Anti-LAG3 monoclonal antibody, method for producing the same, and use thereof

Abstract

The present invention provides anti-LAG3 monoclonal antibodies and uses thereof, as well as nucleotide molecules encoding the antibodies, expression vectors expressing the antibodies, host cells, compositions comprising the antibodies, and uses of the antibodies in the manufacture of medicaments for anti-tumor, treatment of autoimmune diseases, treatment of infectious diseases, and / or anti-transplant rejection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to an anti-LAG3 monoclonal antibody and its production method and use.

Background Art

[0002] LAG3 (Lymphocyte-activation gene 3), also known as CD223, was discovered in 1990, belongs to the immunoglobulin superfamily, and is expressed on activated T cells, NK cells, B cells, and dendritic cells. One of the ligands of LAG3 is the major histocompatibility complex (MHC) class II molecule, and the affinity between LAG3 and MHC II is stronger than that between CD4 and MHC II. In addition, the ligands of LAG3 also include LSECtin, FGL1, etc. The biological function of LAG3 is similar to that of CTLA-4 and PD-1, and plays a role in negatively regulating T cell proliferation and activation. In addition, LAG3 also plays a role in the inhibitory function of Treg cells.

[0003] According to research, it has been found that suppressing LAG3 can reactivate T cells, thereby enhancing the killing effect on tumors. At the same time, suppressing LAG3 can reduce the function of Treg cells to suppress the immune response. Therefore, LAG3 is targeted for cancer immunotherapy, and monoclonal antibodies are used to bind to LAG3, block the binding of LAG3 and its ligand, prevent the generation of inhibitory signals in T cells, thereby promoting the activation and proliferation of T cells and 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. Currently, pharmaceutical companies such as Bristol Myers Squibb, Novartis, and Merck have all developed antibody drugs against LAG3, and currently, they are in the clinical trial stage. The anti-LAG3 antibody BMS986016 (Relatlimab) developed by Bristol Myers Squibb has shown obvious effects in the treatment of advanced melanoma and is currently undergoing phase III clinical trials. In addition, anti-LAG3 antibodies are used in combination with other antibody drugs including anti-PD-1 antibodies, and related clinical trials are also being conducted.

[0004] Therefore, researching and developing new anti-LAG3 monoclonal antibodies for cancer immunotherapy, with lower toxic side effects and better clinical efficacy, has become a current research hotspot, providing more drug options for patients.

Summary of the Invention

[0005] The technical problem to be solved by the present invention is to complete the present invention by providing a new anti-LAG3 monoclonal antibody.

[0006] Therefore, the first object of the present invention is to provide a new anti-LAG3 monoclonal antibody.

[0007] The second object of the present invention is to provide a nucleotide molecule encoding the anti-LAG3 monoclonal antibody.

[0008] The third object of the present invention is to provide an expression vector containing the nucleotide molecule.

[0009] The fourth object of the present invention is to provide a host cell containing the expression vector.

[0010] The fifth object of the present invention is to provide a method for producing the anti-LAG3 monoclonal antibody.

[0011] The sixth object of the present invention is to provide a composition containing the anti-LAG3 monoclonal antibody.

[0012] The seventh object of the present invention is to provide the application of the anti-LAG3 monoclonal antibody in drug production.

[0013] To achieve the above object, the present invention adopts the following technical solutions.

[0014] The first aspect of the present invention provides an anti-LAG3 monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains HCDR1, HCDR2, and HCDR3 regions that are identical to the CDR sequences of the heavy chain variable region shown in SEQ ID NO: 2, 6, or 10. The light chain variable region contains LCDR1, LCDR2, and LCDR3 that are identical to the CDR sequences of the light chain variable region shown in SEQ ID NO: 4, 8, or 12.

[0015] In some embodiments, the anti-LAG3 monoclonal antibody is 1) heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 18, HCDR2 has the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 20 or 21, 2) The light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein 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 has the amino acid sequence shown in SEQ ID NO: 25.

[0016] In some embodiments, the anti-LAG3 monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2, 6, or 10, or a sequence having at least 85% homology with the above sequence, for example, a derived sequence having 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology. The light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, 8, or 12, or a sequence having at least 85% homology with the above sequence, for example, a derived sequence having 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0017] In some embodiments, the anti-LAG3 monoclonal antibody comprises a heavy chain and a light chain. The heavy chain consists of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2, 6, or 10 or a sequence having at least 85% homology with the above sequence, for example, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and a heavy chain constant region shown in SEQ ID NO: 14. The light chain consists of a light chain variable region having the amino acid sequence shown in SEQ ID NO: 4, 8, or 12 or a sequence having at least 85% homology with the above sequence, for example, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and a light chain constant region shown in SEQ ID NO: 16.

[0018] Preferably, the amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.

[0019] Preferably, the 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.

[0020] Preferably, the amino acid sequence of the heavy chain variable region of the anti-LAG3 monoclonal antibody 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.

[0021] The anti-LAG3 monoclonal antibody may be the full-length sequence of the antibody or a fragment of the anti-LAG3 antibody. The fragment may be Fab, Fab’, F(ab’)2, Fv or scFv, etc. Preferably, the anti-LAG3 antibody is an IgG1, IgG2 or IgG4 type antibody.

[0022] 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, or an antibody / antibody fragment-chemical conjugate. The fragment of the anti-LAG3 antibody is Fab, Fab’, F(ab’)2, Fv or scFv, etc.

[0023] The monoclonal antibody described in the present invention can be produced by conventional techniques in the art, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. Preferably, a monoclonal antibody is produced from wild-type or transgenic mice by hybridoma technology.

[0024] The second aspect of the present invention provides an isolated nucleotide molecule encoding the anti-LAG3 monoclonal antibody described above.

[0025] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 3, 7, or 11, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 5, 9, or 13.

[0026] Preferably, the 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 is shown in SEQ ID NO: 5.

[0027] Preferably, the 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 is shown in SEQ ID NO: 9.

[0028] Preferably, the 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 is shown in SEQ ID NO: 13.

[0029] The method for producing the nucleotide molecule is a conventional production method in the art. Preferably, it includes a production method of obtaining the nucleotide molecule encoding the monoclonal antibody by gene cloning techniques such as the PCR method, or a production method of obtaining the nucleotide molecule encoding the monoclonal antibody by artificial complete sequence synthesis.

[0030] As will be understood by those skilled in the art, the nucleotide sequence encoding the amino acid sequence of the monoclonal antibody can be appropriately introduced with substitutions, deletions, modifications, insertions or additions to provide a homolog of the polynucleotide or its conservative variant sequence. The homolog of the polynucleotide or its conservative variant sequence in the present invention can be obtained by substituting, deleting or adding one or more bases encoding the monoclonal antibody gene within the range of maintaining antibody activity.

[0031] The third aspect of the present invention provides an expression vector containing the nucleotide molecule described above.

[0032] The expression vector is a conventional expression vector in the art, and means an expression vector containing appropriate regulatory sequences such as a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene and / or sequence, and other appropriate sequences. The expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid. For further technical details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation can be found in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. The expression vector in the present invention refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art.

[0033] Preferably, the expression vector is selected from one or more of pHLX101, pEE14.4, pCHO 1.0, or pcDNA3.1.

[0034] More preferably, the expression vector is pcDNA3.1.

[0035] The fourth aspect of the present invention provides a host cell containing the expression vector described above.

[0036] The host cell described in the present invention is various conventional host cells in the art, as long as it can stably self-replicate the above recombinant expression vector and can efficiently express the nucleotide it carries. The host cell in the present invention may 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.

[0037] Preferably, the host cell is one or more of COS, CHO (Chinese Hamster Ovary), HeLa cell line, myeloid cell lines such as SP2 / 0 cell line, NS0, sf9, sf21, DH5α, BL21(DE3) or E.coli TG1, YB2 / 0 cell line, etc., and transformed B-cells or hybridoma cells.

[0038] More preferably, 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).

[0039] When the expression vector is transformed into a host cell, a preferred recombinant expression transformant of the present invention can be obtained. Here, the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electrotransformation method.

[0040] The fifth aspect of the present invention is a method for producing the anti-LAG3 monoclonal antibody described above, culturing the host cell described above under expression conditions to express the anti-LAG3 monoclonal antibody in step a); and providing a method for producing an anti-LAG3 monoclonal antibody including step b) of isolating and purifying the anti-LAG3 monoclonal antibody obtained in step a).

[0041] The method for culturing the host cells described in the present invention and the method for separating and purifying the monoclonal anti-LAG3 are ordinary methods in the art. For specific operation methods, refer to the corresponding cell culture technology manuals and monoclonal antibody separation and purification technology manuals.

[0042] All host cells used in the present invention are prior art and can be directly obtained through commercial channels. The media used for culturing are also various ordinary media. Those skilled in the art can select the applicable media based on experience and culture them under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature conversion or chemical induction), and the cells are cultured for a further period. The recombinant polypeptide in the above method may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various isolation methods using its physical, chemical, and other properties. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to, ordinary regeneration treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic shock, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0043] The present invention is obtained by screening the gene sequence of the target antibody from a monoclonal culture cell line, constructing a eukaryotic expression vector, and being able to reconstruct the activity of the antibody after expression to obtain an anti-LAG3 monoclonal antibody.

[0044] The sixth aspect of the present invention provides a composition comprising the anti-LAG3 monoclonal antibody described above and a pharmaceutically acceptable vector.

[0045] The monoclonal antibody of the present invention can be used by preparing a pharmaceutical composition by any means known in the art. Such a composition contains the monoclonal antibody as an active ingredient, and one or more pharmaceutically acceptable vectors, diluents, fillers, binders and other excipients, depending on the mode of administration and the designed dosage form. Therapeutically inert inorganic or organic vectors known to those skilled in the art include lactose, corn starch or its derivatives, talc, vegetable oils, waxes, fats, polyhydroxy compounds such as polyethylene glycol, water, sucrose, ethanol, glycerol, various preservatives, lubricants, dispersants, and flavoring agents, but are not limited thereto. Humectants, antioxidants, sweeteners, coloring agents, stabilizers, salts, buffers, etc. can also be added, and these substances can be used to help the stability of the formulation, improve the activity or its biological effectiveness, or produce an acceptable taste or odor in the case of oral administration. The inhibitors that can be used in such a composition may be in the form of the original compound itself or, optionally, in the form of a pharmaceutically acceptable salt. The monoclonal antibody of the present invention can be administered alone, in various combinations, and in combination with other therapeutic agents. The composition thus prepared can be administered with an inhibitor by selecting any appropriate means known to those skilled in the art, if necessary.

[0046] The anti-LAG3 monoclonal antibody provided by the present invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable vector to more stably exert a therapeutic effect. These preparations can ensure the integrity of the conformation of the amino acid core sequence of the anti-LAG3 monoclonal antibody described in the present invention, and also protect the protein's polyfunctional groups from degradation (including but not limited to coagulation, deamination or oxidation). Usually, in the case of a liquid preparation, it can be stored at 2°C to 8°C for at least one year, and in the case of a lyophilized preparation, it remains stable at 30°C for at least six months. The anti-LAG3 monoclonal antibody preparation is a preparation such as a suspension, a liquid injection, or a lyophilized preparation commonly used in the pharmaceutical field, and a liquid injection or a lyophilized preparation is preferred.

[0047] In the case of an aqueous injection or lyophilized preparation of the anti-LAG3 monoclonal antibody described in the present invention, the pharmaceutically acceptable vector preferably includes, but is not limited to, one or a combination of a surfactant, a solution stabilizer, an isotonicity regulator, and a buffer. Here, as the surfactant, preferably, a nonionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween20 or 80), Poloxamer (e.g., Poloxamer188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, tetradecyl group, linoleic group or octadecyl sarcosine, Pluronics, MONAQUATTM, etc. can be mentioned, but not limited thereto, and the added amount thereof should minimize the granulation tendency of the anti-LAG3 monoclonal antibody. The solution stabilizer preferably includes, but is not limited to, one or a combination of saccharides such as reducing sugars and non-reducing sugars, amino acids such as sodium glutamate or histidine, trivalent alcohols, higher sugar alcohols, alcohols such as propylene glycol and polyethylene glycol, and the added amount of the solution stabilizer should be such that the finally formed preparation maintains a stable state within the time considered by those skilled in the art to reach stability. As the isotonicity regulator, preferably, one or a combination of sodium chloride and mannitol can be mentioned, but not limited thereto. As the buffer, preferably, one or a combination of Tris, histidine buffer, and phosphate buffer can be mentioned, but not limited thereto.

[0048] The seventh aspect of the present invention provides the use of the anti-LAG3 monoclonal antibody or its composition described above in the manufacture of a drug.

[0049] Preferably, the use is for the manufacture of a LAG3 molecule blocker. More preferably, the use for the manufacture of the LAG3 molecule blocker is specifically for the manufacture of an anti-tumor drug or an anti-tumor diagnostic agent.

[0050] The drug described in the present invention is preferably a drug for the treatment of anti-tumor, autoimmune diseases, infectious diseases and / or anti-transplant rejection reaction, more preferably an anti-tumor drug, a drug for the treatment of autoimmune diseases, and even more preferably an anti-tumor drug. The anti-LAG3 monoclonal antibody described in 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 art, including antibody drugs or small molecule anti-tumor drugs. The antibody drug is a conventional antibody drug in the art, preferably including an anti-PD-1 monoclonal antibody. The small molecule anti-tumor drugs are conventional drugs in the art, including paclitaxel, 5-Fu pyrimidine, etc.

[0051] Here, the tumors targeted by the anti-tumor drugs preferably include one or more of melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma, pancreatic cancer, and / or head and neck tumors.

[0052] The anti-tumor drug referred to in the present invention refers to a drug that suppresses and / or treats tumors, including delaying the progression of tumor-related symptoms and / or reducing the severity of these symptoms, further including alleviating existing tumor-related symptoms and preventing the appearance of other symptoms, and further including reducing or preventing the metastasis of tumors.

[0053] When the anti-LAG3 monoclonal antibody and its composition in the present invention are administered to animals including humans, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the administration route. Therefore, the results of animal experiments and various situations can be referred to, and the total dosage does not exceed a certain range. Generally, the dosage for intravenous injection is 1-1800 mg / day.

[0054] On the basis of conforming to the common knowledge in the art, each preferred example of the present invention can be obtained by arbitrarily combining the above-mentioned preferred conditions.

[0055] The beneficial effects of the present invention are as follows: The anti-LAG3 monoclonal antibody of the present invention has good biological activity, has a high expression level in mammalian cells, and has a clear affinity for LAG3 and the ability to inhibit ligand-receptor binding. The monoclonal antibody can be applied alone or in combination with other anti-tumor drugs to the treatment, diagnosis, and screening of tumor immunity, and can be effectively applied to the manufacture of drugs for the treatment of tumors, infectious diseases, autoimmune diseases, and anti-immune rejection, etc.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0057] Hereinafter, the present invention will be further described by examples, but the present invention is not limited to these examples. Although the experimental methods of specific conditions are not specified in the examples, they are selected according to ordinary methods and conditions or according to the product manuals. The room temperature described in the examples is the ordinary room temperature in the technical field, generally 10 - 30°C. Unless otherwise specified, the reagents and raw materials used are commercially available.

[0058] It should be understood that the scope of the present invention is not limited to the following specific embodiments. Also, the terms used in the examples of the present invention are for explaining specific embodiments of the present invention and are not for limiting the scope of the present invention. In this specification and the claims, unless the context clearly indicates otherwise, the singular forms "one", "a", and "the" include the plural forms.

[0059] The term "variable region" of an antibody refers to the variable region (VL) of an antibody light chain or the variable region (VH) of an antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) linked by three complementarity-determining regions (CDRs) (also called hypervariable regions). The CDRs within each chain are held closely together by the 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 the CDRs: (1) a method based on sequence variability among species (i.e., Sequences of Proteins of Immunological Interest by Kabat et al. (5th ed., 1991, National Institutes of Health, Bethesda MD)), and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. 273:927-948 (1997)). As used herein, the CDRs can refer to the CDRs determined by either method or by a combination of both methods.

[0060] The term "antibody framework" or "FR region" refers to a part of the variable domain VL or VH that functions as a scaffold for the antigen-binding loops (CDRs) of the variable domain. In essence, it is a variable domain without CDRs.

[0061] The terms "complementary determining region" and "CDR" refer to one of six hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (Al-Lazikani et al. (1997), JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classical format, according to the Kabat rules, the CDR amino acid residue numbers in the heavy chain variable domain (VH) are 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residue numbers in the light chain variable domain (VL) are 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia rules, 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), 91-96 (LCDR3). By combining both the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.According to the IMGT rules, the CDR amino acid residue numbers in VH are approximately 26 - 35 (HCDR1), 51 - 57 (HCDR2), and 93 - 102 (HCDR3), and the CDR amino acid residue numbers in VL are approximately 27 - 32 (LCDR1), 50 - 52 (LCDR2), and 89 - 97 (LCDR3). According to the IMGT rules, the CDR regions of antibodies can be determined using the program IMGT / DomainGap Align.

[0062] The term "LAG3" refers to lymphocyte activation gene - 3 protein, an immune checkpoint receptor, or T - cell coinhibitory factor, also called CD223. The amino acid sequence of full - length LAG3 is provided in GenBank under accession number NP_002277.4. The term "" includes variants, isoforms, homologs, orthologs, and paralogs. For example, LAG3 protein variants, recombinant LAG3 or fragments thereof, and LAG3 or fragments thereof conjugated to, for example, a histidine tag, mouse or human Fc or signal sequence (e.g., ROR1) are included.

[0063] When numerical ranges are shown in the examples, unless otherwise indicated in the present invention, any of the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, based on the prior art acquisition of those skilled in the art and the description of the present invention, the present invention can be realized using any methods, devices, and materials of the prior art that are similar or equivalent to the methods, devices, and materials described in the examples of the present invention.

[0064] Unless otherwise specified, the experimental methods, detection methods, and manufacturing methods disclosed in the present invention use ordinary techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields that are well known in the art. These techniques have been fully described in the prior literature, specifically Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., 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; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

[0065] Positive control antibody BMS986016: The amino acid sequence is described in the patent application WO 2015 / 042246 Al of Bristol-Myers Squibb. In the present invention, after cloning the nucleotide sequence encoding the amino acid sequence into the pURO vector via restriction endonuclease, the plasmid was transfected into CHO-S cells, and a high-expression cell line was obtained by carbenicillin resistance screening. The cell line was cultured for proliferation, the supernatant of the medium was collected, and the control antibody was obtained by the manufacturing process of protein A affinity chromatography.

[0066] Negative control HLX10 (h1G4): Its sequence was cited from CN109923126A.

[0067] Positive control ab40465: Abcam, catalog number ab40465.

[0068] Isotype - human IgG4 (hIgG4): CrownBio, Cat: AB180018

[0069] Recombinant mouse LAG3-Fc fusion protein: Purchased from Sino Biological Inc., Cat: 53069-M02H.

[0070] Recombinant cynomolgus monkey LAG3-Fc fusion protein: Purchased from Sino Biological Inc., Cat: 90841-C08H Anti-mouse PD1 antibody RMP1-14: BioXCell, Cat: 71791801

[0071] Example 1 Screening of anti-LAG3 antibodies In this example, the purified LAG3-His (the amino acid sequence is shown in SEQ ID NO: 1) protein was self-constructed and discovered. After co-immunizing mice using CHO-S cells expressing the full length of LAG3 and fusing spleen cells with myeloma cells to obtain hybridoma cells, ELISA was used to screen for positive clones.

[0072] Specifically, first, a nucleic acid sequence encoding amino acids 23 to 450 of the extracellular region of LAG3 is cloned into the pEE14.4 vector by a restriction endonuclease. Here, a tag encoding 6xHis is attached to the C-terminus of the LAG3 coding region. Subsequently, the plasmid is transferred into CHO-S cells, and monoclonal clones that highly express the LAG3-his fusion protein are screened by a GS (Glutamine Synthetase) screening system. The monoclonal clones are grown and cultured, the supernatant of the medium is recovered, and the target protein is produced by nickel column affinity chromatography. Next, the protein and CHO-S cells expressing the full-length LAG3 are co-immunized in mice. When screening for positive hybridomas, the LAG3-His recombinant protein is first coated on a 96-well half-area plate at 2 μg / ml (working volume 30 μl) and coated overnight at 4°C. Excess LAG3-His is washed three times with PBS (PBST) containing 0.05% Tween 20. Block with PBS containing 5% skim milk at room temperature for 1 hour. Wash three times with PBST, then add diluted hybridoma cells and culture the supernatant, let it stand at room temperature for 1 hour for reaction, then wash three times with PBST, add 30 μl of a goat anti-mouse IgG secondary antibody (Kangwei Century Co., Ltd.) labeled with horseradish peroxidase diluted with PBS, the dilution ratio is 1:2000, let it stand at room temperature for 1 hour, then wash six times with PBST, add TMB to develop color, and terminate the reaction with 30 μl of 2M H2SO4. Read the light absorption at a wavelength of 450 nm using a microplate reader.

[0073] Furthermore, flow cytometry is used to confirm the positive hybridoma clones of the expressed antibodies obtained by ELISA screening. First, CHO-S cells expressing the full-length human LAG3 gene that are growing normally are washed twice with PBS (2% FBS), and the cell concentration is adjusted to 5×10 5Adjust to 100 μl. Add the hybridoma supernatant and incubate at 4°C for 30 min. Centrifuge the cells at 500 × g for 5 min. Do not add antibody to the negative control group. Wash twice with PBS (2% FBS). Add goat anti-mouse IgG-FITC (Kangwei Century Co., Ltd.) diluted 1:100 and incubate at 4°C for 30 min. Wash the cells twice with PBS (2% FBS). Suspend the cells in 200 μl of PBS and analyze using flow cytometry. As shown in Figure 1, most of the positive clones obtained by ELISA screening can bind to LAG3 on the cell surface.

[0074] Test the ability of the antibody expressed by the hybridoma positive clone obtained by ELISA screening to block the binding of MHC II on the cell surface to LAG3-Fc (Acro Co., Ltd.) by flow cytometry. Wash Daudi cells twice with PBS (2% FBS) and adjust the cell concentration to 3 × 10 5 / 100 μl. Mix the supernatant of the fused tumor with LAG3-Fc at a concentration of 2 μg / ml, then mix with Daudi cells and incubate at 4°C for 30 min. Centrifuge the cells at 500 × g for 5 min. At the same time, use no antibody as the negative control and anti-LAG3 antibody BMS986016 as the positive control. Wash twice with PBS (2% FBS). Add goat anti-human IgG-Fc-FITC (Thermo Co., Ltd.) diluted 1:300 and incubate at 4°C for 30 min. Wash the cells twice with PBS (2% FBS). Suspend the cells in 200 μl of PBS and analyze using flow cytometry. As shown in Figure 2, finally, through screening, multiple monoclonal hybridoma cell lines with the ability to block the binding of MHCII and LAG3-Fc on the cell surface are obtained, and the optimal monoclonal hybridoma cell line is 6H11B10. The amino acid sequence of the heavy chain variable region of 6H11B10 is shown in SEQ ID NO:2, 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, and the nucleotide sequence is shown in SEQ ID NO:5.

[0075] Example 2 Measurement of Affinity of Anti-LAG3 Chimeric Antibody The amplification of mouse light and heavy chain variable regions is carried out according to the method described by Anke Krebber et al. (Journal of Immunological Methods 201.1997.35 - 55). The general method includes producing total RNA from the monoclonal hybridoma cell line 6H11B10 and producing single-stranded cDNA using a reverse transcription kit (TaKaRa). As described by Anke Krebber et al., primers that can complementarily pair with the mouse light and heavy chain variable regions are utilized, and the genes of the antibody heavy chain variable region (SEQ ID NO:3) and light chain variable region (SEQ ID NO:5) are amplified by PCR reaction. Therefore, the light chain variable region and heavy chain variable region genes of 6H11B10 are fused to the constant region gene of heavy chain IgG4 (SEQ ID NO:15) and the constant region gene of light chain (SEQ ID NO:17) to obtain the chimeric antibody heavy chain gene and light chain gene of 6H11B10.

[0076] The chimeric antibody gene of 6H11B10 is transfected into CHO-S cells respectively and expressed to obtain the chimeric antibody protein c6H11B10. The affinity of the chimeric antibody for LAG3 is measured using ELISA technology. The LAG3-Fc recombinant protein is coated on an ELISA plate (working volume 30 μl) at 2 μg / ml and left standing overnight at 4°C. Wash three times with PBS (PBST) containing 0.05% Tween 20. Block with PBS containing 5% skim milk at room temperature for 1 hour. After washing three times with PBST, add serially diluted c6H11B10 and leave standing at room temperature for 1 hour. At the same time, BMS986016 is used as a positive control. Wash three times with PBST, add 30 μl of a 1:4000 diluted horseradish peroxidase-labeled goat anti-human IgGkappa light chain secondary antibody (Millipore) and leave standing at room temperature for 1 hour. Wash six times with PBST, add TMB to develop color, stop the reaction with 2M H2SO4, and read at 450 nm with a microplate reader. As shown in Figure 3, the anti-LAG3 chimeric antibody c6H11B10 has a very strong affinity for LAG3. At low concentrations, c6H11B10 has a higher affinity than the control antibody BMS986016.

[0077] Example 3 Measurement of the Affinity of the Anti-LAG3 Chimeric Antibody The affinity of the chimeric antibody protein c6H11B10 for cell surface LAG3 is measured using flow cytometry. Jurkat cells transfected and expressing the full-length human LAG3 gene are collected, washed twice with PBS (2% FBS), and the cell concentration is adjusted to 2×10 5Adjust to 100 μl. Add the serially diluted chimeric antibody c6H11B10 and mouse antibody m6H11B10, and incubate at 4°C for 30 min. Centrifuge the cells at 500 × g for 5 min. At the same time, use BMS986016 as a positive control. Wash twice with PBS (2% FBS). Add goat anti-human IgG Fab-FITC (Thermo Fisher) diluted 1:300 for the chimeric antibody and BMS986016, and add goat anti-mouse IgG-FITC (ComWin Biotech) diluted 1:100 for the mouse antibody, and incubate at 4°C for 30 min. Wash the cells twice with PBS (2% FBS). Suspend the cells in 200 μl of PBS and analyze using flow cytometry. As shown in Fig. 4, the chimeric antibody c6H11B10 can strongly bind to LAG3 on the cell surface, and the affinity of c6H11B10 is superior to that of the control antibody BMS986016.

[0078] Example 4 Humanization of Anti-LAG3 Mouse Antibody 6H11B10 For the humanization of mouse antibody 6H11B10, the framework regions in mouse antibody 6H11B10 were replaced using the human antibody light chain variable region germline gene IGKV2-28*01 and the heavy chain variable region germline gene IGHV3-11*06, leaving the original complementarity-determining regions (CDRs) intact, to obtain 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. Furthermore, by fusing with the constant region gene of heavy chain IgG4 (SEQ ID NO:15) and the constant region gene of light chain (SEQ ID NO:17), the heavy chain gene and light chain gene of the anti-LAG3 humanized antibody h6H11B10 were obtained. 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), respectively, and the three CDRs of the light chain are LCDR1 (SEQ ID NO:22), LCDR2 (SEQ ID NO:24), and LCDR3 (SEQ ID NO:25), respectively.

[0079] The anti-LAG3 humanized antibody h6H11B10 was affinity matured using phage display technology. Primers with point mutations were designed, and the light-chain CDR and heavy-chain CDR of h6H11B10 were each mutated by PCR to obtain a mutant library. The phage display vector was transformed into E. coli TG1 or SS320 cells to generate a phage library. The phage library was screened twice using streptavidin-conjugated magnetic beads M-280 (Thermo Fisher) and biotinylated LAG3 protein. Furthermore, the 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. Furthermore, they were each fused with the constant region gene of heavy-chain IgG4 (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 h6H11B10#40 heavy chain are HCDR1 (SEQ ID NO:18), HCDR2 (SEQ ID NO:19), and HCDR3 (SEQ ID NO:21), respectively, and the three CDRs of the light chain are LCDR1 (SEQ ID NO:23), LCDR2 (SEQ ID NO:24), and LCDR3 (SEQ ID NO:25), respectively.

[0080] Example 5 Measurement of the Affinity of the Anti-LAG3 Humanized Antibody for LAG3 h6H11B10 and the h6H11B10#40 anti-LAG3 humanized antibody gene are each transfected into CHO-S cells for expression to obtain the h6H11B10 and h6H11B10#40 antibody proteins. The affinity of the humanized antibody for LAG3 is measured using ELISA technology. The LAG3-Fc recombinant protein is coated onto an ELISA plate (working volume 30 μl) at 2 μg / ml and left to stand overnight at 4°C. Wash three times with PBS (PBST) containing 0.05% Tween 20. Block with PBS containing 5% skim milk at room temperature for 1 hour. Wash three times with PBST, then add the serially diluted h6H11B10 and h6H11B10#40 humanized antibody proteins and leave to stand at room temperature for 1 hour. At the same time, BMS986016 is used as a positive control. Wash three times with PBST, add 30 μl of a 1:4000 diluted horseradish peroxidase-labeled goat anti-human IgG kappa light secondary antibody (Millipore) and leave to stand at room temperature for 1 hour. Wash six times with PBST, add TMB to develop color, and stop the reaction with 2 M H2SO4. Read using a microplate reader at 450 nm. As shown in the results in Figure 5, the anti-LAG3 humanized antibody has a strong affinity for LAG3. Here, h6H11B10#40 is superior to h6H11B10, and both are superior to the positive control BMS986016.

[0081] The binding of the humanized antibody to cell surface LAG3 is measured using flow cytometry. CHO-S or Jurkat cells transfected to express the full-length human LAG3 gene are harvested, the cells are washed twice with PBS (2% FBS), and the cell concentration is adjusted to 5×10 5Adjust to 100 μl. Add the anti-LAG3 humanized antibody diluted in a 1:3 stepwise manner and incubate at 4°C for 30 min. At the same time, use BMS986016 as a positive control. Centrifuge the cells at 500×g for 5 min. Wash twice with PBS (2% FBS). Add goat anti-human IgG Fab-FITC (Thermo Fisher) diluted 1:300 and incubate at 4°C for 30 min. Wash the cells twice with PBS (2% FBS). Suspend the cells in 200 μl of PBS and analyze using flow cytometry. As shown in Fig. 6, the anti-LAG3 humanized antibody has a strong affinity for LAG3 on the cell membrane, and both h6H11B10#40 and h6H11B10 are superior to the positive control BMS986016.

[0082] Using surface plasmon resonance (SPR), measure the binding affinity and kinetic constants of the anti-LAG3 humanized antibody for the LAG3 protein. First, immobilize the LAG3-Fc protein on the chip, and serially dilute the anti-LAG3 humanized antibodies h6H11B10#40 and BMS986016 from 80 μg / ml to 2.5 μg / ml in HBS-P buffer and flow them over the chip respectively. The data obtained in the experiment is analyzed using evaluation software, and the curves are fitted using a 1:1 Langmuir binding model. The binding and dissociation kinetics and the calculated affinity constant (KD) are shown in Table 1. As shown in Fig. 7, after affinity maturation, the affinity of the anti-LAG3 humanized antibody h6H11B10#40 for LAG3 is significantly improved, and both the binding and dissociation constants are superior to those of the positive control antibody BMS986016.

[0083] [Table 1]

[0084] Example 6 Ability of anti-LAG3 humanized antibody to block the binding of LAG3 to its ligand Using the flow cytometry method, the ability of the anti-LAG3 humanized antibody to block the binding of MHC II and LAG3-Fc on the surface of Raji cells is measured. Raji cells are washed twice with PBS (2% FBS), and the cell concentration is adjusted to 2×10 5 / 100 μl. After serially diluting the anti-LAG3 humanized antibody h6H11B10#40 in a 1:3 stepwise manner and mixing it with LAG3-Fc at a concentration of 2 μg / ml, hIgG4 protein is used as a negative control and the anti-LAG3 antibody BMS986016 is used as a positive control while allowing it to stand at room temperature for 30 min. Further, it is mixed with Raji cells and incubated at 4°C for 60 min. The cells are centrifuged at 500×g for 5 min. They are washed twice with PBS (2% FBS). Goat anti-human IgG-Fc-Alexa Fluor 488 (Jackson) diluted 1:500 is added and incubated at 4°C for 60 min. The cells are washed twice with PBS (2% FBS). The cells are suspended in 200 μl of PBS and analyzed using flow cytometry. As shown in Fig. 8, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of MHC II and LAG3-Fc on the cell surface, similar to BMS986016.

[0085] Using ELISA technology, measure the ability of a humanized antibody to block the binding of LAG3-Fc to its ligand LSECtin. Coat an enzyme plate (working volume 100 μl) with 1 μg / ml of LAG3-Fc recombinant protein and let it stand overnight at 4°C. Wash it three times with PBS (PBST) containing 0.05% Tween 20. Block it with PBS containing 5% skim milk at room temperature for 1 hour and wash it three times with PBST. Dilute the humanized antibody protein of h6H11B10#40 in a 1:5 stepwise manner. At the same time, use hIgG4 protein as a negative control and anti-LAG3 antibody BMS986016 as a positive control. Also, produce LSECtin (R&D) at 1 μg / ml, mix it with the antibody at a 1:1 ratio, and let it stand at room temperature for 30 min. Subsequently, add it to the ELISA plate and let it stand at room temperature for 1 hour. Wash it three times with PBST, add 100 μl of a 1:5000 diluted horseradish peroxidase-labeled mouse anti-His-labeled secondary antibody (Genscript Biotech) and let it stand at room temperature for 1 hour. Wash it six times with PBST, add TMB to develop color, and stop the reaction with 2M H2SO4. Read it using a microplate reader at 450 nm. As shown in the results in Figure 9, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding of LAG3-Fc to its ligand LSECtin.

[0086] Example 7 Ability of an anti-LAG3 humanized antibody to block the binding of LAG3 to its ligand Using a commercially available kit (purchased from Cisbio, product number: 63ADK000CB10PEG) for measuring the blockade of the binding between LAG3 and FGL1 by an anti-LAG3 antibody, the ability of the anti-LAG3 humanized antibody to block the binding between LAG3 and FGL1 was tested. In this experiment, first, 4 μL of Tag1-LAG3 protein and 4 μL of Tag2-FGL1 protein were mixed into the test samples at the dilution concentration, and after reacting at room temperature for 15 minutes, 10 μL of pre-mixed anti-Tag1-Tb3+ and anti-Tag2-XL665 were added. After sealing this test plate and reacting overnight at room temperature, the fluorescence absorbance at 665 / 620 nm was read using a microplate reader. As shown in Figure 10 for the results, the anti-LAG3 humanized antibody h6H11B10#40 has the ability to block the binding between LAG3 and its ligand FGL1.

[0087] Example 8 Cross-species Reactivity of Anti-LAG3 Humanized Antibody The recombinant mouse and cynomolgus monkey LAG3-Fc fusion proteins are purchased from Sino Biological Inc. Coat the enzyme plate (working volume 30 μl) with the LAG3-Fc recombinant protein at 1 μg / ml and let it stand overnight at 4°C. Wash it three times with PBS (PBST) containing 0.05% Tween 20. Block it with PBS containing 5% skim milk at room temperature for 1 hour. Wash it three times with PBST, add the serially diluted anti-LAG3 humanized antibodies h6H11B10 and h6H11B10#40, and let it stand at room temperature for 1 hour. At the same time, use BMS986016 as a positive control. Wash it three times with PBST, add 30 μl of 1:2000 diluted horseradish peroxidase-labeled goat anti-human IgG kappa light secondary antibody (Millipore), and let it stand at room temperature for 1 hour. Wash it six times with PBST, add TMB to develop color, and stop the reaction with 2 M H2SO4. Read it using a microplate reader at 450 nm. As shown in Figure 11, both the anti-LAG3 humanized antibodies h6H11B10 and h6H11B10#40 have very high affinity for cynomolgus monkey LAG3 compared to BMS986016, while BMS986016 has low LAG3 affinity for cynomolgus monkey. BMS986016 and h6H11B10 both have very weak affinity for mouse LAG3 (not shown).

[0088] The binding of the humanized antibody to cynomolgus monkey LAG3 on the cell surface was measured using flow cytometry. CHO-S transfected to express the full-length cynomolgus monkey LAG3 gene was collected, and the cells were washed twice with PBS (2% FBS). The cell concentration was adjusted to 5×10 5Adjust to 100 μl. Add anti-LAG3 humanized antibody serially diluted 1:3, and incubate at 4 °C for 30 min. At the same time, use BMS986016 as a positive control. Centrifuge the cells at 500 × g for 5 min. Wash twice with PBS (2% FBS). Add goat anti-human IgG Fab-FITC (Thermo Fisher) diluted 1:300, and incubate at 4 °C for 30 min. Wash the cells twice with PBS (2% FBS). Suspend the cells in 200 μl PBS and analyze using flow cytometry. As shown in Fig. 12, the anti-LAG3 humanized antibodies h6H11B10#40 and h6H11B10 have strong affinity for cynomolgus LAG3 on the cell membrane, and both h6H11B10#40 and h6H11B10 are superior to the positive control BMS986016.

[0089] Using surface plasmon resonance (SPR), measure the binding affinity and kinetic constants of anti-LAG3 humanized antibodies for cynomolgus LAG3 protein. First, immobilize cynomolgus LAG3-Fc protein on the chip, serially dilute anti-LAG3 humanized antibodies h6H11B10#40 and BMS986016 from 80 μg / ml to 2.5 μg / ml in HBSPE buffer, and flow them over the chip respectively. The data obtained from the experiment is analyzed using evaluation software, and the curves are fitted using a 1:1 Langmuir binding model. The binding and dissociation kinetics and calculated affinity constants (KD) are shown in Table 2. As a result, as shown in Fig. 13, the anti-LAG3 humanized antibody h6H11B10#40 has high affinity for cynomolgus LAG3.

[0090]

Table 2

[0091] Example 9 Ability of anti-LAG3 humanized antibody to activate T cells Take Raji cells, centrifuge at 500 × g for 5 min, and adjust the cell concentration to 1.2 × 10 6Adjust to / ml. Add SEE (Toxin Technology) to a final concentration of 0.024 ng / ml and incubate at 37 °C for 30 min. Harvest Jurkat cells (Promega) transfected with the LAG3 gene and the NFAT-Luc reporter gene, centrifuge at 500 × g for 5 min, and adjust the cell concentration to 1.6 × 10 6 Adjust to / ml. Take 25 μl each of the anti-LAG3 humanized antibody diluted 1:3 in three steps, using BMS986016 as a positive control, mix with an equal volume of Jurkat cells, then add 25 μl of SEE-treated Raji cells, and incubate at 37 °C for 6 h. Add Bio-Glo (Promega) and read using a multifunctional microplate reader. As shown in Fig. 14, the anti-LAG3 humanized antibody activates Jurkat cells to express luciferase, and its effect is similar to that of BMS986016.

[0092] Example 10 Inhibitory activity of anti-LAG3 humanized antibody against tumor growth in hLAG3 KI mouse model Using hLAG3 KI mice, measure the ability of the anti-LAG3 humanized antibody to inhibit tumor cell growth in vivo. Inoculate mouse colon cancer cells M38 subcutaneously into the back of hLAG3 KI mice at 1 × 10 6 cells per mouse. When the tumor volume reaches approximately 80 - 120 mm 3 initiate intraperitoneal injection of anti-mouse PD-1 antibody RMP1-14 (CrownBio) at a dose of 0.5 mg / kg, and inject the anti-LAG3 humanized antibody h6H11B10#40 at two doses of 30 mg / kg and 10 mg / kg, respectively, twice a week for approximately 2 - 3 weeks. At the same time, inject PBS as a negative control and anti-mouse PD-1 antibody RMP1-14 and anti-LAG3 antibody BMS986016 as positive controls. Observe the size of tumor formation in each mouse twice a week. Tumor volume calculation method: V (mm 3) = 0.5×(length (mm) × width (mm) × width (mm)). As shown in Figure 15, when anti-LAG3 antibody and anti-mouse PD-1 antibody were injected, the tumor growth could be significantly inhibited compared with the control group injected with PBS. Here, in the high-dose group, h6H11B10#40 was used in combination with anti-mouse PD-1 antibody, and the therapeutic effect was better than that of the single antibody.

[0093] Example 11 Reactivation of Depleted T Cells Mediated by Anti-LAG3 Antibody 500 g of human PBMC cells were centrifuged at 10 min, the supernatant was removed, washed twice with culture buffer (RPMI-1640 medium + 2% FBS), counted, and then resuspended with stimulation buffer (RPMI-1640 medium + 2% FBS + 20 ng / mL SEB), and inoculated into a 96-well cell culture plate (5×10 5 cells / well), and cultured and stimulated overnight in a 37 °C, 7% CO2 incubator. On the second day, the desired test samples were serially diluted with culture buffer. In the IL-2 and IFN-γ tests, the test groups were the equally serially diluted h6H11B10#40 sample group (the highest test concentration was 10 μg / mL), the equally serially diluted HLX10 group (the highest test concentration was 10 μg / mL). Here, in the combination group, h6H11B10#40 was diluted with HLX10 solutions at different concentrations (1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, and 0.001 μg / mL), and the diluted test samples were sequentially added to the PBMCs stimulated overnight, placed in a 37 °C, 7% CO2 incubator, and the culture was continued for 2 - 3 days. The supernatant was collected and the release amounts of IL-2 and IFN-γ in each test group were measured using IL-2 and IFN-γ kits.

[0094] As shown in the data of FIGS. 16 and 17, the amounts of IL-2 and IFN-γ in the group treated with h6H11B10#40 or HLX10 alone were low. When the HLX10 concentration reached about 1 μg / mL, it was used in combination with h6H11B10#40, and the release amounts of IL-2 and IFN-γ were higher than those in the h6H11B10#40 single-agent and HLX10 single-agent groups. For the combined groups (when the HLX10 concentration was 1 μg / mL and 0.1 μg / mL), h6H11B10#40 showed a concentration-dependent effect. This in vitro experiment indicates that the combination of h6H11B10#40 and HLX10 enables the reactivation of depleted T cells and increases cytokine release.

[0095] Example 12 Evaluation of Therapeutic Effect in hLAG-3 / hPD-1 Transgenic Mouse MC38 or A20 Subcutaneous Transplant Tumor Model MC38 cells were cultured in a growth medium (RPMI-1640 + 10% FBS). Cells in the logarithmic growth phase were collected before inoculation, resuspended in PBS and Matrigel (0.1 mL / mouse) for subcutaneous inoculation of mice. For the experimental mice to be inoculated (6 - 8 weeks old), MC38 cells (1×10 6 / mouse) were subcutaneously inoculated into the lower posterior part of the right back. When the average tumor volume reached 78.03 mm 3 , they were randomly grouped according to tumor size. The coefficient of variation (CV) of tumor volume between groups was calculated by the formula CV = SD / MTV×100% and should be less than 40%. The day of grouping was defined as day 0, and administration started from day 0. All clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 ) = 1 / 2×(a×b 2 ) (where a is the major axis and b is the minor axis). As shown in the results in FIG. 18, in the MC38 model, during the experimental process, when administered on day 17, the mouse tumors in the control group were 2500 mm 3To reach this, the mice were euthanized and analyzed with D17 data. The TGI of the single-agent groups HLX10 and h6H11B10#40 were 35.22% and 4.88% respectively. In the combination group, when the HLX10 dosage was 1.5 mg / kg and the h6H11B10#40 dosages were set at 10 mg / kg and 30 mg / kg respectively, the TGI% were 62.2% and 56.7% respectively, both higher than those of the single-agent groups. This data indicates that the combination of h6H11B10#40 and HLX10 has a better tumor suppression effect than the single agents.

[0096] A20 cells were cultured in medium RPMI-1640 + 10% FBS, A20 cells in the exponential growth phase were collected, resuspended with PBS to an appropriate concentration (0.1 mL / mouse), and used for subcutaneous inoculation of mice. Experimental mice were subcutaneously inoculated with A20 cells (5×10 5 / mouse) on the right anterior scapula. When the average tumor volume is 90.44 mm 3 , they are randomly grouped according to tumor size. The coefficient of variation (CV) of tumor volume between each group is calculated by the formula CV = SD / MTV×100% and should be less than 40%. The grouping day is defined as day 0, and administration starts from day 0. In the A20 model, on day 14 during the administration process, since the tumor volume exceeded 2500 mm 3 , the number of mice was euthanized, the number of mice was reduced, and TGI analysis was performed on day 14. As shown in Figure 19, the TGI of the single-agent groups of 3 mg / kg HLX10, 10 mg / kg h6H11B10#40, and 30 mg / kg h6H11B10#40 of the single agent were 13.36%, 12.83%, and 22.20% respectively. In the combination group, when the dosage of HLX10 was 3 mg / kg and h6H11B10#40 was 10 mg / kg and 30 mg / kg respectively, the TGI% were 25.9% and 62.4% respectively, both superior to those of the single-agent groups, indicating the anti-tumor effect of the combination group.

[0097] Aspects of the present invention are further described in the following clauses: [Clause 1] An anti-LAG3 monoclonal antibody, wherein the anti-LAG3 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 regions identical to the CDR sequences of the heavy chain variable region shown in SEQ ID NO: 2, 6, or 10, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 identical to the CDR sequences of the light chain variable region shown in SEQ ID NO: 4, 8, or 12. An anti-LAG3 monoclonal antibody characterized by this. [Clause 2] The anti-LAG3 monoclonal antibody is 1) heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 18, HCDR2 has the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 20 or 21, and 2) light chain complementarity determining regions LCDR1, LCDR2, LCDR3, wherein LCDR1 has the amino acid sequence shown in SEQ ID NO: 22 or 23, LCDR2 has the amino acid sequence shown in SEQ ID NO: 24, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 25. The anti-LAG3 monoclonal antibody according to item 1 above, characterized by including this. [Clause 3] The anti-LAG3 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2, 6, or 10, or a sequence having at least 85% homology to the above sequence, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, 8, or 12, or a sequence having at least 85% homology to the above sequence. The anti-LAG3 monoclonal antibody according to item 2 above, characterized by this. [Clause 4] The anti-LAG3 monoclonal antibody comprises a heavy chain and a light chain. The heavy chain has a heavy chain variable region having an amino acid sequence shown in SEQ ID NO: 2, 6 or 10 or a sequence having at least 85% homology to the above sequence, and a heavy chain constant region shown in SEQ ID NO: 14. The light chain has a light chain variable region having an amino acid sequence shown in SEQ ID NO: 4, 8 or 12 or a sequence having at least 85% homology to the above sequence, and a light chain constant region shown in SEQ ID NO: 16. The anti-LAG3 monoclonal antibody according to item 2 above is characterized by this. [Item 5] The anti-LAG3 monoclonal antibody is the full-length sequence of the antibody or an antigen-binding fragment containing the anti-LAG3 antibody. The antigen-binding fragment of the anti-LAG3 antibody is Fab, Fab’, F(ab’)2, Fv or scFv. The anti-LAG3 monoclonal antibody according to item 1 above is characterized by this. [Item 6] A nucleotide molecule, which encodes the anti-LAG3 monoclonal antibody according to any one of items 1 to 5 above. [Item 7] In the nucleotide molecule, the nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 3, 7 or 11, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 5, 9 or 13. The nucleotide molecule according to item 6 above is characterized by this. [Item 8] An expression vector, which contains the nucleotide molecule according to item 6 or 7 above. [Item 9] The expression vector is one or more selected from the group consisting of pHLX101, pEE14.4, pCHO 1.0 or pcDNA3.1. The expression vector according to item 8 above is characterized by this. [Item 10] A host cell, which contains the expression vector according to item 8 or 9 above. [Item 11] The host cell is one or more selected from the group consisting of COS, CHO, HeLa cell line, myeloid cell lines such as SP2 / 0 cell line, NS0, sf9, sf21, DH5α, BL21(DE3) or E.coli TG1, YB2 / 0 cell line, and transformed B-cells or hybridoma cells. The host cell according to item 10 above is characterized by this. [Item 12] A method for producing an anti-LAG3 monoclonal antibody according to any one of items 1 to 5 above, Under expression conditions, culturing the host cell according to item 10 or 11 above to express an anti-LAG3 monoclonal antibody, step a); And step b) of isolating and purifying the anti-LAG3 monoclonal antibody obtained in step a). A method for producing an anti-LAG3 monoclonal antibody. [Item 14] A composition comprising the anti-LAG3 monoclonal antibody according to any one of items 1 to 5 above and a pharmaceutically acceptable vector. [Item 14] Use of the anti-LAG3 monoclonal antibody according to any one of items 1 to 5 above or the composition according to item 13 above in the manufacture of a drug for LAG3 molecule blockade, particularly an anti-tumor drug, treatment of autoimmune diseases, treatment of infectious diseases and / or prevention of transplant rejection. [Item 15] The anti-LAG3 monoclonal antibody is used alone or in combination with other anti-tumor drugs, and the other anti-tumor drugs are selected from antibody drugs such as anti-PD-1 monoclonal antibodies or small molecule anti-tumor drugs such as paclitaxel and 5-Fu pyrimidine. The use according to item 14 above, characterized by this. After reading the above content of the present invention, those skilled in the art should understand that various changes or modifications can be made to the present invention, and these equivalent forms are also included within the scope defined by the appended claims of this application.

[0098] Sequence Listing SEQ ID NO:1 (LAG3-His Amino Acid Sequence) TIFF0007692918000003.tif45161

[0099] SEQ ID NO:2 (6H11B10 Heavy Chain Variable Region Amino Acid Sequence) TIFF0007692918000004.tif13161

[0100] SEQ ID NO:3 (6H11B10 Heavy Chain Variable Region Nucleotide Sequence) TIFF0007692918000005.tif25161

[0101] SEQ ID NO:4 (6H11B10 Light Chain Variable Region Amino Acid Sequence) TIFF0007692918000006.tif12160

[0102] SEQ ID NO:5 (6H11B10 Light Chain Variable Region Nucleotide Sequence) TIFF0007692918000007.tif26160

[0103] SEQ ID NO:6 (h6H11B10 Heavy Chain Variable Region Amino Acid Sequence) TIFF0007692918000008.tif13161

[0104] SEQ ID NO:7 (h6H11B10 Heavy Chain Variable Region Nucleotide Sequence) TIFF0007692918000009.tif26161

[0105] SEQ ID NO:8 (h6H11B10 Light Chain Variable Region Amino Acid Sequence) TIFF0007692918000010.tif13160

[0106] SEQ ID NO:9 (h6H11B10 Light Chain Variable Region Nucleotide Sequence) TIFF0007692918000011.tif25160

[0107] SEQ ID NO:10 (Amino acid sequence of the heavy chain variable region of h6H11B10#40) TIFF0007692918000012.tif14161

[0108] SEQ ID NO:11 (Nucleotide sequence of the heavy chain variable region of h6H11B10#40) TIFF0007692918000013.tif26160

[0109] SEQ ID NO:12 (Amino acid sequence of the light chain variable region of h6H11B10#40) TIFF0007692918000014.tif13161

[0110] SEQ ID NO:13 (Nucleotide sequence of the light chain variable region of h6H11B10#40) TIFF0007692918000015.tif18160 TIFF0007692918000016.tif6161

[0111] SEQ ID NO:14 (Amino acid sequence of the heavy chain constant region) TIFF0007692918000017.tif32160

[0112] SEQ ID NO:15 (Nucleotide sequence of the heavy chain constant region) TIFF0007692918000018.tif65161

[0113] SEQ ID NO:16 (Amino acid sequence of the light chain constant region) TIFF0007692918000019.tif13161

[0114] SEQ ID NO:17 (Nucleotide sequence of the light chain constant region) TIFF0007692918000020.tif19161

[0115] SEQ ID NO:18 (h6H11B10 and h6H11B10#40 HCDR1 amino acid sequence) TIFF0007692918000021.tif6161

[0116] SEQ ID NO:19 (h6H11B10 and h6H11B10#40 HCDR2 amino acid sequence) TIFF0007692918000022.tif7161

[0117] SEQ ID NO:20 (h6H11B10 HCDR3 amino acid sequence) TIFF0007692918000023.tif7160

[0118] SEQ ID NO:21 (h6H11B10#40 HCDR3 amino acid sequence) TIFF0007692918000024.tif6161

[0119] SEQ ID NO:22 (h6H11B10 LCDR1 amino acid sequence) TIFF0007692918000025.tif6161

[0120] SEQ ID NO:23 (h6H11B10#40 LCDR1 amino acid sequence) TIFF0007692918000026.tif7160

[0121] SEQ ID NO:24 (h6H11B10 and h6H11B10#40 LCDR2 amino acid sequence) TIFF0007692918000027.tif7160

[0122] SEQ ID NO:25 (h6H11B10 and h6H11B10#40 LCDR3 amino acid sequence) TIFF0007692918000028.tif7160

Claims

1. An anti-LAG3 monoclonal antibody, wherein the anti-LAG3 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 regions identical to the CDR sequences of the heavy chain variable region shown in SEQ ID NO: 2, 6, or 10, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 identical to the CDR sequences of the light chain variable region shown in SEQ ID NO: 4, 8, or 12. An anti-LAG3 monoclonal antibody characterized by this.

2. The anti-LAG3 monoclonal antibody is 1) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 18, HCDR2 has the amino acid sequence shown in SEQ ID NO: 19, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 20 or 21; and 2) a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 has the amino acid sequence shown in SEQ ID NO: 22 or 23, LCDR2 has the amino acid sequence shown in SEQ ID NO: 24, and LCDR3 has the amino acid sequence shown in SEQ ID NO:

25. The anti-LAG3 monoclonal antibody according to claim 1, characterized by this.

3. The anti-LAG3 monoclonal antibody according to claim 1 or 2, characterized in that it comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 2, 6, or 10, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, 8, or 12.

4. The anti-LAG3 monoclonal antibody according to claim 1 or 2, characterized in that it comprises a heavy chain and a light chain, the heavy chain consists of a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 2, 6, or 10 and a heavy chain constant region shown in SEQ ID NO: 14, and the light chain consists of a light chain variable region having the amino acid sequence shown in SEQ ID NO: 4, 8, or 12 and a light chain constant region shown in SEQ ID NO:

16.

5. The anti-LAG3 monoclonal antibody is the full-length sequence of the antibody or an antigen-binding fragment containing the anti-LAG3 antibody, and the antigen-binding fragment of the anti-LAG3 antibody is Fab, Fab’, F(ab’)2, Fv or scFv. The anti-LAG3 monoclonal antibody according to claim 1 or 2, characterized in that.

6. A nucleotide molecule encoding the anti-LAG3 monoclonal antibody according to any one of claims 1 to 5. The nucleotide molecule is characterized in that.

7. In the nucleotide molecule, the nucleotide sequence encoding the heavy chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 3, 7 or 11, and the nucleotide sequence encoding the light chain variable region of the anti-LAG3 monoclonal antibody is shown in SEQ ID NO: 5, 9 or 13. The nucleotide molecule according to claim 6, characterized in that.

8. An expression vector containing the nucleotide molecule according to claim 6 or 7. The expression vector is characterized in that.

9. The expression vector is one or more selected from the group consisting of pHLX101, pEE14.4, pCHO 1.0 or pcDNA3.

1. The expression vector according to claim 8, characterized in that.

10. A host cell containing the expression vector according to claim 8 or 9. The host cell is characterized in that.

11. The host cell is one or more selected from the group consisting of COS, CHO, HeLa cell line, myeloid cell lines such as SP2 / 0 cell line, NS0, sf9, sf21, DH5α, BL21(DE3) or E. coli TG1, YB2 / 0 cell line, and transformed B-cells or hybridoma cells. The host cell according to claim 10.

12. A method for producing the anti-LAG3 monoclonal antibody according to any one of claims 1 to 5, Culturing the host cell according to claim 10 or 11 under conditions suitable for expression to express the anti-LAG3 monoclonal antibody in step a), And isolating and purifying the anti-LAG3 monoclonal antibody from the host cell in step b). A method for producing an anti-LAG3 monoclonal antibody, comprising.

13. A composition comprising the anti-LAG3 monoclonal antibody according to any one of claims 1 to 5 and a pharmaceutically acceptable vector.

14. The anti-LAG3 monoclonal antibody according to any one of claims 1 to 5 or the composition according to claim 13 for use as a LAG3 molecule blocker.

15. The anti-LAG3 monoclonal antibody according to any one of claims 1 to 5 or the composition according to claim 13 for use as a drug for anti-tumor agents, treatment of autoimmune diseases, treatment of infectious diseases and / or anti-transplant rejection.

16. The anti-LAG3 monoclonal antibody according to claim 14 or 15, wherein the anti-LAG3 monoclonal antibody is used alone or in combination with other anti-tumor agents, and the other anti-tumor agents are selected from antibody drugs such as anti-PD-1 monoclonal antibodies or small molecule anti-tumor agents such as paclitaxel and 5-Fu pyrimidine.

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