WO2019238074A1 - Anticorps lag-3 ayant une affinité élevée et une haute activité biologique, et utilisation associée - Google Patents

Anticorps lag-3 ayant une affinité élevée et une haute activité biologique, et utilisation associée Download PDF

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WO2019238074A1
WO2019238074A1 PCT/CN2019/090979 CN2019090979W WO2019238074A1 WO 2019238074 A1 WO2019238074 A1 WO 2019238074A1 CN 2019090979 W CN2019090979 W CN 2019090979W WO 2019238074 A1 WO2019238074 A1 WO 2019238074A1
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antibody
lag
variable region
chain variable
heavy chain
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Chinese (zh)
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蔡明清
彭国媛
俞玲
冯玉杰
于海佳
朱向阳
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华博生物医药技术（上海）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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the field of medicine, in particular to a LAG-3 antibody with high affinity and high biological activity and application thereof.
  • Lymphocyte activation gene 3 (LAG-3, also referred to as CD223) protein is a member of the immunoglobulin superfamily and was first discovered by Triebel et al. In 1990 (J Exp Exp Med, 1990, 171 (5) : 1393-405).
  • the gene of LAG-3 is located at the tip of the short arm of human chromosome 12, next to the CD4 gene.
  • LAG-3 protein is a class I transmembrane protein, which consists of three parts: extracellular region, transmembrane region and cytoplasmic region.
  • the mature LAG-3 molecule includes 498 amino acids and has a relative molecular weight of approximately 55 kDa.
  • LAG-3 plays an important role in immune system dysfunction caused by viral infections, autoimmune diseases, and tumors. In some diseases, LAG-3 expression is elevated and consequently immunosuppression occurs. See et al. Found that in the blood and tumor tissues of patients with Hodgkin lymphoma, lymphocytes highly express LAG-3; the function of specific CD8 + T cells in tumor tissues is significantly impaired. If LAG-3 positive T cells are removed, their resistance Tumor function can be restored and cytokine secretion increased. Based on this, it is speculated that the expression of LAG-3 is related to the negative immune regulatory function of specific T cells. Inhibiting the function of LAG-3 molecule can enhance the anti-tumor effect of T cells. This molecule may be a potential target for tumor immunotherapy ( Blood, 2006, 108 (7) 2280-9). Several multinational pharmaceutical companies such as BMS and Novartis are developing monoclonal antibodies against LAG-3.
  • the purpose of the present invention is to provide a LAG-3 antibody with high affinity and high biological activity and its application.
  • a heavy chain variable region of an antibody includes the following three complementary determining regions CDRs:
  • any one of the above amino acid sequences further includes at least one (e.g., 1-3, preferably 1-2, more preferably) optionally added, deleted, modified, and / or substituted.
  • the heavy chain variable region further includes a human-derived FR region or a mouse-derived FR region.
  • the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.:1.
  • variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO :: 8.
  • a heavy chain of an antibody said heavy chain having the heavy chain variable region according to 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 includes the following three complementary determining regions (CDRs):
  • amino acid sequence is CDR2 'of LVS
  • any one of the above amino acid sequences further includes at least one (e.g., 1-3, preferably 1-2, more preferably) optionally added, deleted, modified, and / or substituted.
  • the light chain variable region further includes a human-derived FR region or a mouse-derived FR region.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO :: 2.
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO :: 9.
  • a light chain of an antibody having the light chain variable region according to 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 according to the second aspect of the invention; and / or a light chain according to the fourth aspect of the invention.
  • the antibody affinity to human LAG-3 protein (preferably wild type) of the EC 50 is 40-80ng / ml.
  • the antibody affinity to human LAG-3 protein (preferably wild type) of the EC 50 of 63.3ng / ml.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • sequence of the heavy chain variable region of the antibody is shown in SEQ ID No .: 1 or 8; and / or the sequence of the light chain variable region of the antibody is shown in SEQ ID No .: 2 Or as shown in 9.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.:1; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.:2.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.:8; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.:9.
  • the antibody is in the form of a drug conjugate.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a 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 LAG-3, and the scFv has The heavy chain variable region according to the first aspect of the present invention and the light chain variable region according to the third aspect of the present invention.
  • an eighth aspect of the present invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the seventh aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are derived from a human or non-human mammal (such as a mouse).
  • an antibody drug conjugate is provided.
  • the antibody drug conjugate contains:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody portion and the coupling portion are coupled by a chemical bond or a linker.
  • a tenth aspect of the present invention provides the use of an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present invention, and a heavy chain according to the second aspect of the present invention.
  • Chain, a light chain variable region according to the third aspect of the present invention, a light chain according to the fourth aspect of the present invention, or an antibody according to the fifth aspect of the present invention, a recombinant according to the sixth aspect of the present invention A protein, an immune cell according to the eighth aspect of the present invention, an antibody drug conjugate according to the ninth aspect of the present invention, or a combination thereof, the active ingredient is used for
  • the LAG-3 related disease is selected from the group consisting of a tumor, an inflammatory reactive disease, or a combination thereof.
  • the drug or preparation is a LAG-3 inhibitor.
  • the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.
  • the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • Hodgkin's lymphoma or a combination thereof.
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small
  • the tumor is a tumor that highly expresses LAG-3.
  • the medicament or preparation is used for preparing a medicament or preparation for preventing and / or treating a disease related to LAG-3 (positive expression).
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is used to diagnose LAG-3 related diseases.
  • the detection reagent or kit is used for detecting LAG-3 protein in a sample.
  • the detection reagent is a detection sheet.
  • a pharmaceutical composition is provided, and the pharmaceutical composition contains:
  • an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, and a third aspect of the invention
  • Light chain variable region, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, or the eighth aspect of the present invention Immune cells, the antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used to inhibit LAG-3, preferably to down regulate or block the immunosuppressive effect of LAG-3.
  • the pharmaceutical composition is used to enhance immunity, preferably to stimulate the activation, proliferation, and secretion of cytokines of immune cells (such as T cells).
  • the pharmaceutical composition is used to improve the immune response to tumors, preferably to enhance the killing effect of immune cells on tumor cells.
  • the pharmaceutical composition is used for treating tumors.
  • the tumor is a tumor that highly expresses LAG-3.
  • a polynucleotide encoding a polypeptide selected from the group consisting of:
  • a vector containing the polynucleotide according to the twelfth aspect of the present invention is provided.
  • the vector includes: a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • a fourteenth aspect of the present invention there is provided a genetically engineered host cell containing the vector according to the thirteenth aspect of the present invention or a genome integrated with the vector according to the twelfth aspect of the present invention.
  • a method for in vitro detection (including diagnostic or non-diagnostic) of a LAG-3 protein in a sample comprising the steps:
  • a detection plate includes: a substrate (support plate) and a test strip, wherein the test strip contains the antibody according to the fifth aspect of the present invention, or The immunoconjugate according to the ninth aspect of the present invention.
  • a seventeenth aspect of the present invention provides a kit, wherein the kit includes:
  • the kit contains a detection plate according to the sixteenth aspect of the present invention.
  • An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, wherein the method includes:
  • a method for a LAG-3 related disease characterized in that the method includes: administering to a subject in need the antibody according to the fifth aspect of the present invention, the antibody of the antibody -A drug conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.
  • Figure 1 shows the binding of a humanized LAG-3 antibody to CHO-K1 cells overexpressing human LAG-3.
  • Figure 2 shows the blocking effect of humanized LAG-3 antibody on the binding of human LAG-3 antibody to MHC II molecules.
  • FIG. 3 shows the activation of T cell activity by humanized LAG-3 antibodies.
  • the antibody of the present invention can bind to the LAG-3 antigen with high specificity, has high affinity and biological activity, and significantly blocks the binding of LAG-3 to MHC-II without any visible toxic side effects on the mammal itself.
  • the antibody of the invention stimulates the antigen-specific T cell response and enhances the anti-tumor effect of T cells, thereby maximizing the patient's own immune system response to the tumor and achieving the purpose of killing tumor cells.
  • the present invention has been completed on this basis.
  • amino acid three-letter codes and single-letter codes used in the present invention are described in J. biol. Chem, 243, p3558 (1968).
  • administering refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering and “treatment” may refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes the contact of reagents with cells, the contact of reagents with fluids, and the contact of fluids with cells.
  • administering and “treating” also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition or by another cell.
  • Treatment when applied to humans, animals, or research subjects refers to the treatment, prevention, or prophylactic measures, research, and diagnosis; including anti-human LAG-3 antibodies and humans or animals, subjects, cells, tissues , Contact with physiological compartments or physiological fluids.
  • treatment refers to the administration of an internal or external therapeutic agent to a patient, comprising any one of the anti-human LAG-3 antibodies of the present invention and a composition thereof, said patient having one or more symptoms of a disease and known
  • the therapeutic agent has a therapeutic effect on these symptoms.
  • a patient is administered in an amount (a therapeutically effective amount) of a therapeutic agent effective to alleviate the symptoms of one or more diseases.
  • an antibody heavy chain variable region of a specific sequence may be, but is not required to be, one, two, or three.
  • sequence identity means the degree of identity between two nucleic acids or two amino acid sequences when there is optimal alignment and comparison in the case of mutations such as appropriate substitutions, insertions, or deletions.
  • sequence identity between the sequence described in the present invention and the sequence having the same identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 100%.
  • LAG-3 refers to lymphocyte activating gene 3, which LAG-3 includes variants, isoforms, homologs, orthologs, and paralogs.
  • human LAG-3 refers to the complete amino acid sequence of human sequence LAG-3, such as human LAG-3 with Uniprot number P18627.
  • LAG-3 is also known in the art, such as CD223.
  • the human LAG-3 sequence may differ from human LAG-3 of Uniprot: P18627 in that it has, for example, a conservative mutation or a mutation in a non-conserved region, and that human LAG-3 of LAG-3 and Uniprot: P18627 has substantial On the same biological function.
  • the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3, and the epitope is specifically bound by the antibody disclosed in this patent, or the biological function of human LAG-3 is to bind MHC-II molecule.
  • the specific human LAG-3 sequence is generally at least 90% identical to the human LAG-3 of Uniprot No. P18627 in the amino acid sequence, and contains a human amino acid sequence that is identified when compared to the LAG-3 amino acid sequence of other species (e.g., rodents). Amino acid residues.
  • human LAG-3 may be at least 85% or even at least 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to LAG-3 of Uniprot No .: P18627.
  • the human LAG-3 sequence shows no more than 10 amino acid differences from the LAG-3 sequence of Uniprot number: P18627.
  • human LAG-3 may show no more than 5 or even no more than 4, 3, 2 or 1 amino acid difference compared to Uniprot: P18627LAG-3 sequence. Percent identity can be determined as set forth herein.
  • LAG-3 and CD4 have a high structural similarity, and both can bind to MHC-II (main histocompatibility complex) molecules. Compared with CD4 molecules, LAG-3 binds more to MHC-II molecules. In order to be compact, it can compete with CD4 molecules for binding to MHC-II molecules (Curr, Opin, Immunol, 2009, 21 (2): 179-86; Eur, Immunol, 2003, 33 (4): 970-9). LAG-3 is mainly expressed on activated T lymphocytes, NK cells, B lymphocytes, Treg cells, and dendritic cells.
  • LAG-3 is a negative co-stimulatory molecule that can negatively regulate lymphocyte function.
  • LAG-3 can inhibit the antigen-induced T lymphocyte proliferation response, block LAG-3, T lymphocyte activation and proliferation, and type 1 helper T cells (Th1 helper cells, Th1) secretion
  • Th1 helper cells Th1 helper cells
  • anti-LAG-3 antibodies can also maintain the homeostasis of CD4 + and CD8 + T lymphocytes, and the ability of CD8 + T lymphocytes to kill tumor cells is significantly enhanced after blocking LAG-3 (Jlin Clin Invest, 2007, 117 (11): 3383-92).
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond.
  • the amino acid composition and arrangement of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , Alpha, and epsilon chains.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain according to the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the antibody light chain of the present invention may further comprise a light chain constant region, and the light chain constant region comprises a human or murine ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the present invention may further include a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four backbone regions (FR) whose sequences are relatively conserved. Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions.
  • the sequence from amino terminal to end is: FR1, CDR1, FR2 , CDR2, FR3, CDR3 and FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, and preferably humanized antibodies.
  • the term "murine antibody” in the present invention is a monoclonal antibody against human LAG-3 prepared according to the knowledge and skills in the art. Test subjects are injected with LAG-3 antigen during preparation, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.
  • the mouse-derived LAG-3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived ⁇ , ⁇ chain or a variant thereof, or further include a mouse-derived IgG1 , IgG2, IgG3, or a variant of the heavy chain constant region thereof.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can alleviate the immune response response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibodies Antibodies produced in framework sequences. Humanized antibodies can overcome the heterogeneous response induced by chimeric antibodies because they carry a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence may be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, LAG-3). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of the antibody. Examples of binding fragments included in the term “antigen-binding fragment of an antibody” include
  • Fab fragment a monovalent fragment consisting of VL, VH, CL, and CH1 domains
  • the Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but has no constant region, and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a LAG-3 molecule).
  • An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation.
  • the terms “specifically bind”, “selectively bind”, “selectively bind” and “specifically bind” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is less than about 10 -7 M, such as about less than 1O -8 M, 1O -9 M or lO -10 M or smaller affinity (KD) binding.
  • competitive binding refers to an antibody that recognizes the same epitope (also called an epitope) or a part of the same epitope on the extracellular region of human LAG-3 as the monoclonal antibody of the present invention and binds to the antigen .
  • An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human LAG-3 recognized by the monoclonal antibody of the present invention.
  • KD dissociation equilibrium constant of a particular antibody-antigen interaction.
  • the antibodies of the present invention is less than about 10 -7 M, such as less than about 1O -8 M, 1O -9 M or lO -10 M or less, a dissociation equilibrium constant (KD) combined LAG-3, by the use of surface Plasma resonance (SPR) technology was measured in a BIACORE instrument.
  • SPR surface Plasma resonance
  • antigenic determinant refers to a three-dimensional spatial site on an antigen that is discontinuous and is recognized by an antibody or antigen-binding fragment of the invention.
  • the present invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed between antibodies and other sequences. Therefore, the invention also includes fragments, derivatives and analogs of said antibodies.
  • 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 prepared using DNA recombinant techniques well known in the art.
  • the term "monoclonal antibody” refers to antibodies secreted from clones derived from a single cell. Monoclonal antibodies are highly specific, being directed against a single epitope.
  • the cells may be eukaryotic, prokaryotic or phage cloned cell lines.
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibody of the present invention also includes a conservative variant thereof, which means that there are at most 10, preferably at most 8 and more preferably at most 5 compared with the amino acid sequence of the antibody of the present invention Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservatively mutated polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention provides an anti-human LAG-3 antibody (hereinafter referred to as LAG-3 antibody).
  • LAG-3 antibody provides a highly specific and high-affinity antibody against LAG-3, which includes a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (VH) amino acid sequence, the light chain Contains the light chain variable region (VL) amino acid sequence.
  • the LAG-3 antibody of the present invention stimulates antigen-specific T cell responses and enhances the anti-tumor effect of T cells, thereby maximizing the patient's own immune system response to tumors and achieving the purpose of killing tumor cells.
  • the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the following group:
  • any one of the above amino acid sequences is added, deleted, modified and / or substituted for at least one (such as 1-5, 1-3, preferably 1-2, more preferably 1) amino acid A sequence with LAG-3 binding affinity.
  • the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90 %, Optimally at least 95% of the amino acid sequence.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully human Sourced antibodies.
  • the antibody derivatives of the present invention may be single-chain antibodies and / or antibody fragments, such as: Fab, Fab ', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, Any one or more of IgE, IgG, and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a rat.
  • the antibody of the present invention may be a murine antibody, a chimeric antibody, a humanized antibody, a CDR grafted and / or modified antibody that targets human LAG-3.
  • VL light chain variable region
  • VH, CDR1, CDR2, and CDR3 are independently selected from any one or several sequences of SEQ ID Nos .: 3, 4, and 5, or they are added, deleted, or modified. And / or a sequence having LAG-3 binding affinity that replaces at least one amino acid; VL, CDR1, CDR2, and CDR3 are independently selected from SEQ ID NO .: 6, amino acid sequence: LVS and SEQ ID NO .: 7 Or several sequences, or sequences with LAG-3 binding affinity that have been added, deleted, modified, and / or substituted for at least one amino acid.
  • the number of amino acids added, deleted, modified, and / or substituted is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, and more preferably 1-33%. , More preferably 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the number of said added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, Optimally one.
  • LAG-3 antibody of the present invention Any method suitable for producing monoclonal antibodies can be used to produce the LAG-3 antibody of the present invention.
  • animals can be immunized with a linked or naturally occurring LAG-3 protein or fragment thereof.
  • Appropriate immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • LAG-3 can be used as an immunogen (antigen) for generating non-human antibodies specific to LAG-3, and screening the antibodies for biological activity.
  • the immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells.
  • the DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin.
  • the DNA encoding the immunogen can be expressed using a suitable genetic vector including, but not limited to, an adenoviral vector, a baculovirus vector, a plasmid, and a non-viral vector.
  • Example 1 An exemplary method for producing a LAG-3 antibody of the present invention is described in Example 1.
  • the humanized antibody may be selected from any kind of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody, and the IgG1 subtype is used.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains, or variants thereof are useful in the compounds and methods of the invention.
  • Example 4 An exemplary method for humanizing a LAG-3 antibody of the invention is described in Example 4.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as using PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can be fused together to form a single chain antibody.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic methods can also be used to synthesize related sequences, especially when the fragment length is short.
  • long fragments can be obtained by synthesizing multiple small fragments first and then ligating them.
  • This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art.
  • nucleic acid molecule refers to both DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operatively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which an additional DNA segment can be ligated.
  • the invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the term "host cell” refers to a cell into which an expression vector has been introduced.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant or animal cell (such as a mammalian cell).
  • the steps of transforming host cells with recombinant DNA described in the present invention can be performed using techniques well known in the art.
  • the obtained transformant can be cultured by a conventional method, and the transformant expresses a polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present 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.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • the antibodies of the present invention can be purified by conventional isolation and purification means well known to those skilled in the art.
  • the obtained monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or an active fragment thereof or a fusion protein thereof or an ADC or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably about 6-8, although the pH may vary with The nature of the formulation and the condition to be treated will vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.
  • the antibody of the present invention may also be used for cell therapy by expressing a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T-cell immunotherapy (CAR-T).
  • CAR-T chimeric antigen receptor T-cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used to bind LAG-3 protein molecules, and thus can be used to prevent and treat LAG-3 related diseases.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99% by weight, preferably 0.01-90% by weight, more preferably 0.1-80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and a pharmacy Acceptable carrier or excipient.
  • a safe and effective amount such as 0.001-99% by weight, preferably 0.01-90% by weight, more preferably 0.1-80% by weight
  • Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention may be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1
  • a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, Preferably the dose is from about 10 micrograms / kg body weight to about 20 mg / kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health, and other factors, which are all within the skill of a skilled physician.
  • the antibodies of the invention can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples, and biopsy specimens.
  • biopsy as used in the present invention shall include all kinds of biopsy known to those skilled in the art.
  • the biopsy used in the present invention may thus include, for example, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing an antibody (or a fragment thereof) of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the antibody of the present invention may be immobilized on a detection plate.
  • the antibody of the present invention has excellent biological activity and specificity.
  • the humanized antibodies of the present invention have lower immunogenicity while retaining comparable affinity to LAG-3.
  • the antibody of the present invention can significantly inhibit the binding of LAG-3 to the MHC-II receptor, and its inhibitory effect is better than that of the positive control BMS mAb.
  • the antibody of the present invention and LAG-3 of certain non-human mammals have comparable affinity to human LAG-3, which is convenient for testing and quality control testing in animal models.
  • the antibody of the present invention can effectively activate the activity of antigen-specific T cells by releasing LAG-3's inhibition of the activity of antigen-specific T cells (its effect is better than the control antibody BMSmAb), and significantly enhance the anti-tumor effect of T cells , So as to improve the patient's own immune system response to the tumor, to achieve the purpose of killing tumor cells.
  • the method for preparing mouse monoclonal antibodies uses the hybridoma preparation technology invented by Kohler and Milstein in 1975. The specific steps are briefly described as follows: First, human LAG-3-Fc protein (Recombinant Human LAG-3-Fc), Sino Biological, # 16498-H05H is emulsified with Freund's adjuvant, and then Five BALB / c mice were immunized at multiple points subcutaneously. After three rounds of immunization, sera were collected and the titer was measured by ELISA. After the titer reached the predetermined standard, splenocytes were taken and fused with SP2 / 0 myeloma cells.
  • Hybridoma polyclonal cells were screened by HAT, and polyclonal cell lines that specifically bound human LAG-3 were screened by ELISA. Monoclonalization was then performed, and specific binding monoclonal cell lines were screened again using the ELISA method. The selected monoclonals were screened by flow cytometry, and the monoclonal cells 75J19 and other monoclonal cells expressing human LAG-3 antibodies were finally obtained.
  • the indirect ELSIA method was selected to screen highly specific binding mouse hybridoma cells.
  • Recombinant human LAG-3-Fc was prepared into a 1 ⁇ g / ml coating solution with CBS, added to a microtiter plate, and coated at 2-8 ° C for more than 12 hours. The remaining liquid of the coating plate was discarded, and 3% milk was added to 200 ⁇ L per well. Block for 1 hour at room temperature. Add not less than 200 ⁇ L of PBST to each well and wash once, dilute the hybridoma supernatant to 100 ⁇ g / ml, and then dilute 10 gradients 10 times, and add 100 ⁇ L / well to the microtiter plate. Incubate for 1 hour at room temperature. Add not less than 200 ⁇ L of PBST to each well.
  • hybridoma 75J19 (or the antibody produced by it) has the highest binding activity to human LAG-3 protein.
  • the antibody variable region gene was amplified using 5'RACE technology. Briefly, gene-specific cDNAs for heavy and light chains were prepared using SMART 5'RACE Synthesis Kit (TAKARA, # 634859) according to the manufacturer's instructions, and the corresponding PCRs were then amplified. The PCR product was cloned into the vector pEASY-Blunt Simple plasmid (Beijing Quanshijin, # CB111-02), and transformed into Stellar E. coli competent cells (TAKARA, # 636763). Clones were screened by colony PCR using universal M13 forward or reverse primers, and 6-8 clones were selected from each reaction for DNA sequencing analysis. The sequencing results showed that the sequence of the V region of the anti-LAG-3 antibody expressed by 75J19 was as follows:
  • underlined areas are CDRs (IMGT definition, a single column is shown in Table 2 below):
  • the mouse cloned 75J19 VH and VL region cDNAs were ligated to the constant regions of human IgG1 and K chain to construct the chimeric heavy and light chain expressed sequences.
  • PCR primers were used to modify the 5 'and 3' ends of the mouse cDNA sequence, and the primers were designed to add appropriate leader sequences to each chain and add restriction sites that allowed cloning to existing recombinant antibody expression vectors pHB-Fc .
  • the host cells used for protein expression were CHO-K1 cells (# CCL-61) purchased from ATCC. The cells undergo a series of domestication steps to domesticate CHO-K1 cells that can be cultured in suspension in serum-free medium (EX-CELLTM302). The constructed light and heavy chain recombinant expression plasmids were transferred into CHO-K1 cells by electroporation. Place in an incubator for 3-5 days. The antibody concentration from CHO-K1 transfected supernatant was measured by indirect ELISA. The results showed that the transfected CHO-K1 cells secreted about 30 mg / L of chimeric antibodies.
  • the indirect ELSIA method was selected to analyze the binding characteristics of the chimeric antibody (referred to as Chi75J19, hereinafter the same) in Example 3.1 of the present invention to ensure that the selected antibody recognizes the human LAG3 conformational epitope.
  • Humanization of antibodies was performed by the following method. The sequence of the variable region of the antibody was compared with the available sequences in the NCBI protein database. Through identification and analysis, the human framework regions suitable for constructing CDR-grafted heavy and light chains were finally determined.
  • the humanized point mutation antibody expression plasmid was expressed by CHO-K1 (ATCC, NO. CCL-61) cells, respectively, and the humanized antibody protein was obtained after purification.
  • CHO-K1 ATCC, NO. CCL-61 cells, respectively, and the humanized antibody protein was obtained after purification.
  • ELISA receptor binding inhibition experiments, Biacore, and cell viability assays, a highly humanized LAG-3 monoclonal antibody (hereinafter also referred to as B5D1) was obtained.
  • VH and VL sequences of the obtained humanized LAG-3 antibody are shown in SEQ ID NO .: 8 and 9, respectively:
  • BMS humanized LAG-3 antibody (BMS mAb) was cloned according to the humanized sequence provided by US20140093511, and transiently transfected for expression.
  • the binding specificity of humanized LAG-3 antibody B5D1 to human LAG-3 was determined by ELSIA method.
  • Recombinant human LAG-3 was diluted by CBS to obtain 1 ⁇ g / ml coating solution.
  • 50 ⁇ L / well was added to the microtiter plate and coated at 2 ⁇ 8 ° C for more than 12 hours. The remaining liquid of the coating plate was discarded and 3% milk-PBST was added.
  • the present invention is humanized monoclonal antibodies B5D1 low EC 50 and EC 90 values, the recombinant human LAG3 high binding activity.
  • the ELSIA method is used to determine the antigen-antibody binding ability of the anti-LAG-3 antibody to LAG-3 of different species.
  • Recombinant human LAG-3 and recombinant Cynomolgus LAG-3 were diluted by CBS to prepare 1 ⁇ g / ml coating solution, 50 ⁇ L / well, and each of the microtiter plates was coated, 2 ⁇ 8 °C Coat for more than 12 hours; discard the remaining liquid from the plate, add 3% milk-PBST, 200 ⁇ L / well, and block at room temperature for 1 hour; wash once with PBST, 200 ⁇ L / well; dilute B5D1 to 100 ⁇ g / ml, then dilute 5 times 10 Add 100 ⁇ L / well to the microtiter plate and incubate for 1 hour at room temperature; wash 4 times with PBST, 200 ⁇ L / well; then add 3% milk-PBST to dilute 25000-fold GOXHU Fc HRP AFFINITY, 100 ⁇ L / well, and incubate for 1 hour at room temperature; Wash 6 times with PBST, 200 ⁇ L /
  • the humanized LAG-3 monoclonal antibody B5D1 of the present invention in addition to binding to recombinant human LAG3, also binds to recombinant cynomolgus macaque LAG3, which facilitates clinical animal testing.
  • This example uses the SPR method to determine antibody-antigen binding kinetics and affinity.
  • the anti-Human Capture-CM5 chip was prepared according to the amino coupling method of Human Antibody Kit. The chip was equilibrated at room temperature for 20-30 minutes and loaded into a Biacore 8K instrument; B5D1 was diluted to the experimental working concentration with the equilibration buffer; the antigen was diluted to 50 nM with the equilibration buffer, and then three dilutions of 6 concentration gradients were set, and 2 were set Zero concentration (ie, equilibration buffer) and one repetitive concentration (generally the lowest concentration repetitive); 10 antigen concentrations (2 zero concentrations, 7 gradient concentrations, and 1 repetition) in the order of antibodies, antigens, and regeneration Concentration) for experimental analysis.
  • B5D1 was diluted to the experimental working concentration with the equilibration buffer
  • the antigen was diluted to 50 nM with the equilibration buffer, and then three dilutions of 6 concentration gradients were set, and 2 were set Zero concentration (ie, equilibration buffer)
  • the injection rate of the antigen is 30 ⁇ L / min, the binding time is 120 seconds, and the dissociation time is 600 seconds.
  • the corresponding analysis program is used to analyze the data to confirm that there is no obvious reference binding, and kinetics is selected.
  • 1: 1 binding model (Kinetics, 1: 1 binding modle), fitting the data to obtain the B5D1 kinetic parameters Ka, Kd and KD values.
  • the antibody was co-incubated with CHO cells overexpressing human LAG-3 on the cell membrane, and detected by flow cytometry.
  • the antibodies to be detected were diluted with flow buffer (1 ⁇ PBS + 1% BSA) in a gradient.
  • flow buffer (1 ⁇ PBS + 1% BSA
  • 20 ⁇ L cell suspension of 2 ⁇ 10 4 human LAG-3 expressing CHO cells was plated in each well, 20 ⁇ L of antibody dilution was added, mixed, and incubated at room temperature for 30 minutes. Cells were washed twice with flow buffer.
  • the results are shown in Table 8 and Figure 1.
  • the results show that the B5D1 antibody of the present invention has good biological activity and strong binding ability to LAG-3 protein on the cell surface, and its EC 50 is 2.059 ⁇ g / ml.
  • Recombinant human LAG-3-Fc (Beijing Yiqiao Shenzhou, Cat. No. 16498-H05H-200) was reconstituted with double distilled water for future use.
  • Recombinant human LAG-3-Fc was diluted to 6 ⁇ g / ml with PBS solution containing 1% BSA (1% BSA / PBS), 20 ⁇ L per well was added to a 96-well U-shaped plate, and anti-LAG-3 was serially diluted with 3 times than Humanized antibody B5D1 was reacted at a volume ratio of 1: 1 in a 96-well U-shaped plate at room temperature for 20 min.
  • the 96-well U-shaped plate after the reaction was resuspended in 1% BSA / PBS, and the supernatant was discarded by centrifugation (300g ⁇ 3min), and then washed twice, and then added 1: 300 diluted Alexa488-Goat anti mouse- Fc (Jackson ImmunoResearch, Cat. No. 115-545-071), react at room temperature for 15min, resuspend the 96-well U-shaped plate after the reaction with 1% BSA / PBS, and centrifuge (300g ⁇ 3min) to discard the supernatant, and wash it 3 times. Finally, 100 ⁇ L of 1% BSA / PBS was resuspended in each well, and the fluorescence intensity of the first channel was detected by flow cytometry (BD, Accuri C6).
  • BD flow cytometry
  • the effector cell is a murine hybridoma T cell 3A9 cell (purchased from ATCC), and its specific antigen is the polypeptide HEL48-62.
  • the human LAG-3 protein was overexpressed in 3A9 cells, and a 3A9-huLAG-3 stably transformed cell line was constructed.
  • LK35.2 cells with MHC-II molecules expressed on the cell surface as antigen-presenting cells presented the polypeptide HEL48-62 to 3A9 cells or 3A9-huLAG-3 cells. By detecting the secretion of IL-2 by 3A9-huLAG-3 cells, it was shown that the antibody stimulated T cell activity.
  • LK35.2 cells (96 2.5x10 4 cells / well) were incubated with 200nM polypeptide HEL48-62 30 minutes at 37 °C, the 3A9-huLAG-3 cells (96 5.0x10 4 cells / Well) and a gradient dilution of the anti-LAG-3 antibody to be tested for a total of 15 minutes at 37 ° C.
  • the two cell systems were then mixed and cultured in a 37 ° C incubator for 24 hours.
  • the supernatant was collected, and the expression of IL-2 was detected by ELISA detection kit (mouse IL-2 OptEIA kit, BD Bioscience, # 555148).
  • BMS humanized LAG-3 antibody (BMS mAb) was used as a positive control.

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Abstract

L'invention concerne un anticorps LAG-3 ayant une affinité élevée et une haute activité biologique, ainsi qu'une utilisation de celui-ci. En particulier, l'invention concerne un nouvel anticorps monoclonal anti-LAG-3. L'anticorps est capable de se lier à un antigène LAG-3 avec une spécificité élevée, a une affinité élevée et une haute activité biologique, a une faible immunogénicité, et est utilisé pour préparer un médicament destiné à prévenir ou à traiter des maladies associées à LAG-3.
PCT/CN2019/090979 2018-06-14 2019-06-12 Anticorps lag-3 ayant une affinité élevée et une haute activité biologique, et utilisation associée WO2019238074A1 (fr)

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CN113348182B (zh) * 2019-12-30 2022-07-12 上海海路生物技术有限公司 Lag-3抗体及其医药用途
CN114621345B (zh) * 2020-12-10 2022-11-29 北京东方百泰生物科技股份有限公司 一种抗lag-3的单克隆抗体、其抗原结合片段及其应用

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