WO2021147837A1 - 抗lag3单克隆抗体及其制备方法和应用 - Google Patents

抗lag3单克隆抗体及其制备方法和应用 Download PDF

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WO2021147837A1
WO2021147837A1 PCT/CN2021/072610 CN2021072610W WO2021147837A1 WO 2021147837 A1 WO2021147837 A1 WO 2021147837A1 CN 2021072610 W CN2021072610 W CN 2021072610W WO 2021147837 A1 WO2021147837 A1 WO 2021147837A1
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Prior art keywords
lag3
monoclonal antibody
seq
variable region
antibody
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English (en)
French (fr)
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宋戈
肖辉
何虹霖
许旭
姜伟东
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Shanghai Henlius Biopharmaceutical Co Ltd
Shanghai Henlius Biotech Inc
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Shanghai Henlius Biopharmaceutical Co Ltd
Shanghai Henlius Biotech Inc
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Priority to AU2021210029A priority Critical patent/AU2021210029A1/en
Priority to JP2022544146A priority patent/JP7692918B6/ja
Priority to KR1020227028342A priority patent/KR20220131279A/ko
Priority to EP21744582.4A priority patent/EP4095159A4/en
Priority to BR112022013544A priority patent/BR112022013544A2/pt
Priority to US17/759,180 priority patent/US20230074657A1/en
Application filed by Shanghai Henlius Biopharmaceutical Co Ltd, Shanghai Henlius Biotech Inc filed Critical Shanghai Henlius Biopharmaceutical Co Ltd
Priority to CN202180008891.3A priority patent/CN114945595A/zh
Priority to CA3165211A priority patent/CA3165211A1/en
Publication of WO2021147837A1 publication Critical patent/WO2021147837A1/zh
Anticipated expiration legal-status Critical
Priority to ZA2022/08339A priority patent/ZA202208339B/en
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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 biomedicine, in particular to an anti-LAG3 monoclonal antibody and its preparation method and application.
  • LAG3 (Lymphocyte-activation gene 3), also called CD223, was discovered in 1990. It belongs to the immunoglobulin superfamily and is expressed on activated T cells, NK cells, B cells and dendritic cells.
  • MHC major histocompatibility complex
  • MHC II major histocompatibility complex
  • the affinity of LAG3 and MHC II is even stronger than that of CD4 and MHC II.
  • the ligands of LAG3 also include LSECtin and FGL1.
  • the biological function of LAG3 is similar to CTLA-4 and PD-1, and it plays a role in negatively regulating the cell proliferation and activation of T cells.
  • LAG3 also plays a role in the suppressive function of Treg cells.
  • T cells can be reactivated, thereby enhancing the killing effect on tumors.
  • inhibiting LAG3 can also reduce the function of Treg cells to suppress the immune response. Therefore, using LAG3 as a target for cancer immunotherapy, the use of monoclonal antibodies to bind LAG3 can block the binding of LAG3 to its ligand and prevent the generation of T cell suppression signals. Thereby promoting the activation and proliferation of T cells, as well as the expression of cytokines, up-regulating the monitoring activity of the immune system against tumor cells, enhancing the specific anti-tumor immune response, and achieving the purpose of treating tumors.
  • anti-LAG3 antibody BMS986016 researched and developed by Bristol-Myers Squibb has achieved significant results in the treatment of advanced melanoma and is currently undergoing phase III clinical trials.
  • anti-LAG3 antibodies are used in combination with other antibody drugs including anti-PD-1 antibodies, and related clinical trials are also being carried out.
  • the technical problem to be solved by the present invention is to provide a new anti-LAG3 monoclonal antibody, thereby completing the present invention.
  • the first objective of the present invention is to provide a new anti-LAG3 monoclonal antibody.
  • the second object of the present invention is to provide a nucleotide molecule encoding the anti-LAG3 monoclonal antibody.
  • the third object of the present invention is to provide an expression vector containing the nucleotide molecule.
  • the fourth object of the present invention is to provide a host cell containing the expression vector.
  • the fifth object of the present invention is to provide a method for preparing the anti-LAG3 monoclonal antibody.
  • the sixth object of the present invention is to provide a composition containing the anti-LAG3 monoclonal antibody.
  • the seventh objective of the present invention is to provide the application of the anti-LAG3 monoclonal antibody in the preparation of medicines.
  • the first aspect of the present invention provides an anti-LAG3 monoclonal antibody, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 2, 6
  • the heavy chain variable region shown in 10 has HCDR1, HCDR2, and HCDR3 regions with the same CDR sequence, and the light chain variable region includes the same as those shown in SEQ ID NO: 4, 8 or 12.
  • the anti-LAG3 monoclonal antibody includes:
  • the HCDR1 has the amino acid sequence shown in SEQ ID No: 18, the HCDR2 has the amino acid sequence shown in SEQ ID No: 19, and the HCDR3 has the amino acid sequence shown in SEQ ID No: 19, The amino acid sequence shown in SEQ ID No: 20 or 21;
  • the light chain complementarity determining regions LCDR1, LCDR2, LCDR3, the LCDR1 has the amino acid sequence shown in SEQ ID No: 22 or 23, the LCDR2 has the amino acid sequence shown in SEQ ID No: 24, and the LCDR3 It has the amino acid sequence shown in SEQ ID No: 25.
  • the anti-LAG3 monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 2, 6, or 10. Or a sequence having at least 85% homology with the aforementioned sequence, such as a derivative sequence with 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology;
  • the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 4, 8 or 12, or a sequence with at least 85% homology with the above sequence, such as 85%, 90%, 92%, 94%, 95 %, 96%, 97%, 98%, or 99% homology of derived sequences.
  • the anti-LAG3 monoclonal antibody includes a heavy chain and a light chain, and the heavy chain is composed of an amino acid sequence as shown in SEQ ID NO: 2, 6 or 10 or at least 85% identical to the above sequence.
  • Source sequences such as heavy chain variable regions of 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and such as SEQ ID NO:
  • the heavy chain constant region shown in 14 is composed;
  • the light chain is composed of an amino acid sequence shown in SEQ ID NO: 4, 8 or 12, or a sequence having at least 85% homology with the above sequence, such as 85%,
  • amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.
  • amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12.
  • the anti-LAG3 monoclonal antibody may be the full-length sequence of the antibody, or may be a fragment of an anti-LAG3 antibody, and the fragment is Fab, Fab', F(ab')2, Fv, scFv, or the like.
  • the anti-LAG3 antibody is an IgG1, IgG2 or IgG4 type antibody.
  • the present invention further provides a derivative of the anti-LAG3 antibody, the derivative is a fragment of the LAG3 antibody, an antibody/antibody fragment-factor fusion protein, an antibody/antibody fragment-chemical conjugate; the fragment of the anti-LAG3 antibody is Fab, Fab', F(ab')2, Fv or scFv, etc.
  • the monoclonal antibodies of the present invention can be prepared by conventional techniques in the art, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • monoclonal antibodies are prepared from wild-type or transgenic mice by hybridoma technology.
  • the second aspect of the present invention provides an isolated nucleotide molecule encoding the anti-LAG3 monoclonal antibody as described above.
  • the nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is as shown in SEQ ID NO: 3, 7 or 11, which encodes the light chain variable region of the anti-LAG3 monoclonal antibody
  • the nucleotide sequence of is shown in SEQ ID NO: 5, 9 or 13.
  • nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody As shown in SEQ ID NO: 5.
  • nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody As shown in SEQ ID NO: 9.
  • nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 11, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody As shown in SEQ ID NO: 13.
  • the preparation method of the nucleotide molecule is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the monoclonal antibody by gene cloning technology, such as PCR method, or by artificial full sequence
  • the synthetic method obtains the nucleotide molecule encoding the above-mentioned monoclonal antibody.
  • nucleotide sequence encoding the amino acid sequence of the monoclonal antibody can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homologue or conservative variant sequence thereof.
  • the homologue of the polynucleotide of the present invention or its conservative variant sequence can be prepared by replacing, deleting or adding one or more bases encoding the monoclonal antibody gene within the scope of maintaining antibody activity.
  • the third aspect of the present invention provides an expression vector containing the nucleotide molecule as described above.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate Sequence expression vector.
  • the expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid.
  • a virus or a plasmid such as an appropriate phage or phagemid.
  • Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • the expression vector in the present invention refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art.
  • the expression vector is selected from one or more of pHLX101, pEE14.4, pCHO 1.0 or pcDNA3.1.
  • the expression vector is pcDNA3.1.
  • the fourth aspect of the present invention provides a host cell containing the expression vector as described above.
  • the host cell of the present invention is a variety of conventional host cells in the field, as long as it can make the above-mentioned recombinant expression vector stably replicate itself and the nucleotides carried can be effectively expressed.
  • the host cell in the present invention 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 mammalian cell.
  • the host cell is: COS, CHO (Chinese Hamster Ovary), HeLa cell line, bone marrow cell line such as SP2/0 cell line, NS0, sf9, sf21, DH5 ⁇ , BL21(DE3) or E .coli TG1, YB2/0 cell line, etc. and one or more of transformed B-cells or hybridoma cells.
  • the host cell is E. coli TG1, BL21 cell (expressing single-chain antibody or Fab antibody) or CHO-K1 cell (expressing full-length IgG antibody).
  • the expression vector is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the fifth aspect of the present invention provides a method for preparing the anti-LAG3 monoclonal antibody as described above, including the following steps:
  • the isolation and purification method of the anti-LAG3 monoclonal antibody is a conventional method in the art.
  • specific operation method please refer to the corresponding cell culture technology manual and the monoclonal antibody isolation and purification technology manual.
  • the host cells used in the present invention are all existing technologies and can be directly obtained through commercial channels.
  • the culture medium used in the culture is also various conventional culture media. Those skilled in the art can select the appropriate medium based on experience.
  • the host cell is cultured under conditions where it grows. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art.
  • Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention screens and obtains the gene sequence of the target antibody from a monoclonal cultured cell line, and is used to construct a eukaryotic expression vector. After expression, the activity of the antibody can be reconstructed to obtain an anti-LAG3 monoclonal antibody.
  • the sixth aspect of the present invention provides a composition comprising the anti-LAG3 monoclonal antibody as described above and a pharmaceutically acceptable carrier.
  • the monoclonal antibody of the present invention can be used by formulating a pharmaceutical composition in any manner known in the art.
  • This composition takes the monoclonal antibody as the active ingredient, plus one or more pharmaceutically acceptable carriers, diluents, fillers, binding agents and other excipients, depending on the mode of administration and the design The dosage form.
  • the therapeutically inert inorganic or organic carriers known to those skilled in the art include (but are not limited to) lactose, corn starch or its derivatives, talc, vegetable oils, waxes, fats, polyanthocyanidin compounds such as polyethylene glycol, water , Sucrose, ethanol, glycerin, etc., various preservatives, lubricants, dispersing agents, flavoring agents.
  • Moisturizers antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like can also be added. These substances are used as needed to help the stability of the formulation or help increase the activity or its biological effectiveness or In the case of oral administration, it produces acceptable taste or smell.
  • the inhibitor may be used in the form of its original compound itself, or optionally in the form of its pharmaceutically acceptable salt.
  • the cloned antibody can be administered alone, or in various combinations, and in combination with other therapeutic agents.
  • the composition thus formulated can be used to administer the inhibitor in any appropriate manner known to those skilled in the art as required.
  • the anti-LAG3 monoclonal antibody provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to achieve a more stable therapeutic effect.
  • These preparations can ensure the amino acid core sequence of the anti-LAG3 monoclonal antibody of the present invention Conformational integrity, while also protecting the protein's multifunctional groups to prevent its degradation (including but not limited to aggregation, deamination or oxidation).
  • it can be stored at 2°C-8°C for at least one year, and for freeze-dried preparations, it can be kept stable at 30°C for at least six months.
  • the anti-LAG3 monoclonal antibody preparation can be a suspension, water injection, freeze-dried and other preparations commonly used in the pharmaceutical field, preferably a water injection or freeze-dried preparation,
  • the pharmaceutically acceptable carrier preferably includes, but is not limited to: one of surfactants, solution stabilizers, isotonic regulators and buffers or Its combination.
  • the surfactants preferably include but are not limited to: non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80); Poloxamer (such as Poloxamer 188); Triton; sodium dodecyl sulfate (SDS) ); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount added should minimize the tendency of anti-LAG3 monoclonal antibody to granulate.
  • non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80); Poloxamer (such as Poloxamer 188); Triton; sodium dodecyl sulfate (SDS) ); sodium lauryl sulfate; tetradecyl, linoleyl or octa
  • the solution stabilizer preferably includes but is not limited to one or a combination of the following: sugars, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, for example : Triols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc.
  • the amount of solution stabilizer added should enable the final formulation to maintain a stable state within a time period considered by those skilled in the art to be stable.
  • the isotonicity adjusting agent preferably includes, but is not limited to, one of sodium chloride, mannitol, or a combination thereof.
  • the buffer preferably includes but is not limited to one of Tris, histidine buffer, phosphate buffer, or a combination thereof.
  • the seventh aspect of the present invention provides the application of the above-mentioned anti-LAG3 monoclonal antibody or its composition in the preparation of medicines.
  • the application is for preparing LAG3 molecular blocker drugs. More preferably, the use of the preparation of LAG3 molecular blocker drugs is specifically the preparation of tumor treatment or tumor diagnosis drugs.
  • the drug of the present invention is preferably an anti-tumor, treatment of autoimmune diseases, treatment of infectious diseases and/or anti-transplant rejection, more preferably anti-tumor drugs, treatment of autoimmune diseases, and more preferably For anti-tumor drugs.
  • the anti-LAG3 monoclonal antibody of the present invention can be used alone or in combination with other anti-tumor drugs.
  • the other anti-tumor drugs are conventional anti-tumor drugs in the field, including antibody drugs or small molecule anti-tumor drugs.
  • the antibody drugs are conventional antibody drugs in the art, and preferably include anti-PD-1 monoclonal antibodies.
  • the small molecule anti-tumor drugs are conventional drugs in the field, including paclitaxel, 5-Fu pyrimidine and the like.
  • the tumors targeted by the anti-tumor drugs preferably include but are not limited to: melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, One or more of esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, and/or head and neck tumors.
  • the anti-tumor drugs referred to in the present invention refer to drugs capable of inhibiting and/or treating tumors, which may include delays in the development of tumor-related symptoms and/or reduction in the severity of these symptoms, and further include alleviation of symptoms associated with existing tumors. And to prevent the appearance of other symptoms, including reducing or preventing tumor metastasis and so on.
  • the dosage of administration varies with the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. You can refer to the animal As a result of the experiment and various circumstances, the total dose should not exceed a certain range. Generally, the dose for intravenous injection is 1-1800 mg/day.
  • the anti-LAG3 monoclonal antibody of the present invention has good biological activity, high expression in mammalian cells, and has obvious affinity for LAG3 and the ability to inhibit ligand-receptor binding.
  • the monoclonal antibody can be used alone or in combination with other anti-tumor drugs in tumor immunotherapy, diagnosis and screening, and can be effectively used in the preparation of drugs for the treatment of tumors, infectious diseases, autoimmune diseases, and anti-immune rejection. .
  • Figure 1 shows the experimental results of the binding ability of anti-LAG3 antibodies to LAG3 on the cell surface
  • Figure 2 shows the experimental results of anti-LAG3 antibody blocking the binding of LAG3 to its ligand MHC II
  • Figure 3 shows the experimental results of the binding ability of anti-LAG3 chimeric antibodies to LAG3
  • Figure 4 shows the experimental results of the ability of anti-LAG3 chimeric antibody to bind to LAG3 on the cell surface
  • Figure 5 shows the experimental results of the binding ability of anti-LAG3 humanized antibodies to LAG3;
  • Figure 6 shows the experimental results of the binding ability of anti-LAG3 humanized antibodies to LAG3 on the cell surface
  • Figure 7 shows the experimental results of surface plasmon resonance of the affinity of anti-LAG3 humanized antibody to LAG3;
  • Figure 8 shows the experimental results of anti-LAG3 humanized antibodies blocking the binding of LAG3 to its ligand MHC II;
  • Figure 9 shows the experimental results of anti-LAG3 humanized antibodies blocking the binding of LAG3 to its ligand LSECtin
  • Figure 10 shows the experimental results of anti-LAG3 humanized antibodies blocking the binding of LAG3 to its ligand FGL1;
  • Figure 11 shows the results of an ELISA experiment of species cross-reaction of anti-LAG3 humanized antibody
  • Figure 12 shows the results of a flow cytometry experiment of species cross-reaction of anti-LAG3 humanized antibody
  • Figure 13 shows the experimental results of surface plasmon resonance of the species cross-reaction of anti-LAG3 humanized antibody
  • Figure 14 shows the experimental results of the activation of T cells by anti-LAG3 humanized antibodies
  • Figure 15 shows the experimental results of anti-LAG3 humanized antibodies inhibiting tumor growth in the hLAG3 KI mouse model
  • Figure 16 shows that the combination of h6H11B10#40 and HLX10 activates the release of IL-2;
  • Figure 17 shows that the combination of h6H11B10#40 and HLX10 activates the release of IFN- ⁇ ;
  • Figure 18 shows the growth curve of the tumor volume of each group of mice in the MC38 model
  • Figure 19 shows the growth curve of the tumor volume of each group of mice in the A20 model
  • the following examples further illustrate the present invention, but the present invention is not limited to the scope of the described examples.
  • the experimental methods without specific conditions are selected in accordance with conventional methods and conditions, or in accordance with the product specification.
  • the room temperature described in the embodiment is a conventional room temperature in the art, and is generally 10-30°C. Unless otherwise specified, all reagents and raw materials used are commercially available.
  • variable region of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination.
  • VL variable region
  • VH variable region
  • the variable regions of the heavy chain and the light chain each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDR) (also called hypervariable regions).
  • FR framework regions
  • CDR complementarity determining regions
  • the CDRs in each chain are held together tightly by FRs and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody.
  • There are at least two techniques for determining CDRs (1) Methods based on cross-species sequence variability (ie, Kabat et al.
  • CDR may refer to a CDR determined by either method or a combination of the two methods.
  • antibody framework or "FR region” refers to a part of a variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • CDR-H1, CDR-H2, CDR-H3 three CDRs
  • CDR-L1, CDR-L2, CDR-H3 three CDRs
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • CDR is defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-35 in human VL.
  • 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute.
  • the CDR amino acid residue numbers in VH are roughly 26-35 (HCDR1), 51-57 (HCDR2) and 93-102 (HCDR3)
  • the CDR amino acid residue numbers in VL are roughly 27-32 (LCDR1) ), 50-52 (LCDR2) and 89-97 (LCDR3).
  • the CDR region of an antibody can be determined using the program IMGT/DomainGap Align.
  • LAG3 refers to the lymphocyte activation gene-3 protein, an immune checkpoint receptor or T cell co-suppressor, also known as CD223.
  • the amino acid sequence of the full length LAG3 is provided in GenBank under the accession number NP_002277.4.
  • the term "" includes variants, isoforms, homologs, orthologs and paralogues.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. Conventional technology. These technologies have been fully explained in the existing literature.
  • Positive control antibody BMS986016 The amino acid sequence is recorded in Bristol-Myers Squibb's patent application WO 2015/042246 Al.
  • the present invention clones the nucleotide sequence encoding the amino acid sequence into the pURO vector by restriction endonucleases, transfects the plasmid into CHO-S cells, and obtains high-expressing cell strains by screening for carbenicillin resistance. Expand the culture of the cell line, collect the medium supernatant, and obtain the control antibody through the preparation process of protein A affinity chromatography.
  • Negative control HLX10 (h1G4): its sequence is quoted from CN109923126A.
  • Positive control ab40465 Abcam company, item number ab40465.
  • Isotype-Human IgG4 (hIgG4): Sino-American Crown Corporation, Cat: AB180018
  • Recombinant mouse LAG3-Fc fusion protein purchased from Yiqiao Shenzhou, Cat:53069-M02H.
  • Recombinant cynomolgus monkey LAG3-Fc fusion protein purchased from Yiqiao Shenzhou, Cat:90841-C08H
  • Anti-mouse PD1 antibody RMP1-14 BioXCell, Cat: 717918O1
  • LAG3-His amino acid sequence shown in SEQ ID No: 1
  • CHO-S cells expressing the full length of LAG3 were used to co-immunize mice, and spleen cells and myeloma cells were fused to obtain After hybridoma cells, positive clones were then screened by ELISA.
  • the nucleic acid sequence encoding the 23-450 amino acids of the extracellular region of LAG3 was cloned into the pEE14.4 vector by restriction endonuclease, wherein the C-terminus of the LAG3 coding region had a tag encoding 6xHis, and then the plasmid It was electroporated into CHO-S cells, and a single clone with high expression of LAG3-his fusion protein was screened by the GS (Glutamine Synthetase) screening system. The clone was expanded and cultured, and the culture supernatant was collected and passed through nickel. The method of column affinity chromatography prepares the target protein.
  • the protein and CHO-S cells expressing the full length of LAG3 were co-immunized with mice.
  • the LAG3-His recombinant protein was first coated in a 96-well half-well plate (working volume 30 ⁇ l) at 2 ⁇ g/ml, and coated overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20 to remove excess LAG3-His. Block with 5% skim milk in PBS for 1 hour at room temperature.
  • the flow cytometry method was further used to determine the positive hybridoma clones that were screened by ELISA and express antibodies.
  • the normally grown CHO-S cells expressing the full-length human LAG3 gene were washed twice with PBS (2% FBS), and the cell concentration was adjusted to 5 ⁇ 10 5 /100 ⁇ l.
  • the cells were washed twice with PBS (2% FBS).
  • the cells were suspended in 200 ⁇ l PBS and analyzed by flow cytometry. The results are shown in Figure 1. Most of the positive clones screened by ELISA can bind to LAG3 on the cell surface.
  • Flow cytometry was used to test the ability of the antibodies expressed by the positive clones of hybridomas screened by ELISA to block the binding of MHC II on the cell surface to LAG3-Fc (Acro). Wash Daudi cells twice with PBS (2% FBS) and adjust the cell concentration to 3 ⁇ 10 5 /100 ⁇ l. The fusion tumor supernatant was mixed with LAG3-Fc at a concentration of 2 ⁇ g/ml, then mixed with Daudi cells, and incubated at 4°C for 30min. Centrifuge the cells at 500 ⁇ g for 5 min. At the same time, no antibody was used as a negative control, and the anti-LAG3 antibody BMS986016 was used as a positive control. Wash twice with PBS (2% FBS).
  • amino acid sequence of the heavy chain variable region of 6H11B10 is shown in SEQ ID No: 2, and the nucleotide sequence is shown in SEQ ID No: 3; the amino acid sequence of the light chain variable region is shown in SEQ ID No: 4.
  • the nucleotide sequence is shown in SEQ ID No:5.
  • the amplification of the variable regions of the mouse light chain and heavy chain was carried out according to the method introduced by Anke Krebber et al. (Journal of Immunological Methods 201.1997.35-55).
  • the general method includes preparing total RNA from the monoclonal hybridoma cell line 6H11B10, and preparing single-stranded cDNA using a reverse transcription kit (TaKaRa).
  • TaKaRa reverse transcription kit
  • the heavy chain variable region SEQ ID No: 3
  • light chain variable region of the antibody are amplified by PCR. Region (SEQ ID No: 5).
  • the light chain variable region and heavy chain variable region genes of 6H11B10 were fused with the heavy chain IgG4 constant region gene (SEQ ID No: 15) and the light chain constant region gene (SEQ ID No: 17) respectively. , Thereby obtaining the 6H11B10 chimeric antibody heavy chain gene and light chain gene.
  • the chimeric antibody genes of 6H11B10 were respectively transfected into CHO-S cells and expressed to obtain the chimeric antibody protein c6H11B10.
  • ELISA technique was used to detect the affinity of the chimeric antibody to LAG3.
  • the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 30 ⁇ l) at 2 ⁇ g/ml, and allowed to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20. Block with 5% skim milk in PBS for 1 hour at room temperature. Wash with PBST 3 times, then add c6H11B10 with gradient dilution, and let stand at room temperature for 1 hour.
  • PBST PBS
  • BMS986016 was used as a positive control. Wash with PBST 3 times, add 30 ⁇ l of horseradish peroxidase labeled goat anti-human IgG kappa light chain secondary antibody (Millipore company) diluted 1:4000, and let stand at room temperature for 1 hour. Wash 6 times with PBST, add TMB for color development, and stop the reaction with 2M H 2 SO 4 , and read at 450 nm with a microplate reader. The results are shown in Figure 3, the anti-LAG3 chimeric antibody c6H11B10 has a strong affinity for LAG3, and at low concentrations, the affinity of c6H11B10 is higher than that of the control antibody BMS986016.
  • Flow cytometry was used to determine the affinity of the chimeric antibody protein c6H11B10 to LAG3 on the cell surface. Take the Jurkat cells transfected and express the full-length human LAG3 gene, wash the cells twice with PBS (2% FBS), and adjust the cell concentration to 2 ⁇ 10 5 /100 ⁇ l. Add serially diluted chimeric antibody c6H11B10 and murine antibody m6H11B10, and incubate at 4°C for 30 min. Centrifuge the cells at 500 ⁇ g for 5 min. At the same time, BMS986016 was used as a positive control. Wash twice with PBS (2% FBS).
  • chimeric antibodies and BMS986016 For chimeric antibodies and BMS986016, add 1:300 diluted goat anti-human IgG Fab-FITC (Thermo Fisher), and for mouse antibodies add 1:100 diluted goat anti-mouse IgG-FITC (Kangwei Century), Incubate for 30 min at 4°C. The cells were washed twice with PBS (2% FBS). The cells were suspended in 200 ⁇ l PBS and analyzed by flow cytometry. The results are shown in Figure 4, the chimeric antibody c6H11B10 can bind strongly to LAG3 on the cell surface, and the affinity of c6H11B10 is better than that of the control antibody BMS986016.
  • the human antibody light chain variable region germline gene IGKV2-28*01 and the heavy chain variable region germline gene IGHV3-11*06 were used to convert the framework of the murine antibody 6H11B10 Region replacement, retaining the original complementarity determining region (CDR), thereby obtaining the anti-6H11B10 humanized antibody h6H11B10.
  • the amino acid sequence of the heavy chain variable region of h6H11B10 is shown in SEQ ID No: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 8.
  • the gene sequence of the heavy chain variable region of h6H11B10 is shown in SEQ ID No: 7, and the gene sequence of the light chain variable region is shown in SEQ ID No: 9, respectively. : 15) and the constant region gene of the light chain (SEQ ID No: 17) to obtain the heavy chain gene and the light chain gene of the anti-LAG3 humanized antibody h6H11B10.
  • the three CDRs of the h6H11B10 heavy chain are: HCDR1 (SEQ ID No: 18), HCDR2 (SEQ ID No: 19) and HCDR3 (SEQ ID No: 20); the three CDRs of the light chain are: LCDR1 (SEQ ID No: 22), LCDR2 (SEQ ID No: 24) and LCDR3 (SEQ ID No: 25).
  • the humanized anti-LAG3 antibody h6H11B10 uses phage display technology for affinity maturation, designs primers with point mutations, and uses PCR to mutate the light chain CDRs and heavy chain CDRs of h6H11B10 to obtain a mutation library.
  • the phage display vector Transform into E. coli TG1 or SS320 cells to generate a phage library.
  • streptavidin-coupled magnetic beads M-280 Thermo Fisher
  • biotin-labeled LAG3 protein the phage library was screened in two rounds. One clone with the highest affinity, h6H11B10#40, was screened by ELISA.
  • the amino acid sequence of the heavy chain variable region of h6H11B10#40 is shown in SEQ ID No: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 12.
  • the heavy chain variable region gene of h6H11B10#40 is shown in SEQ ID No: 11, and the light chain variable region gene is shown in SEQ ID No: 13, and the heavy chain IgG4 constant region gene (SEQ ID No: 15 ) And the constant region gene of the light chain (SEQ ID No: 17) to obtain the heavy chain gene and light chain gene of the anti-LAG3 humanized antibody h6H11B10#40.
  • the three CDRs of the heavy chain of h6H11B10#40 are: HCDR1 (SEQ ID No: 18), HCDR2 (SEQ ID No: 19) and HCDR3 (SEQ ID No: 21); the three CDRs of the light chain are: LCDR1 ( SEQ ID No: 23), LCDR2 (SEQ ID No: 24) and LCDR3 (SEQ ID No: 25).
  • the h6H11B10 and h6H11B10#40 anti-LAG3 humanized antibody genes were respectively transfected into CHO-S cells and expressed to obtain h6H11B10 and h6H11B10#40 antibody proteins.
  • ELISA technology was used to detect the affinity of humanized antibody to LAG3.
  • the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 30 ⁇ l) at 2 ⁇ g/ml, and allowed to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20. Block with 5% skim milk in PBS for 1 hour at room temperature.
  • the cells were washed twice with PBS (2% FBS). The cells were suspended in 200 ⁇ l PBS and analyzed by flow cytometry. The results are shown in Figure 6, the anti-LAG3 humanized antibody has a strong affinity for LAG3 on the cell membrane, and both h6H11B10#40 and h6H11B10 are better than the positive control BMS986016.
  • SPR Surface plasmon resonance
  • Example 6 The ability of anti-LAG3 humanized antibody to block the binding of LAG3 to its ligand
  • Flow cytometry was used to measure the ability of anti-LAG3 humanized antibodies to block the binding of MHC II on the surface of Raji cells to LAG3-Fc. Wash Raji cells twice with PBS (2% FBS) and adjust the cell concentration to 2 ⁇ 10 5 /100 ⁇ l. After the humanized anti-LAG3 antibody h6H11B10#40 is diluted 1:3, it is diluted with 2 ⁇ g/ml LAG3- After mixing the Fc, place it at room temperature for 30 minutes, while using hIgG4 protein as a negative control and anti-LAG3 antibody BMS986016 as a positive control. Then mix with Raji cells and incubate at 4°C for 60 min. Centrifuge the cells at 500 ⁇ g for 5 min.
  • the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of MHC II on the cell surface to LAG3-Fc, similar to BMS986016.
  • ELISA technology was used to detect the ability of humanized antibodies to block the binding of LAG3-Fc to its ligand LSECtin.
  • the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 100 ⁇ l) at 1 ⁇ g/ml, and left to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween 20. Block with 5% skim milk in PBS for 1 hour at room temperature, and wash 3 times with PBST.
  • the humanized antibody protein of h6H11B10#40 was diluted 1:5 with hIgG4 protein as a negative control and anti-LAG3 antibody BMS986016 as a positive control.
  • LSECtin (R&D company) was prepared with 1 ⁇ g/ml, mixed with antibody at a ratio of 1:1, and allowed to stand at room temperature for 30min. Then add it to the microtiter plate and let stand at room temperature for 1 hour. Wash 3 times with PBST, add 100 ⁇ l horseradish peroxidase labeled mouse anti-His tag secondary antibody (GenScript) diluted 1:5000, and let stand at room temperature for 1 hour. Wash 6 times with PBST, add TMB for color development, and terminate the reaction with 2M H 2 SO 4. Use a microplate reader to read at 450nm. The results are shown in Figure 9, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of LAG3-Fc to its ligand LSECtin.
  • Example 7 The ability of anti-LAG3 humanized antibody to block the binding of LAG3 to its ligand
  • a commercial kit for determining the binding of LAG3 to FGL1 by anti-LAG3 antibodies (purchased from Cisbio, catalog number: 63ADK000CB10PEG) was used to test the ability of anti-LAG3 humanized antibodies to block the binding of LAG3 and FGL1.
  • This experiment first mixes 4 ⁇ L of Tag1-LAG3 protein and 4 ⁇ L of Tag2-FGL1 protein with the diluted test sample, reacts at room temperature for 15 minutes, then adds 10 ⁇ L of pre-mixed anti-Tag1-Tb3+ and anti-Tag2-XL665, and seals the test. After reacting overnight at room temperature, read the fluorescence absorbance value of 665/620nm with a microplate reader. The results are shown in Figure 10, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of LAG3 to its ligand FGL1.
  • the recombinant mouse and cynomolgus LAG3-Fc fusion protein was purchased from Yiqiao Shenzhou.
  • the LAG3-Fc recombinant protein was coated into an enzyme-labeled plate (working volume 30 ⁇ l) at 1 ⁇ g/ml, and allowed to stand overnight at 4°C. Wash 3 times with PBS (PBST) containing 0.05% Tween20. Block with 5% skim milk in PBS for 1 hour at room temperature. Wash 3 times with PBST, add gradiently diluted anti-LAG3 humanized antibodies h6H11B10 and h6H11B10#40, and let stand at room temperature for 1 hour. At the same time, BMS986016 was used as a positive control.
  • Flow cytometry was used to measure the binding of humanized antibodies to the cynomolgus monkey LAG3 on the cell surface, and CHO-S cells transfected and expressing the cynomolgus monkey LAG3 full-length gene were taken, and the cells were washed with PBS (2% FBS) Twice, adjust the cell concentration to 5 ⁇ 10 5 /100 ⁇ l. Add 1:3 gradient dilution of anti-LAG3 humanized antibody, and incubate at 4°C for 30min. At the same time, BMS986016 was used as a positive control. Centrifuge the cells at 500 ⁇ g for 5 min. Wash twice with PBS (2% FBS).
  • SPR Surface plasmon resonance
  • Example 9 The ability of anti-LAG3 humanized antibodies to activate T cells
  • Example 10 Inhibitory activity of anti-LAG3 humanized antibody on tumor growth of hLAG3 KI mouse model
  • hLAG3 KI mice Using hLAG3 KI mice, the ability of humanized anti-LAG3 antibodies to inhibit tumor cell growth in vivo was tested.
  • Mouse colon cancer cells M38 were inoculated into the back of hLAG3 KI mice subcutaneously at 1 ⁇ 10 6 cells per mouse.
  • the antibody has two doses of 30mg/kg and 10mg/kg, injected twice a week, about 2 to 3 weeks.
  • the test groups include: isocratically diluted h6H11B10#40 sample group (the highest test concentration is 10 ⁇ g/mL) , Isocratic dilution HLX10 group (the highest test concentration is 10 ⁇ g/mL), in which the combination group is diluted with different concentrations of HLX10 solution (1 ⁇ g/mL, 0.1 ⁇ g/mL, 0.01 ⁇ g/mL and 0.001 ⁇ g/mL) h6H11B10# 40.
  • the amount of IL-2 and IFN- ⁇ in the group treated with h6H11B10#40 or HLX10 alone is less.
  • concentration of HLX10 reaches about 1 ⁇ g/mL
  • IL-2 The release amount of and IFN- ⁇ is higher than that of h6H11B10#40 single-drug and HLX10 single-drug group, combined drug group (when HLX10 concentration is 1 ⁇ g/mL and 0.1 ⁇ g/mL), h6H11B10#40 shows a concentration-dependent effect.
  • This in vitro experiment shows that the combination of h6H11B10#40 and HLX10 can reactivate exhausted T cells and increase the release of cytokines.
  • CV coefficient of variation
  • the TGI% is 62.2% and 56.7%, which are higher than the single-drug group.
  • the A20 cells were cultured in the medium RPMI-1640+10% FBS, and the A20 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration (0.1 mL/mouse) for subcutaneous inoculation of mice.
  • the experimental mice were subcutaneously inoculated with A20 cells (5 ⁇ 10 5 /mouse) on the right anterior scapula.
  • the day of grouping was defined as day 0, and the administration started on day 0.
  • the tumor volume was greater than 2500mm 3 and euthanized, resulting in a decrease in the number of mice.
  • the 14th day was used as a TGI analysis.
  • the TGI of h6H11B10#40 per kg and 30mg/kg h6H11B10#40 were 13.36%, 12.83% and 22.20%, respectively.
  • the TGI% are 25.9% and 62.4%, respectively, which are better than the single-drug group, showing the combination group’s Anti-tumor effect.

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Abstract

本发明提供了一种抗LAG3单克隆抗体及其用途。还提供了编码该抗体的核苷酸分子，表达该抗体的表达载体、宿主细胞，含有该抗体的组合物，以及该抗体在制备抗肿瘤、治疗自身免疫性疾病、治疗感染性疾病和/或抗移植排斥反应的药物中的用途。

Description

抗LAG3单克隆抗体及其制备方法和应用 技术领域
本发明涉及生物医药领域，具体涉及抗LAG3单克隆抗体及其制备方法和应用。
背景技术
LAG3(Lymphocyte-activation gene 3)，也叫CD223，于1990年被发现，属于免疫球蛋白超家族，在活化的T细胞、NK细胞、B细胞以及树突状细胞上表达。LAG3的配体之一是主要组织相容性复合体MHC(major histocompatibility complex)II类分子，LAG3与MHC II的亲和力甚至强于CD4与MHC II。另外，LAG3的配体还包括LSECtin和FGL1等。LAG3的生物学功能与CTLA-4和PD-1类似，起到负调节T细胞的细胞增殖和活化的作用。另外，LAG3也在Treg细胞的抑制功能中发挥作用。
研究发现，通过抑制LAG3能够让T细胞重新被激活，从而增强对肿瘤的杀伤效果。同时抑制LAG3还能够降低Treg细胞抑制免疫反应的功能，因此以LAG3作为癌症免疫治疗的靶点，利用单克隆抗体结合LAG3，可以阻断LAG3与其配体的结合，阻止T细胞抑制信号的产生，从而促进T细胞的活化与增殖，以及细胞因子表达，上调免疫系统针对肿瘤细胞的监控活性，增强特异性抗肿瘤的免疫反应，达到治疗肿瘤的目的。目前，包括百时美施贵宝、诺华、默沙东等制药公司都在开展各自的针对LAG3的抗体药物，目前均在临床试验阶段。由百时美施贵宝公司研究开发的抗LAG3抗体BMS986016(Relatlimab)在晚期黑色素瘤的治疗中取得了明显的效果，目前正在开展III期临床试验。另外，抗LAG3抗体与包括抗PD-1抗体在内的其他抗体药物联用，也正在开展相关的临床试验。
因此研发新型的抗LAG3单克隆抗体，用于癌症的免疫治疗，使其具有更低的毒副作用，更佳的临床药效，成为目前的研究热点，也将给患者提供更多的药物选择。
发明内容
本发明所要解决的技术问题是提供了新的抗LAG3单克隆抗体，从而完成了本发明。
因此，本发明的第一个目的在于提供新的抗LAG3单克隆抗体。
本发明的第二个目的在于提供编码所述抗LAG3单克隆抗体的核苷酸分子。
本发明的第三个目的在于提供包含所述核苷酸分子的表达载体。
本发明的第四个目的在于提供包含所述表达载体的宿主细胞。
本发明的第五个目的在于提供一种所述抗LAG3单克隆抗体的制备方法。
本发明的第六个目的在于提供包含所述抗LAG3单克隆抗体的组合物。
本发明的第七个目的在于提供所述抗LAG3单克隆抗体在制备药物中的应用。
为了实现上述目的，本发明采取了如下技术方案：
本发明的第一个方面提供了一种抗LAG3单克隆抗体，该单克隆抗体包含重链可变区和轻链可变区，所述重链可变区包含与SEQ ID NO:2、6或10所示重链可变区具有的CDR序列相同的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO:4、8或12所示轻链可变区具有的CDR序列相同的LCDR1、LCDR2和LCDR3。
在一些实施方式中，所述抗LAG3单克隆抗体包括：
1)重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1具有如SEQ ID No:18所示的氨基酸序列，所述HCDR2具有如SEQ ID No:19所示的氨基酸序列，所述HCDR3具有如SEQ ID No:20或21所示的氨基酸序列；
2)轻链互补决定区LCDR1、LCDR2、LCDR3，所述LCDR1具有如SEQ ID No:22或23所示的氨基酸序列，所述LCDR2具有如SEQ ID No:24所示的氨基酸序列，所述LCDR3具有如SEQ ID No:25所示的氨基酸序列。
在一些实施方式中，所述抗LAG3单克隆抗体包括重链可变区和轻链可变区，所述重链可变区具有如SEQ ID NO:2、6或10所示的氨基酸序列，或者与上述序列具有至少85％同源性的序列，例如85％、90％、92％、94％、95％、96％、97％、98％或99％同源性的衍生序列；所述轻链可变区具有如SEQ ID NO:4、8或12所示的氨基酸序列，或者与上述序列具有至少85％同源性的序列，例如85％、90％、92％、94％、95％、96％、97％、98％或99％同源性的衍生序列。
在一些实施方式中，所述抗LAG3单克隆抗体包括重链和轻链，所述重链由具有如SEQ ID NO:2、6或10所示的氨基酸序列或者与上述序列具有至少85％同源性的序列，例如85％、90％、92％、94％、95％、96％、97％、98％或99％同源性的序列的重链可变区，和如SEQ ID NO:14所示的重链恒定区组成；所述轻链由具有如SEQ ID NO:4、8或12所示的氨基酸序列，或者与上述序列具有至少85％同源性的序列，例如85％、90％、92％、94％、95％、96％、97％、98％或99％同源性的序列的轻链可变区，和如SEQ ID NO:16所示的轻链恒定区组成。
优选地，所述抗LAG3单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:4所示。
优选地，所述抗LAG3单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。
优选地，所述抗LAG3单克隆抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示。
所述抗LAG3单克隆抗体可以是抗体的全长序列，也可以是抗LAG3抗体的片段，所述片段为Fab、Fab’、F(ab’)2、Fv或scFv等。优选的，所述抗LAG3抗体为IgG1、IgG2或IgG4型抗体。
本发明进一步提供所述抗LAG3抗体的衍生物，所述衍生物为LAG3抗体的片段、抗体/抗体片段-因子融合蛋白、抗体/抗体片段-化学偶联物；所述抗LAG3抗体的片段为Fab、Fab’、F(ab’)2、Fv或scFv等。
本发明所述单克隆抗体可以由本领域常规技术制备，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，优选地是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
本发明的第二个方面提供了一种分离的核苷酸分子，所述核苷酸分子编码如上所述的抗LAG3单克隆抗体。
在一些实施方式中，编码所述抗LAG3单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:3、7或11所示，编码抗LAG3单克隆抗体的轻链可变区的核苷酸序列如SEQ ID NO:5、9或13所示。
优选地，编码所述抗LAG3单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:3所示，编码所述抗LAG3单克隆抗体的轻链可变区的核苷酸序列如SEQ ID NO:5所示。
优选地，编码所述抗LAG3单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:7所示，编码所述抗LAG3单克隆抗体的轻链可变区的核苷酸序列如SEQ ID NO:9所示。
优选地，编码所述抗LAG3单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:11所示，编码所述抗LAG3单克隆抗体的轻链可变区的核苷酸序列如SEQ ID NO:13所示。
所述核苷酸分子的制备方法为本领域常规制备方法，优选地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码所述单克隆抗体的核苷酸分子，或者通过人工全序列合成的方法得到编码上述单克隆抗体的核苷酸分子。
本领域技术人员知晓，编码上述单克隆抗体的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物或其保守型变异序列。本发明中多聚核苷酸的同系物或其保守型变异序列，可以通过对编码该单克隆抗体基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明的第三个方面提供了一种表达载体，所述表达载体含有如上所述的核苷酸分子。
所述表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明中的表达载体指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。
优选地，所述表达载体选自pHLX101、pEE14.4、pCHO 1.0或pcDNA3.1中的一种或多种。
更优选地，所述表达载体为pcDNA3.1。
本发明的第四个方面提供了一种宿主细胞，所述宿主细胞含有如上所述的表达载体。
本发明所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。本发明中的宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
优选地，所述宿主细胞为：COS、CHO(中国仓鼠卵巢，Chinese Hamster Ovary)、HeLa细胞系、骨髓细胞系如SP2/0细胞系、NS0、sf9、sf21、DH5α、BL21(DE3)或E.coli TG1、YB2/0细胞系等以及转化的B-细胞或杂交瘤细胞中的一种或多种。
更优选地，所述宿主细胞为E.coli TG1、BL21细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。
将表达载体转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，优选地为化学转化法，热激法或电转法。
本发明的第五个方面提供了一种制备如上所述的抗LAG3单克隆抗体的方法，包括以下步骤：
a)在表达条件下，培养如上所述的宿主细胞，表达抗LAG3单克隆抗体；
b)分离并纯化步骤a)所得的抗LAG3单克隆抗体。
本发明所述的宿主细胞的培养方法，所述抗LAG3的单克隆的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及单克隆抗体分离纯化技术手册。
本发明中所用的宿主细胞均为现有技术，可通过商业途径直接获取，培养 中所用的培养基亦为各种常规培养基，本领域技术人员可根据经验选择适用的培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其他特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其他各种液相层析技术及这些方法的结合。
本发明从单克隆培养的细胞株中筛选获取目的抗体的基因序列，用以构建真核表达载体，表达后即可重建抗体的活性，获得抗LAG3单克隆抗体。
本发明的第六个方面提供了一种组合物，其包含如上所述的抗LAG3单克隆抗体和药学上可接受的载体。
本发明的单克隆抗体可以通过本领域任何已知的方式配制药物组合物来使用。这种组合物以所述单克隆抗体为活性成分，加上一种或多种药物可接受的载体、稀释剂、填充剂、结合剂及其他赋形剂，这依赖于给药方式及所设计的剂量形式。本领域枝术人员已知的治疗惰性的无机或有机的载体包括(但不限于)乳糖、玉米淀粉或其衍生物、滑石、植物油、蜡、脂肪、多羚基化合物例如聚乙二醇、水、蔗糖、乙醇、甘油，诸如此类，各种防腐剂、润滑剂、分散剂、矫味剂。保湿剂、抗氧化剂、甜味剂、着色剂、稳定剂、盐、缓冲液诸如此类也可加入其中，这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味，在这种组合物中可以使用抑制剂可是其原始化合物本身的形式，或任选地使用其药物学可接受的盐的形式，本发明的单克隆抗体可以单独给药，或以各种组合给药，以及与其他治疗药剂一起结合形式给药。如此配制的组合物根据需要可选择本领域技术人员已知的任何适当的方式把抑制剂进行给药。
本发明提供的抗LAG3单克隆抗体，可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明所述的抗LAG3单克隆抗体的氨基酸核心序列的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。通常情况下，对于液体制剂，通常可以在2℃-8℃条件下保存至少稳定一年，对于冻干制剂，在30℃至少六个月保持稳定。所述抗LAG3单克隆抗体制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂，
对于本发明所述的抗LAG3单克隆抗体的水针或冻干制剂，药学上可以接 受的载体优选地包括但不限于：表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中表面活性剂优选地包括但不限于：非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(Tween 20或80)；Poloxamer(如Poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使抗LAG3单克隆抗体的颗粒化趋势最小。溶液稳定剂优选地包括但不限于以下列举之一或其组合：糖类，例如，还原性糖和非还原性糖；氨基酸类，例如，谷氨酸单钠或组氨酸；醇类，例如：三元醇、高级糖醇、丙二醇、聚乙二醇等，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态。等渗调节剂优选地包括但不限于氯化钠、甘露醇之一或其组合。缓冲液优选地包括但不限于：Tris、组氨酸缓冲液、磷酸盐缓冲液之一或其组合。
本发明的第七个方面提供了如上述抗LAG3单克隆抗体或其组合物在制备药物中的应用。
优选地，所述应用为制备LAG3分子阻滞药物上的用途。更优选地，所述制备LAG3分子阻滞药物上的用途具体为制备肿瘤治疗或肿瘤诊断类药物。
本发明所述的药物优选地为抗肿瘤、治疗自身免疫性疾病、治疗感染性疾病和/或抗移植排斥反应的药物，更优选地为抗肿瘤药物、治疗自身免疫性疾病药物，进一步优选地为抗肿瘤药物。本发明所述抗LAG3单克隆抗体可以单独使用或与其他抗肿瘤药物联合使用。所述其他抗肿瘤药物为本领域常规抗肿瘤药物，包括抗体类药物或小分子抗肿瘤药物。所述抗体类药物为本领域常规抗体类药物，优选地包括抗PD-1单克隆抗体。所述小分子抗肿瘤药物为本领域常规药物，包括紫杉醇、5-Fu嘧啶等。
其中所述抗肿瘤药物所针对的肿瘤优选地包括但不限于：黑色素瘤、肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、结肠癌、神经胶质瘤、膀胱癌、乳腺癌、肾癌、食道癌、胃癌、口腔鳞状细胞癌、尿道上皮细胞癌、胰腺癌和/或头颈肿瘤中的一种或多种。
本发明所称的抗肿瘤药物，指具有抑制和/或治疗肿瘤的药物，可以包括伴随肿瘤相关症状发展的延迟和/或这些症状严重程度的降低，进一步还包括已存在的肿瘤伴随症状的减轻并防止其他症状的出现，还包括减少或防止肿瘤的转移等。
本发明中抗LAG3单克隆抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。一般地，静脉注射的剂量是1-1800mg/天。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明 各优选实例。
本发明的有益效果：
本发明的抗LAG3单克隆抗体具有良好的生物活性，在哺乳动物细胞中具有较高的表达量，并且对LAG3具有明显的亲和力和抑制配体-受体结合的能力。该单克隆抗体能够单独，或与其他抗肿瘤药物联合应用在肿瘤免疫治疗以及诊断和筛查中，能够有效应用于治疗肿瘤、感染性疾病、自身免疫性疾病以及抗免疫排斥等药物的制备中。
附图说明
图1为抗LAG3抗体与细胞表面的LAG3结合能力实验结果；
图2为抗LAG3抗体阻断LAG3与其配体MHC II结合的实验结果；
图3为抗LAG3嵌合抗体与LAG3结合能力实验结果；
图4为抗LAG3嵌合抗体与细胞表面的LAG3结合能力实验结果；
图5为抗LAG3人源化抗体与LAG3结合能力实验结果；
图6为抗LAG3人源化抗体与细胞表面的LAG3结合能力实验结果；
图7为抗LAG3人源化抗体与LAG3亲和力的表面等离子共振的实验结果；
图8为抗LAG3人源化抗体阻断LAG3与其配体MHC II结合的实验结果；
图9为抗LAG3人源化抗体阻断LAG3与其配体LSECtin结合的实验结果；
图10为抗LAG3人源化抗体阻断LAG3与其配体FGL1结合的实验结果；
图11为抗LAG3人源化抗体物种交叉反应的ELISA实验结果；
图12为抗LAG3人源化抗体物种交叉反应的流式细胞仪实验结果；
图13为抗LAG3人源化抗体物种交叉反应的表面等离子共振的实验结果；
图14为抗LAG3人源化抗体激活T细胞的实验结果；
图15为抗LAG3人源化抗体在hLAG3 KI小鼠模型中抑制肿瘤生长的实验结果；
图16显示h6H11B10#40与HLX10联合用药激活IL-2的释放；
图17显示h6H11B10#40与HLX10联合用药激活IFN-γ的释放；
图18显示在MC38模型中各组小鼠肿瘤体积的生长曲线；
注：数据以“平均值±标准误差”表示；
图19显示在A20模型中各组小鼠肿瘤体积的生长曲线；
注：1.数据以“平均值±标准误差”表示。
具体实施方式
下面通过实施例进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。实施例中所述的室温为本领域常规的室温，一般为 10-30℃。除非另外说明，所用试剂和原料均为市售可得。
应理解，本发明的保护范围不局限于下述特定的具体实施方案；并且本发明实施例中使用的术语是为了描述本发明的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
术语抗体的“可变区”是指单独的或组合的抗体轻链的可变区(VL)或抗体重链的可变区(VH)。如在本领域中已知的，重链和轻链的可变区各自由通过3个互补决定区(CDR)(也称为高变区)连接的4个框架区(FR)组成。每一条链中的CDR通过FR紧密地保持在一起并且与来自另一条链的CDR一起促成抗体的抗原结合部位的形成。存在至少2个用于确定CDR的技术：(1)基于跨种序列变异性的方法(即，Kabat等Sequences of Proteins of Immunological Interest(第5版，1991,National Institutes of Health,Bethesda MD))；和(2)基于抗原-抗体复合物的晶体学研究的方法(Al-Lazikani等，J.Molec.Biol.273:927-948(1997))。如本文中所用，CDR可指由任一方法或由两种方法的组合确定的CDR。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”和“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(CDR-H1、CDR-H2、CDR-H3)和每个轻链可变区中存在三个CDR(CDR-L1、CDR-L2、CDR-L3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(Al-Lazikani等(1997)，JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist,7,132-136(1999)；Lefranc,M.P.等，Dev.Comp.Immunol.,27,55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97 (LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(HCDR1)、51-57(HCDR2)和93-102(HCDR3)，VL中的CDR氨基酸残基编号大致为27-32(LCDR1)、50-52(LCDR2)和89-97(LCDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“LAG3”指淋巴细胞活化基因-3蛋白，一种免疫检验点受体或T细胞共抑制子，也称为CD223。全长LAG3的氨基酸序列在GenBank中以登录号NP_002277.4提供。术语“”包括变体、同工型、同系物、直系同源物和旁系同源物。例如LAG3蛋白变体、重组LAG3或其片段，以及与例如组氨酸标签、小鼠或人Fc或信号序列(如ROR1)偶联的LAG3或其片段。
当实施例给出数值范围时，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等,MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989 and Third edition,2001；Ausubel等,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley&Sons,New York,1987 and periodic updates；the series METHODS IN ENZYMOLOGY,Academic Press,San Diego；Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998；METHODS IN ENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),Academic Press,San Diego,1999；和METHODS IN MOLECULAR BIOLOGY,Vol.119,Chromatin Protocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
阳性对照抗体BMS986016：氨基酸序列记载于百时美施贵宝公司专利申请WO 2015/042246 Al。本发明将编码该氨基酸序列的核苷酸序列通过限制性内切酶克隆到pURO载体后，将该质粒转染至CHO-S细胞中，经羧汴青霉素抗性筛选得到高表达细胞株，通过扩大培养该细胞株，收集培养基上清，通过protein A亲和层析的制备工艺得到该对照抗体。
阴性对照HLX10(h1G4)：其序列引自CN109923126A。
阳性对照ab40465：Abcam公司，货号ab40465.
同种型-人IgG4(hIgG4)：中美冠科公司，Cat:AB180018
重组小鼠LAG3-Fc融合蛋白：购自义翘神州，Cat:53069-M02H。
重组食蟹猴LAG3-Fc融合蛋白：购自义翘神州，Cat:90841-C08H
抗小鼠PD1抗体RMP1-14：BioXCell公司，Cat:717918O1
实施例1 抗LAG3抗体的筛选
本实施例采用自主构建并表达纯化的LAG3-His(氨基酸序列如SEQ ID No:1所示)蛋白以及表达LAG3全长的CHO-S细胞共同免疫小鼠，将脾脏细胞与骨髓瘤细胞融合得到杂交瘤细胞后，然后采用ELISA筛选阳性克隆。
具体地，首先将编码LAG3胞外区的23-450氨基酸的核酸序列通过限制性内切酶克隆到pEE14.4载体中，其中在LAG3编码区C端带有编码6xHis的标签，然后将该质粒电转至CHO-S细胞中，通过GS(Glutamine Synthetase，谷氨酰胺合成酶)筛选系统筛选出高表达LAG3-his融合蛋白的单克隆，将该单克隆扩大培养，收集培养基上清，通过镍柱亲和层析的方法制备目的蛋白。随后，将该蛋白以及表达LAG3全长的CHO-S细胞共同免疫小鼠。在筛选阳性杂交瘤的过程中，首先将LAG3-His重组蛋白按2μg/ml包被96孔半孔板中(工作体积30μl)，4℃包被过夜。用含有0.05％的Tween 20的PBS(PBST)洗3次洗掉过量的LAG3-His。用5％的脱脂牛奶的PBS室温封闭1小时。用PBST洗3次，接着加入稀释后的杂交瘤细胞培养上清液，室温静置反应1小时后用PBST洗3次，加入30μl用PBS稀释的辣根过氧化物酶标记的山羊抗鼠IgG二抗(康为世纪公司)，稀释比例为1:2000，室温静置1小时后，后用PBST洗6次，加入TMB显色，并用30μl 2M的H 2SO 4终止反应。用酶标仪读取在450nm的波长的光吸收。
进一步采用流式细胞仪方法确定经ELISA筛选得到的表达抗体的阳性杂交瘤克隆。首先，将正常生长的表达人LAG3全长基因的CHO-S细胞，用PBS(2％FBS)清洗细胞两次，将细胞浓度调整到5×10 5/100μl。添加杂交瘤上清液，在4℃下孵育30min。在500×g下将细胞离心5min。阴性对照组不加入抗体。用PBS(2％FBS)清洗两次。添加经1:100稀释的山羊抗鼠IgG-FITC(康为世纪公司)，在4℃下孵育30min。用PBS(2％FBS)清洗细胞两次。将细胞悬浮于200μl PBS中，用流式细胞仪进行分析。结果如图1所示，经ELISA筛选得到的阳性克隆大多数都能与细胞表面的LAG3结合。
采用流式细胞仪方法测试以上经ELISA筛选得到的杂交瘤阳性克隆表达的抗体阻断细胞表面的MHC II与LAG3-Fc(Acro公司)结合的能力。用PBS(2％FBS)清洗Daudi细胞两次，将细胞浓度调整到3×10 5/100μl。将融合瘤上清液，与2μg/ml浓度的LAG3-Fc混合后，再与Daudi细胞混合，在4℃下孵育30min。在500×g下将细胞离心5min。同时用无抗体作为阴性对照，抗LAG3抗体 BMS986016作为阳性对照。用PBS(2％FBS)清洗两次。添加经1:300稀释的山羊抗人IgG-Fc-FITC(Thermo公司)，在4℃下孵育30min。用PBS(2％FBS)清洗细胞两次。将细胞悬浮于200μl PBS中，用流式细胞仪进行分析。结果如图2所示，最终筛选得到了多株具有阻断细胞表面的MHC II与LAG3-Fc结合的能力的单克隆杂交瘤细胞系，最优的一株单克隆杂交瘤细胞系为6H11B10。6H11B10的重链可变区的氨基酸序列如SEQ ID No:2所示，核苷酸序列如SEQ ID No:3所示；轻链可变区的氨基酸序列如SEQ ID No:4所示，核苷酸序列如SEQ ID No:5所示。
实施例2 抗LAG3嵌合抗体的亲和力测定
小鼠轻链和重链可变区的扩增，按照Anke Krebber等(Journal of Immunological Methods 201.1997.35-55)所介绍的方法进行。大致方法包括，将单克隆杂交瘤细胞系6H11B10制备总RNA，在利用反转录试剂盒(TaKaRa公司)制备单链cDNA。根据Anke Krebber等提到的利用能与小鼠轻链和重链可变区互补配对的引物，通过PCR反应扩增出抗体的重链可变区(SEQ ID No:3)和轻链可变区(SEQ ID No:5)的基因。于是，再分别将6H11B10的轻链可变区和重链可变区基因分别与重链IgG4的恒定区基因(SEQ ID No:15)和轻链的恒定区基因(SEQ ID No:17)融合，从而获得6H11B10的嵌合抗体重链基因和轻链基因。
将6H11B10的嵌合抗体基因分别转染进CHO-S细胞，并进行表达，从而获得嵌合抗体蛋白c6H11B10。采用ELISA技术检测嵌合抗体对LAG3的亲和力。将LAG3-Fc重组蛋白按2μg/ml包被到酶标板中(工作体积30μl)，4℃静置过夜。用含有0.05％的Tween 20的PBS(PBST)洗3次。用5％的脱脂牛奶的PBS室温封闭1小时。用PBST洗3次，接着加入梯度稀释的c6H11B10，室温静置1小时。同时用BMS986016作为阳性对照。用PBST洗3次，加入30μl 1:4000稀释的辣根过氧化物酶标记的山羊抗人IgGkappa light chain二抗(Millipore公司)，室温静置1小时。用PBST洗6次，加入TMB显色，并用2M的H 2SO 4终止反应，用酶标仪在450nm下读数。结果如图3所示，抗LAG3嵌合抗体c6H11B10对LAG3有很强的亲和力，且在低浓度时，c6H11B10的亲和力高于对照抗体BMS986016。
实施例3 抗LAG3嵌合抗体的亲和力测定
采用流式细胞仪方法测定嵌合抗体蛋白c6H11B10对于细胞表面的LAG3的亲和力。取经过转染并表达人LAG3全长基因的Jurkat细胞，用PBS(2％FBS)清洗细胞两次，将细胞浓度调整到2×10 5/100μl。添加梯度稀释的嵌合抗体 c6H11B10，以及鼠源抗体m6H11B10，在4℃下孵育30min。在500×g下将细胞离心5min。同时用BMS986016作为阳性对照。用PBS(2％FBS)清洗两次。对于嵌合抗体和BMS986016添加经1:300稀释的山羊抗人IgG Fab-FITC(Thermo Fisher公司)，对于鼠源抗体添加经1:100稀释的山羊抗鼠IgG-FITC(康为世纪公司)，在4℃下孵育30min。用PBS(2％FBS)清洗细胞两次。将细胞悬浮于200μl PBS中，用流式细胞仪进行分析。结果如图4所示，嵌合抗体c6H11B10能与细胞表面的LAG3有较强的结合，且c6H11B10的亲和力优于对照抗体BMS986016。
实施例4 抗LAG3鼠源抗体6H11B10的人源化
对于鼠源抗体6H11B10人源化，使用人的抗体轻链可变区的种系基因IGKV2-28*01和重链可变区的种系基因IGHV3-11*06将鼠源抗体6H11B10中的框架区替换，保留原来的互补决定区(CDR)，从而得到抗6H11B10人源化抗体h6H11B10。h6H11B10的重链可变区的氨基酸序列如SEQ ID No:6所示，轻链可变区的氨基酸序列如SEQ ID No:8所示。h6H11B10的重链可变区的基因序列如SEQ ID No:7所示，轻链可变区的基因序列如SEQ ID No:9所示，再分别与重链IgG4的恒定区基因(SEQ ID No:15)和轻链的恒定区基因(SEQ ID No:17)融合，从而获得抗LAG3人源化抗体h6H11B10的重链基因和轻链基因。h6H11B10重链的三个CDR分别为：HCDR1(SEQ ID No:18)、HCDR2(SEQ ID No:19)和HCDR3(SEQ ID No:20)；轻链的三个CDR分别为：LCDR1(SEQ ID No:22)、LCDR2(SEQ ID No:24)和LCDR3(SEQ ID No:25)。
抗LAG3人源化抗体h6H11B10采用噬菌体展示技术对其进行亲和力成熟，设计带有点突变的引物，利用PCR对h6H11B10的轻链CDR以及重链CDR分别进行突变，从而得到一个突变文库，将噬菌体展示载体转化进大肠杆菌TG1或SS320细胞，从而产生噬菌体文库。利用偶合链霉亲和素的磁珠M-280(Thermo Fisher公司)与生物素标记的LAG3蛋白，对噬菌体文库进行两轮筛选。再经由ELISA筛选出1个亲和力最高的克隆，h6H11B10#40。h6H11B10#40的重链可变区的氨基酸序列如SEQ ID No:10所示，轻链可变区的氨基酸序列如SEQ ID No:12所示。h6H11B10#40的重链可变区基因如SEQ ID No:11所示，轻链可变区基因如SEQ ID No:13所示，再分别与重链IgG4的恒定区基因(SEQ ID No:15)和轻链的恒定区基因(SEQ ID No:17)融合，从而获得抗LAG3人源化抗体h6H11B10#40的重链基因和轻链基因。h6H11B10#40重链的三个CDR分别为：HCDR1(SEQ ID No:18)、HCDR2(SEQ ID No:19)和HCDR3(SEQ ID No:21)；轻链的三个CDR分别为：LCDR1(SEQ ID No:23)、LCDR2(SEQ ID No:24)和LCDR3(SEQ ID No:25)。
实施例5 抗LAG3人源化抗体对LAG3的亲和力检测
将h6H11B10和h6H11B10#40抗LAG3人源化抗体基因分别转染进CHO-S细胞，并进行表达，从而获得h6H11B10和h6H11B10#40抗体蛋白。采用ELISA技术检测人源化抗体对LAG3的亲和力。将LAG3-Fc重组蛋白按2μg/ml包被到酶标板中(工作体积30μl)，4℃静置过夜。用含有0.05％的Tween 20的PBS(PBST)洗3次。用5％的脱脂牛奶的PBS室温封闭1小时。用PBST洗3次，接着加入梯度稀释的h6H11B10和h6H11B10#40的人源化抗体蛋白，室温静置1小时。同时用BMS986016作为阳性对照。用PBST洗3次，加入30μl 1:4000稀释的辣根过氧化物酶标记的山羊抗人IgG kappa light二抗(Millipore公司)，室温静置1小时。用PBST洗6次，加入TMB显色，并用2M的H 2SO 4终止反应。用酶标仪在450nm下读数。结果如图5所示，抗LAG3人源化抗体对于LAG3均有很强的亲和力，其中，h6H11B10#40要优于h6H11B10，两者均优于阳性对照BMS986016。
采用流式细胞仪方法测人源化抗体对细胞表面的LAG3的结合，取经过转染并表达人LAG3全长基因的CHO-S或者Jurkat细胞，用PBS(2％FBS)清洗细胞两次，将细胞浓度调整到5×10 5/100μl。加入1:3梯度稀释的抗LAG3人源化抗体，在4℃下孵育30min。同时用BMS986016作为阳性对照。在500×g下将细胞离心5min。用PBS(2％FBS)清洗两次。添加经1:300稀释的山羊抗人IgG Fab-FITC(Thermo Fisher公司)，在4℃下孵育30min。用PBS(2％FBS)清洗细胞两次。将细胞悬浮于200μl PBS中，用流式细胞仪进行分析。结果如图6所示，抗LAG3人源化抗体对于细胞膜上的LAG3均有很强的亲和力，h6H11B10#40和h6H11B10均要优于阳性对照BMS986016。
利用表面等离子共振(SPR)测量抗LAG3人源化抗体与LAG3蛋白的结合亲和力及动力学常数。先将LAG3-Fc蛋白首先固定于芯片上，再将抗LAG3人源化抗体h6H11B10#40以及BMS986016用HBSPE缓冲液中从80μg/ml至2.5μg/ml梯度稀释，分别流过芯片。实验所获得的数据采用评估软体分析，曲线用1:1朗缪尔(Langmuir)结合模型拟合。结合及解离动力学及算得的亲和力常数(KD)如表1所示。结果如图7所示，经过亲和力成熟后，抗LAG3人源化抗体h6H11B10#40对于LAG3的亲和力均有大幅度的提高，结合及解离常数均要优于阳性对照抗体BMS986016。
表1
样品 ka(1/Ms) kd(1/s) KD(M)
BMS986016 3.22E+04 2.04E-04 6.35E-09
h6H11B10#40 4.35E+04 1.37E-05 3.13E-10
实施例6 抗LAG3人源化抗体对于阻断LAG3与其配体结合的能力
采用流式细胞仪方法测抗LAG3人源化抗体阻断Raji细胞表面的MHC II与LAG3-Fc结合的能力。用PBS(2％FBS)清洗Raji细胞两次，将细胞浓度调整到2×10 5/100μl，抗LAG3人源化抗体h6H11B10#40按1:3梯度稀释后，与2μg/ml浓度的LAG3-Fc混合后，室温放置30min，同时用hIgG4蛋白作为阴性对照，抗LAG3抗体BMS986016作为阳性对照。再与Raji细胞混合，在4℃下孵育60min。在500×g下将细胞离心5min。用PBS(2％FBS)清洗两次。添加经1:500稀释的山羊抗人IgG-Fc-Alexa Fluor 488(Jackson公司)，在4℃下孵育60min。用PBS(2％FBS)清洗细胞两次。将细胞悬浮于200μl PBS中，用流式细胞仪进行分析。结果如图8所示，抗LAG3人源化抗体h6H11B10#40具有阻断细胞表面的MHC II与LAG3-Fc结合的能力，能力与BMS986016相似。
采用ELISA技术检测人源化抗体阻断LAG3-Fc与其配体LSECtin结合的能力。将LAG3-Fc重组蛋白按1μg/ml包被到酶标板中(工作体积100μl)，4℃静置过夜。用含有0.05％的Tween 20的PBS(PBST)洗3次。用5％的脱脂牛奶的PBS室温封闭1小时，用PBST洗3次。按1:5梯度稀释的h6H11B10#40的人源化抗体蛋白，同时用hIgG4蛋白作为阴性对照，抗LAG3抗体BMS986016作为阳性对照。另外将LSECtin(R&D公司)配制成用1μg/ml，与抗体按1:1混合，室温静置30min。然后加入到酶标板中，室温静置1小时。PBST洗3次，加入100μl 1:5000稀释的辣根过氧化物酶标记的小鼠抗His标签二抗(金斯瑞公司)，室温静置1小时。用PBST洗6次，加入TMB显色，并用2M的H 2SO 4终止反应。用酶标仪在450nm下读数。结果如图9所示，抗LAG3人源化抗体h6H11B10#40具有阻断LAG3-Fc与其配体LSECtin结合的能力。
实施例7 抗LAG3人源化抗体对于阻断LAG3与其配体结合的能力
使用商业化的测定抗LAG3抗体阻断LAG3与FGL1结合的试剂盒(购自Cisbio公司，货号：63ADK000CB10PEG)，测试抗LAG3人源化抗体阻断LAG3和FGL1结合的能力。该实验首先将4μL的Tag1-LAG3蛋白和4μL的Tag2-FGL1蛋白混合与浓度稀释的测试样品，室温反应15分钟后加入预混合的10μL anti-Tag1-Tb3+和anti-Tag2-XL665，密封该测试板，室温反应过夜后用酶标仪读取665/620nm的荧光吸收值。结果如图10所示，抗LAG3人源化抗体h6H11B10#40具有阻断LAG3与其配体FGL1结合的能力。
实施例8 抗LAG3人源化抗体的物种交叉反应性
重组小鼠及食蟹猴LAG3-Fc融合蛋白购买自义翘神州。将LAG3-Fc重组蛋 白按1μg/ml包被到酶标板中(工作体积30μl)，4℃静置过夜。用含有0.05％的Tween20的PBS(PBST)洗3次。用5％的脱脂牛奶的PBS室温封闭1小时。用PBST洗3次，加入梯度稀释的抗LAG3人源化抗体h6H11B10和h6H11B10#40，室温静置1小时。同时用BMS986016作为阳性对照。用PBST洗3次，加入30μl 1:2000稀释的辣根过氧化物酶标记的山羊抗人IgG kappa light二抗(millipore公司)，室温静置1小时。用PBST洗6次，加入TMB显色，并用2M的H 2SO 4终止反应。用酶标仪在450nm下读数。如图11所示，抗LAG3人源化抗体h6H11B10和h6H11B10#40相比BMS986016，均与食蟹猴的LAG3有很高的亲和力，而BMS986016对于食蟹猴的LAG3亲和力较低。BMS986016和h6H11B10对小鼠LAG3亲和力都很弱(未在图中显示)。
采用流式细胞仪方法测人源化抗体对细胞表面的食蟹猴LAG3的结合，取经过转染并表达食蟹猴LAG3全长基因的CHO-S细胞，用PBS(2％FBS)清洗细胞两次，将细胞浓度调整到5×10 5/100μl。加入1:3梯度稀释的抗LAG3人源化抗体，在4℃下孵育30min。同时用BMS986016作为阳性对照。在500×g下将细胞离心5min。用PBS(2％FBS)清洗两次。添加经1:300稀释的山羊抗人IgG Fab-FITC(Thermo Fisher公司)，在4℃下孵育30min。用PBS(2％FBS)清洗细胞两次。将细胞悬浮于200μl PBS中，用流式细胞仪进行分析。结果如图12所示，抗LAG3人源化抗体h6H11B10#40和h6H11B10对于细胞膜上的食蟹猴LAG3均有很强的亲和力，h6H11B10#40和h6H11B10均要优于阳性对照BMS986016。
利用表面等离子共振(SPR)测量抗LAG3人源化抗体与食蟹猴LAG3蛋白的结合亲和力及动力学常数。先将食蟹猴LAG3-Fc蛋白首先固定于芯片上，再将抗LAG3人源化抗体h6H11B10#40以及BMS986016用HBSPE缓冲液中从80μg/ml至2.5μg/ml梯度稀释，分别流过芯片。实验所获得的数据采用评估软体分析，曲线用1:1朗缪尔(Langmuir)结合模型拟合。结合及解离动力学及算得的亲和力常数(KD)，如表2所示。结果如图13所示，抗LAG3人源化抗体h6H11B10#40对于食蟹猴LAG3具有很高的亲和力。
表2
样品 ka(1/Ms) kd(1/s) KD(M)
h6H11B10#40 2.17E+04 3.09E-05 1.422E-09
实施例9 抗LAG3人源化抗体激活T细胞的能力
取Raji细胞，500×g离心5min，将细胞浓度调整到1.2×10 6/ml。加入SEE(Toxin Technology)，使终浓度为0.024ng/ml，37℃孵育30min。取转染了LAG3基因和NFAT-Luc报告基因的Jurkat细胞(Promega公司)，500×g离心5min， 将细胞浓度调整到1.6×10 6/ml。按1:3梯度稀释的抗LAG3人源化抗体，同时用BMS986016作为阳性对照，分别取25μl与等体积的Jurkat细胞混合，然后再加入25μl的SEE处理过的Raji细胞，37℃孵育6小时。加入Bio-Glo(Promega)，在多功能酶标仪进行读数。结果如图14所示，抗LAG3人源化抗体激活Jurkat细胞表达荧光素酶，其作用与BMS986016相似。
实施例10 抗LAG3人源化抗体对hLAG3 KI小鼠模型肿瘤生长的抑制活性
利用hLAG3 KI小鼠，检测抗LAG3人源化抗体在体内对肿瘤细胞生长的抑制能力。小鼠结肠癌细胞M38按每只小鼠1×10 6个细胞接种到hLAG3 KI小鼠背部皮下。当肿瘤体积达成约80-120mm 3时，开始腹腔注射抗小鼠PD-1抗体RMP1-14(中美冠科公司)，剂量为0.5mg/kg，以及抗LAG3人源化抗体h6H11B10#40，抗体分别有30mg/kg和10mg/kg两个剂量，每周注射两次，大约2至3周。同时注射PBS作为阴性对照，注射抗小鼠PD-1抗体RMP1-14和抗LAG3抗体BMS986016作为阳性对照。观察每只小鼠的肿瘤形成大小，一周两次。肿瘤体积计算方法为：V(mm 3)＝0.5×(长度(mm)×宽度(mm)×宽度(mm))。结果如图15所示，注射抗LAG3抗体与抗小鼠PD-1抗体，相比注射PBS的对照组，均能显著抑制肿瘤的生长，其中在高剂量组中，h6H11B10#40与抗小鼠PD-1抗体联用，疗效要好于单独抗体的效果。
实施例11 抗LAG3抗体介导的耗竭T细胞的重新激活
将人PBMC细胞500g离心10min，去上清，用培养缓冲液(RPMI-1640培养基+2％FBS)洗涤两次，计数后用刺激缓冲液(RPMI-1640培养基+2％FBS+20ng/mL SEB)重悬，接种至96孔细胞培养板中(5×10 5个细胞/孔)，放置37℃、7％CO2培养箱培养刺激过夜。第二天用培养缓冲液依次梯度稀释所需测试样品，在IL-2和IFN-γ测试实验中，测试组别包括：等梯度稀释的h6H11B10#40样品组(最高测试浓度为10μg/mL)，等梯度稀释HLX10组(最高测试浓度为10μg/mL)，其中联合用药组分别用不同浓度的HLX10溶液(1μg/mL，0.1μg/mL，0.01μg/mL和0.001μg/mL)稀释h6H11B10#40，分别将稀释好的检测样品依次加入刺激过夜的PBMC中，放置37℃、7％CO 2培养箱，继续培养2-3天，取上清用IL-2和IFN-γ试剂盒测定各检测组别IL-2和IFN-γ的释放量。
如图16和17数据所示，单独h6H11B10#40或HLX10处理的组别IL-2和IFN-γ的量较少，当HLX10浓度达约1μg/mL时与h6H11B10#40联合用药，IL-2和IFN-γ的释放量高于h6H11B10#40单药和HLX10单药组别，联合用药组(HLX10浓度为1μg/mL和0.1μg/mL时)，h6H11B10#40显示浓度依赖效应。该体外实验说明h6H11B10#40和HLX10联合用药可以使耗竭T细胞重新激活， 增加细胞因子的释放。
实施例12 在hLAG-3/hPD-1转基因小鼠MC38或A20皮下移植肿瘤模型中的疗效评估
MC38细胞培养在生长培养基中(RPMI-1640+10％FBS)，接种前收集对数生长期的细胞，重悬在PBS与基质胶中(0.1mL/只)用于小鼠皮下接种，待接种实验小鼠(周龄6-8周)，右侧背部后下方皮下接种MC38细胞(1×10 6/只)。待肿瘤平均体积78.03mm 3时，根据肿瘤大小随机分组。各组间的肿瘤体积的变异系数(CV)用公式CV＝SD/MTV×100％计算，应小于40％。分组当天定义为第0天，给药开始于第0天。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(公式中a表示长径，b表示短径)。结果如图18所示，MC38模型中，由于在实验过程中用药第17天，对照组小鼠肿瘤达到2500mm 3，对该小鼠执行安乐死，以D17数据分析，单药组HLX10和h6H11B10#40的TGI分别为35.22％和4.88％。，联合用药组中，当HLX10剂量为1.5mg/kg，h6H11B10#40剂量分别设定为10mg/kg和30mg/kg时，TGI％分别为62.2％和56.7％，均高于单药组。该数据说明h6H11B10#40和HLX10联合用药具有优于单药的抑瘤效果。
A20细胞培养在培养基RPMI-1640+10％FBS中，收集指数生长期的A20细胞，用PBS重悬至适合浓度(0.1mL/只)，用于小鼠皮下接种。实验小鼠于右前肩胛处皮下接种A20细胞(5×10 5/只)，待肿瘤平均体积90.44mm 3时，根据肿瘤大小随机分组。各组间的肿瘤体积的变异系数(CV)用公式CV＝SD/MTV×100％计算，应小于40％。分组当天定义为第0天，给药开始于第0天。A20模型中，用药过程中第14天，由于肿瘤体积大于2500mm 3而安乐死，导致小鼠数目减少，第14天作为TGI分析，如图19所示，单药组3mg/kg的HLX10、10mg/kg的h6H11B10#40和单药30mg/kg的h6H11B10#40的TGI分别为13.36％，12.83％和22.20％。联合用药组中，HLX10剂量为3mg/kg，h6H11B10#40分别为10mg/kg和30mg/kg时，其TGI％分别为25.9％和62.4％，均优于单药组，显示了联合用药组的抑瘤效果。
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Figure PCTCN2021072610-appb-000001
Figure PCTCN2021072610-appb-000002
Figure PCTCN2021072610-appb-000003
Figure PCTCN2021072610-appb-000004

Claims (15)

  1. 一种抗LAG3单克隆抗体，其特征在于，所述抗LAG3单克隆抗体包含重链可变区和轻链可变区，所述重链可变区包含与SEQ ID NO:2、6或10所示重链可变区具有的CDR序列相同的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO:4、8或12所示轻链可变区具有的CDR序列相同的LCDR1、LCDR2和LCDR3。
  2. 如权利要求1所述的抗LAG3单克隆抗体，其特征在于，所述抗LAG3单克隆抗体包括：
    1)重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1具有如SEQ ID No:18所示的氨基酸序列，所述HCDR2具有如SEQ ID No:19所示的氨基酸序列，所述HCDR3具有如SEQ ID No:20或21所示的氨基酸序列；
    2)轻链互补决定区LCDR1、LCDR2、LCDR3，所述LCDR1具有如SEQ ID No:22或23所示的氨基酸序列，所述LCDR2具有如SEQ ID No:24所示的氨基酸序列，所述LCDR3具有如SEQ ID No:25所示的氨基酸序列。
  3. 如权利要求2所述的抗LAG3单克隆抗体，其特征在于，所述抗LAG3单克隆抗体包括重链可变区和轻链可变区，所述重链可变区具有如SEQ ID NO:2、6或10所示的氨基酸序列，或者与上述序列具有至少85％同源性的序列；所述轻链可变区具有如SEQ ID NO:4、8或12所示的氨基酸序列，或者与上述序列具有至少85％同源性的序列。
  4. 如权利要求2所述的抗LAG3单克隆抗体，其特征在于，所述抗LAG3单克隆抗体包括重链和轻链，所述重链由具有如SEQ ID NO:2、6或10所示的氨基酸序列或者与上述序列具有至少85％同源性的序列的重链可变区和如SEQ ID NO:14所示的重链恒定区组成；所述轻链由具有如SEQ ID NO:4、8或12所示的氨基酸序列或者与上述序列具有至少85％同源性的序列的轻链可变区和如SEQ ID NO:16所示的轻链恒定区组成。
  5. 如权利要求1所述的抗LAG3单克隆抗体，其特征在于，所述抗LAG3单克隆抗体是抗体的全长序列或包含抗LAG3抗体的抗原结合片段，所述抗LAG3抗体的抗原结合片段为Fab、Fab’、F(ab’)2、Fv或scFv。
  6. 一种核苷酸分子，其特征在于，所述核苷酸分子编码如权利要求1至5中任一项所述的抗LAG3单克隆抗体。
  7. 如权利要求6所述的核苷酸分子，其特征在于，所述核苷酸分子中，编码抗LAG3单克隆抗体的重链可变区的核苷酸序列如SEQ ID NO:3、7或11所示，编码抗LAG3单克隆抗体的轻链可变区的核苷酸序列如SEQ ID NO:5、9或13所示。
  8. 一种表达载体，其特征在于，所述表达载体含有权利要求6或7所述的 核苷酸分子。
  9. 如权利要求8所述的表达载体，其特征在于，所述表达载体选自pHLX101、pEE14.4、pCHO 1.0或pcDNA3.1中的一种或多种。
  10. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求8或9所述的表达载体。
  11. 如权利要求10所述的宿主细胞，其特征在于，所述宿主细胞选自COS、CHO、HeLa细胞系、骨髓细胞系如SP2/0细胞系、NS0、sf9、sf21、DH5α、BL21(DE3)或E.coli TG1、YB2/0细胞系以及转化的B-细胞或杂交瘤细胞中的一种或多种。
  12. 一种制备权利要求1至5任一项所述的抗LAG3单克隆抗体的方法，包括以下步骤：
    a)在表达条件下，培养权利要求10或11所述的宿主细胞，表达抗LAG3单克隆抗体；
    b)分离并纯化步骤a)所得的抗LAG3单克隆抗体。
  13. 一种组合物，其特征在于，所述组合物包含如权利要求1至5任一项所述的抗LAG3单克隆抗体和药学上可接受的载体。
  14. 如权利要求1至5任一项所述的抗LAG3单克隆抗体或权利要求13所述的组合物在制备LAG3分子阻滞药物，尤其是制备抗肿瘤、治疗自身免疫性疾病、治疗感染性疾病和/或抗移植排斥反应的药物中的应用。
  15. 如权利要求14所述的用途，其特征在于，将所述抗LAG3单克隆抗体单独使用或与其他抗肿瘤药物联合使用，所述其他抗肿瘤药物选自抗体类药物，如抗PD-1单克隆抗体，或小分子抗肿瘤药物，如紫杉醇、5-Fu嘧啶。
PCT/CN2021/072610 2020-01-21 2021-01-19 抗lag3单克隆抗体及其制备方法和应用 Ceased WO2021147837A1 (zh)

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BR112022013544A BR112022013544A2 (pt) 2020-01-21 2021-01-19 Anticorpo monoclonal anti-lag3 e método de preparação do mesmo e uso do mesmo
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