-14所示的LCDR1-LCDR3；
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NO:12、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1-LCDR3；
或者
所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:15和SEQ ID NO:14所示的LCDR1-LCDR3。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体或嵌合抗体。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，所述的抗体以小于0.2nM，例如小于0.15nM、小于0.1nM、小于0.08nM、0.06nM或小于0.05nM或更小的EC 50结合人LAG3-mFc；优选地，所述EC 50通过间接ELISA方法测得。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述的抗体，其恒定区来自人抗体；
优选地，所述抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
在本发明的一些实施方式中，所述的抗体或其抗原结合片段，其中，
所述抗LAG3抗体的重链恒定区为Ig gamma-1 chain C region(例如，如SEQ ID NO: 18所示)或Ig gamma-4 chain C region(例如，如SEQ ID NO:20所示)；轻链恒定区为Ig kappa chain C region(例如，如SEQ ID NO:19所示)。
在本发明的一些实施方式中，所述的抗LAG3抗体为单克隆抗体。
在本发明的一些实施方式中，所述的抗LAG3抗体为免疫球蛋白形式。
在本发明的一些实施方式中，所述的抗LAG3抗体为单链抗体。
本发明的另一方面涉及抗体药物偶联物(antibody-drug conjugate，ADC)，其包括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为本发明中任一项所述的抗LAG3抗体或其抗原结合片段；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为肿瘤化疗药物。
所述化疗药物可以是常规的肿瘤化疗药物，例如烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗癌药、激素、免疫制剂等。
在本发明的一个或多个实施方案中，所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段通过连接子与小分子药物连接；所述连接子可以是本领域技术人员知悉的连接子，例如，所述连接子为腙键、二硫键或肽键。
在本发明的一个或多个实施方案中，所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段与小分子药物的摩尔比为1：(2－4)，例如1：2，1：3或1：4。
本发明的再一方面涉及一种分离的核酸分子，其编码本发明中任一项所述的抗LAG3抗体。
本发明的再一方面涉及一种重组载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者包含本发明的重组载体。
本发明的再一方面涉及一种制备本发明中任一项所述的抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段的步骤。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物；可选地，其还包括药学上可接受的辅 料。
本发明的再一方面涉及本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物在制备治疗和/或预防肿瘤或贫血病的药物中的用途；
优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、***癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌；
优选地，所述血液瘤为白血病；
优选地，所述食管癌为食管鳞癌。
根据本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物，其用于治疗和/或预防肿瘤或贫血病；
优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、***癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌；
优选地，所述血液瘤为白血病；
优选地，所述食管癌为食管鳞癌。
本发明的再一方面涉及一种治疗和/或预防肿瘤或贫血病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗体或其抗原结合片段、本发明中任一项所述的抗体药物偶联物的步骤；
优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、***癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
优选地，所述肺癌为非小细胞肺癌；
优选地，所述血液瘤为白血病；
优选地，所述食管癌为食管鳞癌。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本 发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.,Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,(1987 and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature 1989；342:878-883，或者IMGT编号***定义，见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307的定义。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获 得(
Figure PCTCN2022122185-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522 525；Reichmann et al.,Nature,1988；332:323 329；Presta,Curr.Op.Struct.Biol.1992；2:593-596；和Clark,Immunol.Today 2000；21:397 402。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，PD-1蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫 ***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，当提及淋巴细胞活化基因3(lymphocyte-activation gene 3，LAG3)的氨基酸序列时，其包括LAG3蛋白的全长，或者LAG3的胞外片段LAG3 ECD或者包含LAG3 ECD的片段；还包括LAG3蛋白的全长的融合蛋白或LAG3 ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在LAG3蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“添加)，蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述LAG3蛋白的序列片段时，其还包括其天然或人工变体中的相应序列片段。
发明的有益效果
本发明取得了如下效果中的一项或多项：
(1)本发明的抗LAG3抗体具有优越的亲和力和特异性；
(2)本发明的抗LAG3抗体能够有效地阻断LAG3与MHC-II相互作用，特异地解除LAG3对机体免疫抑制。
附图说明
图1：间接ELISA方法测定H7L8(hG1WT)与抗原LAG3-mFc结合活性检测结果。
图2：ELISA方法测定H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)与抗原人LAG3-mFc的结合活性。
图3：FACS检测H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)与293T-LAG3细胞表面抗原LAG3的结合活性。
图4：竞争流式细胞法测定H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)与LAG3-mFc竞争结合293T-LAG3细胞膜表面抗原MHC II检测结果。
图5：混合淋巴细胞反应MLR检测抗LAG3抗体促IFN-γ分泌的生物活性检测结果。
图6：混合淋巴细胞反应MLR检测抗LAG3抗体促IL-2分泌的生物活性检测结果。
图7：抗LAG抗体阻断LAG-3与MHC-II相互作用的生物学活性检测结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。
阳性对照抗体Relatlimab，其序列参考美国专利公开：US20160326248A1。其中重链氨基酸序列参照该专利公开中的SEQ ID NO:1，轻链氨基酸序列参照该专利公开中的SEQ ID NO:2。Relatlimab是抗LAG-3抗体。
Relatlimab的重链氨基酸序列：
Figure PCTCN2022122185-appb-000002
Relatlimab的轻链氨基酸序列：
Figure PCTCN2022122185-appb-000003
对照抗体14C12H1L1(hG1TM)为抗PD-1抗体，由康方生物制备，批号B105Y2080601。
14C12H1L1(hG1TM)的重链氨基酸序列：
Figure PCTCN2022122185-appb-000004
Figure PCTCN2022122185-appb-000005
14C12H1L1(hG1TM)的轻链的氨基酸序列
Figure PCTCN2022122185-appb-000006
293T-LAG3细胞系由中山康方生物医药有限公司构建。293T-LAG3细胞系由HEK293T细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为plenti6.3/V5-huLAG3FL-BSD(其中LAG3，Genebank ID:NM_002277.4；载体plenti6.3/V5-BSD，购自Invitrogen，产品编号：K5315-20)。
Raji-PDL1细胞系由中山康方生物医药有限公司构建。Raji-PDL1细胞系由Raji细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为plenti6.3/V5-PDL1(其中PDL1，Genebank ID:NP_054862.1；载体plenti6.3/V5，购自Invitrogen，货号：K5315-20)。
Jurkat-NFAT-PD1-LAG3细胞系由中山康方生物医药有限公司构建。Jurkat-NFAT-PD1-LAG3细胞系由PD-1效应细胞(PD-1 Effector cells(CPM)，厂家：Promega，货号：J112A)细胞经病毒感染制得，病毒制备使用的是3rd Generation Lentiviral Systems，参见，例如A Third Generation Lentivirus Vector with a Conditional Packaging System.Dull T,Zufferey R,Kelly M,Mandel RJ,Nguyen M,Trono D,and Naldini L.J Virol.1998.72(11):8463-8471.其中所使用的慢病毒表达载体为pCDH-huLAG3FL-RFP-NEO(其中LAG3，Genebank ID:NM_002277.4；载体pCDH-CMV-MCS-EF1-RFP+Neo，购自优宝生物，产品编号：VT9005)。
制备例1：抗LAG3抗体的设计和制备
1.抗体的设计
本发明人根据已有的LAG3蛋白序列(NCBI Reference Sequence:NP_002277.4)及其蛋白三维晶体结构等，创造性地人工设计了一系列的抗体序列。通过大量的筛选和检测，最终得到了与LAG3特异性结合的人源化单克隆抗体，分别命名为H7L8、H7L9、H7L10。该单克隆抗体重链和轻链可变区的氨基酸序列及其编码序列如下。
H7L8的重链可变区H7v的核酸序列(360bp)：
Figure PCTCN2022122185-appb-000007
H7L8的重链可变区H7v的氨基酸序列(120aa)：
Figure PCTCN2022122185-appb-000008
H7L8的轻链可变区L8v的核酸序列(321bp)：
Figure PCTCN2022122185-appb-000009
H7L8的轻链可变区L8v的氨基酸序列(107aa)：
Figure PCTCN2022122185-appb-000010
H7L9的重链可变区H7v的核酸序列与H7L8的重链可变区H7v的核酸序列相同，即SEQ ID NO:1。
H7L9的重链可变区H7v的氨基酸序列与H7L8的重链可变区H7v的氨基酸序列相同，即SEQ ID NO:2。
H7L9的轻链可变区L9v的核酸序列(321bp)：
Figure PCTCN2022122185-appb-000011
H7L9的轻链可变区L9v的氨基酸序列(107bp)：
Figure PCTCN2022122185-appb-000012
H7L10的重链可变区H7v的核酸序列与H7L8的重链可变区H7v的核酸序列相同，即SEQ ID NO:1。
H7L10的重链可变区H7v的氨基酸序列与H7L8的重链可变区H7v的氨基酸序列相同，即SEQ ID NO:2。
H7L10的轻链可变区L10v的核酸序列(321bp)：
Figure PCTCN2022122185-appb-000013
H7L10的轻链可变区L10v的氨基酸序列(107bp)：
Figure PCTCN2022122185-appb-000014
抗体H7L8的CDR的氨基酸序列如下(按照IMGT编号***)：
HCDR1：GGSISDYY(SEQ ID NO:9)；
HCDR2：INHRGTT(SEQ ID NO:10)；
HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:11)；
LCDR1：QTISSY(SEQ ID NO:12)；
LCDR2：DAS(SEQ ID NO:13)；
LCDR3：QQRSNWPIT(SEQ ID NO:14)。
抗体H7L9的CDR的氨基酸序列如下(按照IMGT编号***)：
HCDR1：GGSISDYY(SEQ ID NO:9)；
HCDR2：INHRGTT(SEQ ID NO:10)；
HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:11)；
LCDR1：QTISSY(SEQ ID NO:12)；
LCDR2：DGS(SEQ ID NO:15)；
LCDR3：QQRSNWPLT(SEQ ID NO:16)。
抗体H7L10的CDR的氨基酸序列如下(按照IMGT编号***)：
HCDR1：GGSISDYY(SEQ ID NO:9)；
HCDR2：INHRGTT(SEQ ID NO:10)；
HCDR3：AFGYSDYEYDWFDP(SEQ ID NO:11)；
LCDR1：QSISSY(SEQ ID NO:17)；
LCDR2：DGS(SEQ ID NO:15)；
LCDR3：QQRSNWPIT(SEQ ID NO:14)。
2.人源化抗体H7L8(hG1WT)的表达和纯化
将H7L8(hG1WT)的重链cDNA序列(可变区编码序列如SEQ ID NO:1所示；恒定区是Ig gamma-1 chain C region)和轻链的cDNA序列(可变区编码序列如SEQ ID NO:3所示；恒定区为human Ig kappa chain C region)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-H7和pUC57simple-L8质粒。分别将质粒pUC57simple-H7和pUC57simple-L8进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
H7L8(hG1WT)重链恒定区氨基酸序列
Figure PCTCN2022122185-appb-000015
H7L8(hG1WT)轻链恒定区氨基酸序列
Figure PCTCN2022122185-appb-000016
3.人源化抗体H7L8(hG4WT)、H7L9(hG4WT)以及H7L10(hG4WT)的表达和纯化
将H7L8(hG4WT)、H7L9(hG4WT)以及H7L10(hG4WT)的重链cDNA序列(可变区编码序列如SEQ ID NO:1所示；恒定区是Ig gamma-4 chain C region)和H7L8(hG4WT)轻链的cDNA序列(可变区编码序列如SEQ ID NO:3所示；恒定区为human Ig kappa chain C region)、H7L9(hG4WT)轻链的cDNA序列(可变区编码序列如SEQ ID NO:5所示；恒定区为human Ig kappa chain C region)、H7L10(hG4WT)轻链的cDNA序列(可变区编码序列如SEQ ID NO:7所示；恒定区为human Ig kappa chain C region)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-H7和pUC57simple-L8、pUC57simple-L9、pUC57simple-L10质粒。分别将质粒pUC57simple-H7和pUC57simple-L8、pUC57simple-L9、pUC57simple-L10进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
H7L8(hG4WT)、H7L9(hG4WT)或H7L10(hG4WT)重链恒定区氨基酸序列：
Figure PCTCN2022122185-appb-000017
Figure PCTCN2022122185-appb-000018
制备例2：人抗鸡蛋溶酶体抗体的制备
人抗鸡蛋溶酶体抗体(human anti-Hen Egg Lysozyme IgG，anti-HEL，即human IgG，简称hIgG)，其序列来自于Acierno等人发表的Affinity maturation increases the stabil ity and plasticity of the Fv domain of anti-protein antibodies研究中Fab F10.6.6序列的可变区序列(Acierno等人.J Mol Biol.2007；374(1):130-46.)。制备方法如下：
human IgG委托南京金斯瑞生物对抗体的重轻链(全序列或可变区)基因进行氨基酸的密码子优化和基因合成，参照《分子克隆实验指南(第三版)》介绍的标准技术，采用PCR、酶切、DNA胶回收、连接转化、菌落PCR或酶切鉴定等标准的分子克隆技术将重轻链基因分别亚克隆到哺乳动物表达***的抗体重链表达载体和抗体轻链表达载体，并进一步对重组表达载体的重轻链基因进行测序分析。测序验证正确后，中大量制备去内毒素级别的表达质粒并将重轻链表达质粒瞬时共转染HEK293细胞进行重组抗体的表达。培养7天后收集细胞培养液，进行rProtein A柱(GE)亲和纯化，收获的抗体样品用SDS-PAGE和SEC-HPLC标准分析技术对其进行质量鉴定。
实验例1：ELISA方法测定抗LAG3抗体与抗原的结合活性
将人LAG3-mFc(康方生物自制，批号：20200417)，0.5μg/mL包被酶标板置于4℃孵育过夜，然后使用PBST洗板包被了抗原的酶标板1次，再使用1％BSA的PBS溶液作为封闭液在37℃下对酶标板进行封闭2小时。酶标板封闭结束后用PBST洗板3次，加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表1)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1:5000比例稀释的HRP标记羊抗人IgG FC(H+L)(Jackson,货号：109-035-098)二抗工作液，然后置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表1和图1所示。
表1：ELISA检测Relatlimab、H7L8(hG1WT)与LAG3-mFc的结合
Figure PCTCN2022122185-appb-000019
由图1可知，Relatlimab、H7L8(hG1WT)与抗原人LAG3-mFc能够有效的结合，并且其结合效率呈剂量依赖关系，各剂量的吸光度强度见表1，通过对结合的抗体进行吸光度定量分析，曲线模拟计算获得抗体Relatlimab(作为阳性对照)、H7L8(hG1WT)的结合效率EC 50分别为0.106nM、0.045nM。
以上实验结果表明，在相同实验条件下，H7L8(hG1WT)具有有效结合人LAG3-mFc的活性，且H7L8(hG1WT)结合人LAG3-mFc的活性强于靶点阳性药Relatlimab。
实施例2：ELISA方法测定抗LAG3抗体与抗原的结合活性
将人LAG3-mFc，2μg/mL包被酶标板置于4℃孵育过夜，然后使用PBST洗板包被了抗原的酶标板1次，再使用1％BSA的PBS溶液作为封闭液在37℃下对酶标板进行封闭2小时。酶标板封闭结束后用PBST洗板3次，加入PBST溶液梯度稀释的抗体(抗体稀释梯度详见表1)，加入待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1:5000比例稀释的HRP标记羊抗人IgG(H+L)(Jackson,货号：109-035-088)二抗工作液，然后置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标 板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
检测结果如表2和图2所示。
表2：ELISA检测H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)与抗原人LAG3-mFc的结合
Figure PCTCN2022122185-appb-000020
结果显示：抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)均能有效地结合抗原人LAG3-mFc，结合效率呈剂量依赖关系，且其结合活性与阳性对照抗体Relatlimab相当。
实施例3：流式细胞仪方法检测抗LAG3抗体与细胞表面抗原LAG3的结合活性
1.表达LAG3抗原的宿主细胞293T的构建
具体步骤如下：
表达LAG3抗原的宿主细胞293T的构建：按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将包含LAG3的载体pLenti6.3/V5-huLAG3FL-BSD(载体pLenti6.3购自Invitrogen公司)转染293T细胞，经筛选获得稳定表达LAG3的克隆群体293T-LAG3。
2.抗体对293T-LAG3细胞表面抗原的结合
抗体标记和流式细胞仪检测：采用常规胰酶消化方法上述步骤获得的表达LAG3抗原的宿主细胞293T-LAG3，并使每个收集管细胞数为3×10 5，用1％PBSA(含1％BSA的PBS)配制终浓度分别为0.0123nM，0.123nM，1.23nM，3.7nM，11.1nM，33.3nM，100nM，300nM的LAG3抗体稀释液，冰上与表达LAG3的293T细胞孵育1小时。用1％PBSA离心洗涤数次后，每管加入100μL FITC羊抗人IgG(购自Jackson，货号：109-095-098)(1：500倍稀释)冰上避光孵育40min。用1％PBSA洗1次后加入200μL 1％PBSA重悬细胞，在流式细胞仪上用FITC通道检测荧光信号。
人源化抗LAG3抗体与293T-LAG3细胞的结合结果如图3所示。各抗LAG3抗体与293T-LAG3表面抗原的结合效率EC 50如表3所示。
表3：流式细胞仪检测抗LAG3抗体结合293T-LAG3表面抗原的结合活性
  EC 50(nM)
Relatlimab 4.289
H7L8(hG4WT) 4.929
H7L9(hG4WT) 4.809
H7L10(hG4WT) 4.168
由图3可见，抗LAG3抗体能有效地结合宿主细胞293T-LAG3表面的靶标LAG3蛋白，且抗LAG3抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)与293T-LAG3表面抗原的结合活性和阳性对照抗体Relatlimab相当。
实施例4：竞争流式细胞法测定抗LAG3抗体与LAG3-mFc竞争结合细胞膜表面抗 原MHC II的竞争结合活性
将Raji细胞(培养基：1640+10％FBS)(中国科学院上海生命科学研究院细胞资源中心，货号：TCHu 44)按30万个细胞/样本加入EP管中，加入1000μL 1％PBSA(含1％BSA的PBS)，600×g离心5min弃上清。按实验设计每管加入终浓度为300nM的hIgG1(康方生物制备，批号：20190410)，每管100μL冰上孵育1h；向孵育后的Raji细胞加入200μL 1％PBSA，600×g离心5min，去上清。与此同时，按实验设计另取洁净的EP管，加入相应浓度稀释抗体(300nM、100nM、33.3nM、11.1nM、3.7nM、1.23nM、0.123nM、0.0123nM)，每管60μL，并设计Blank(PBSA+细胞)；然后分别将60μL的LAG3-mFc(康方生物制备，批号：20190508)(终浓度为3nM)加入相应的抗体管中，混匀，冰上预孵育30min。向样本加入预孵育完的100μL的抗体与蛋白混合液，混匀后冰上避光孵育1h；加入200μL 1％PBSA，600×g离心5min，去上清，洗两次；加入100μL的APC anti mouse抗体(购自Biolegend，货号：405308)(1:400倍稀释)，混匀后冰上避光孵育40min；加入200μL 1％PBSA，600×g离心5min，去上清；200μL Washing Buffer/管，重悬细胞，转移至流式上样管，上机测试。
结果如图4、表4所示。通过荧光定量分析和曲线拟合，计算出抗体Relalimab、H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)的竞争结合EC 50分别为1.153nM、1.342nM、1.317nM、1.267nM。
表4：FACS检测Relalimab、H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)竞争结合Raji细胞表面抗原的荧光强度分析
  EC 50(nM)
Relatlimab 1.153
H7L8(hG4WT) 1.342
H7L9(hG4WT) 1.317
H7L10(hG4WT) 1.267
结果显示，抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)能有效地阻断LAG-3与Raji宿主细胞表面的MHC II的结合，且呈剂量依赖关系，其活性与阳性对照抗体Relatlimab相当。
实验例5：混合淋巴细胞反应MLR检测抗LAG3抗体促IFN-γ和IL-2分泌的生物 活性
1.抗LAG3抗体促Raji-PDL1混合淋巴反应体系分泌IFN-γ的生物活性检测
Raji-PDL1细胞常规传代培养；复苏PBMC，用10mL 1640完全培养基培养，0.5μg/mL的SEB(Staphylococcal enterotoxin B)(德诺泰克，货号：S010201)刺激两天。Raji-PDL1细胞用25μg/mL的MMC(Stressmarq，货号：SIH-246-10MG)处理，置于37℃、5％CO 2二氧化碳培养箱中1小时；收集经SEB刺激2天后PBMC和经MMC处理1小时的Raji-PDL1细胞，用PBS洗涤两次后用完全培养基(即RPMI1640+10％FBS)重悬计数。将PBMC和Raji-PDL1各按10×10 4个细胞/孔加入到U型96孔板(Corning，型号：3799)中共培养。并按实验设计加入抗体(单用和联用时各个抗体的终浓度均为300nM、30nM、3nM)，于培养箱共培养3天；3天后，1200rpm离心5min，收集细胞培养上清，ELISA法进行IFN-γ检测。
如图5所示，人PBMC和Raji-PDL1细胞混合培养后对PBMC的IFN-γ的分泌具有促进作用，而在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IFN-γ，在促IFN-γ分泌活性水平方面，抗LAG3抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)分别联用14C12H1L1(hG1TM)、阳性对照抗体Relatlimab联用14C12H1L1(hG1TM)均能促IFN-γ分泌，且其活性相当。
2.抗LAG抗体促Raji-PDL1混合淋巴反应体系分泌IL-2的生物活性检测
Raji-PDL1细胞常规传代培养；复苏PBMC，用10mL 1640完全培养基培养，添加0.5μg/mL的SEB(Staphylococcal enterotoxin B，购自德诺泰克，货号：S010201)刺激两天。Raji-PDL1细胞用25μg/mL的MMC(Stressmarq，货号：SIH-246-10MG)处理，置于37℃、5％CO 2二氧化碳培养箱中1小时。收集经SEB刺激2天后的PBMC和经MMC处理1小时的Raji-PDL1细胞，用PBS洗两次后用完全培养基(即RPMI1640+10％FBS)重悬计数。将PBMC和Raji-PDL1各按10×10 4个细胞/孔加入U型96孔板(Corning，型号：3799)中共培养。并按实验设计加入抗体(单用和联用时各个抗体的终浓度均为300nM、30nM、3nM)，共培养3天；3天后，1200rpm离心5min，收集细胞培养上清，ELISA法进行IL-2检测。
如图6所示，人PBMC(来自健康捐献者)和Raji-PDL1细胞混合培养后对PBMC的IL-2的分泌有一定的促进作用，而在混合培养体系中同时加入抗体能显著诱导PBMC进一步分泌IL-2，具有显著剂量依赖关系，在促IL-2分泌活性水平方面，抗LAG3 抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)分别联用14C12H1L1(hG1TM)、阳性对照Relatlimab联用14C12H1L1(hG1TM)均能促IL-2分泌，且其活性相当。
实验例6：抗LAG抗体阻断LAG-3与MHC-II相互作用的生物学活性评价(报告基 因法)
以Jurkat-NFAT-PD1-LAG3细胞和Raji细胞作为报告基因体系，加入SEE超抗原后，激活TCR-NFAT信号通路，诱导荧光素酶的表达。而Jurkat细胞上的LAG-3与Raji细胞上的MHC-II结合，抑制NFAT信号通路，下调荧光素酶的表达。抗体通过特异性结合LAG-3，解除抑制，上调荧光素酶的表达。
收集Jurkat-NFAT-PD1-LAG3细胞和Raji细胞(购自中国科学院上海生命科学研究院细胞资源中心，货号：TCHu 44)，110xg离心5min去上清，1640+10％FBS培养基重悬细胞，计数。Jurkat-NFAT-PD1-LAG3细胞按10 5个细胞/孔接种至黑底96孔板(Corning，型号：3916)，30μL/孔；按实验设计加入抗体(终浓度为0.3nM、3nM、300nM)，10μL/孔，置于37℃，5％CO 2培养箱中预先孵育30min。与此同时，向Raji细胞加入SEE(Staphylococcal Enterotoxins E，购自Toxin Technology，货号：ET404)(终浓度0.05ng/mL)后置于37℃，5％CO 2培养箱中孵育30min。30min后，将经SEE处理的Raji细胞按2×10 4个细胞/孔(40μL/孔)加至上述含有Jurkat-NFAT-PD1-LAG3细胞的96孔板中，体系终体积为80μL，混匀后置于37℃，5％CO 2培养箱中孵育6h。孵育完毕后取出培养板，平衡至室温，加入Bright-Glo TMLuciferase Assay System(购自Promega，货号：E2650)80μL/孔，避光孵育2min后读取RLU值。其中同型对照hIgG1DM为康方生物自制，批号：20181107；同型对照hG4WT为康方生物自制，批号：20190910。
结果如图7所示，抗LAG抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)、阳性对照抗体Relatlimab均能阻断LAG-3与MHC-II的相互作用，上调荧光素酶的表达，且抗LAG抗体H7L8(hG4WT)、H7L9(hG4WT)、H7L10(hG4WT)的活性均优于对照抗体Relatlimab。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (18)

  1. 抗LAG3抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中，
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NOs:12-14所示的LCDR1-LCDR3；
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NO:12、SEQ ID NO:15和SEQ ID NO:16所示的LCDR1-LCDR3；
    或者
    所述重链可变区包含：氨基酸序列分别如SEQ ID NOs:9-11所示的HCDR1-HCDR3；并且所述轻链可变区包含：氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:15和SEQ ID NO:14所示的LCDR1-LCDR3。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中，
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:4所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
    或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
  3. 根据权利要求1至2中任一权利要求所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体或嵌合抗体。
  4. 根据权利要求1至3中任一权利要求所述的抗体或其抗原结合片段，其中，所述的抗体以小于0.2nM，例如小于0.15nM、小于0.1nM、小于0.08nM、0.06nM或小于0.05nM或更小的EC 50结合人LAG3-mFc；优选地，所述EC 50通过间接ELISA方法测得。
  5. 根据权利要求1至4中任一权利要求所述的抗体或其抗原结合片段，其中，
    所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  6. 根据权利要求1至5中任一权利要求所述的抗体或其抗原结合片段，其中，
    所述的抗体，其恒定区来自人抗体；
    优选地，所述抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
  7. 根据权利要求1至6中任一权利要求所述的抗体或其抗原结合片段，其中，
    所述抗LAG3抗体的重链恒定区为Ig gamma-1 chain C region(例如，如SEQ ID NO:18所示)或Ig gamma-4 chain C region(例如，如SEQ ID NO:20所示)；轻链恒定区为Ig kappa chain C region(例如，如SEQ ID NO:19所示)。
  8. 抗体药物偶联物，其包括抗体或其抗原结合片段以及小分子药物，其中，所述抗体或其抗原结合片段为权利要求1至7中任一权利要求所述的抗体或其抗原结合片段；优选地，所述小分子药物为小分子细胞毒药物；更优选地，所述小分子药物为肿瘤化疗药物。
  9. 根据权利要求8所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段通过连接子与小分子药物连接；例如，所述连接子为腙键、二硫键或肽键。
  10. 根据权利要求8或9所述的抗体药物偶联物，其中，所述抗体或其抗原结合片段与小分子药物的摩尔比为1：(2－4)。
  11. 分离的核酸分子，其编码权利要求1至7中任一权利要求所述的抗LAG3抗体。
  12. 一种重组载体，其包含权利要求11所述的分离的核酸分子。
  13. 一种宿主细胞，其包含权利要求11所述的分离的核酸分子，或者包含权利要求12所述的重组载体。
  14. 制备权利要求1至7中任一权利要求所述的抗体或其抗原结合片段的方法，其包括 在合适的条件下培养权利要求13的宿主细胞，以及从细胞培养物中回收所述抗体或其抗原结合片段的步骤。
  15. 一种药物组合物，其包含权利要求1至7中任一权利要求所述的抗体或其抗原结合片段或者权利要求8至10中任一权利要求所述的抗体药物偶联物；可选地，其还包括药学上可接受的辅料。
  16. 权利要求1至7中任一权利要求所述的抗体或其抗原结合片段或者权利要求8至10中任一权利要求所述的抗体药物偶联物在制备治疗和/或预防肿瘤或贫血病的药物中的用途；
    优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、***癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌；
    优选地，所述血液瘤为白血病；
    优选地，所述食管癌为食管鳞癌。
  17. 根据权利要求1至7中任一权利要求所述的抗体或其抗原结合片段或者权利要求8至10中任一权利要求所述的抗体药物偶联物，其用于治疗和/或预防肿瘤或贫血病；
    优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、肾细胞癌、肺癌、***癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌；
    优选地，所述血液瘤为白血病；
    优选地，所述食管癌为食管鳞癌。
  18. 一种治疗和/或预防肿瘤或贫血病的方法，包括给予有需求的受试者以有效量的权利要求1至7中任一权利要求所述的抗体或其抗原结合片段或者权利要求8至10中任一权利要求所述的抗体药物偶联物的步骤；
    优选地，所述肿瘤为选自卵巢癌、食管癌、黑色素瘤、血液瘤、神经胶质母细胞瘤、 肾细胞癌、肺癌、***癌、膀胱癌、结肠癌、直肠癌、肝癌、胃肠道癌、乳腺癌、脑癌、胰腺癌、甲状腺癌、头颈癌和肾癌中的一种或多种；
    优选地，所述肺癌为非小细胞肺癌；
    优选地，所述血液瘤为白血病；
    优选地，所述食管癌为食管鳞癌。
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