Anti-human BTNL2 antibody, biological detection product and application of anti-human BTNL2 antibody in anti-tumor drugs

Abstract

The invention discloses an anti-human BTNL2 antibody, a biological detection product and application of the anti-human BTNL2 antibody in anti-tumor drugs, and relates to the technical field of biomedicine. The antibody comprises a heavy chain complementarity determining region in a heavy chain variable region as shown in SEQ ID NO: 1, and a light chain complementarity determining region in a light chain variable region as shown in SEQ ID NO: 2. The antibody can specifically recognize and block human BTNL2, so that BTNL2 cannot inhibit the functions of T cells, and the tumor killing function of the T cells is enhanced. Therefore, the anti-human BTNL2 specific antibody provided by the invention has a good prevention or treatment application prospect in tumor treatment or BTNL2-related autoimmune diseases. In addition, the antibody can also be used for diagnosis or auxiliary diagnosis of BTNL2 marker related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an anti-human BTNL2 antibody, a biological detection product and application thereof in anti-tumor drugs. Background Art

[0002] Tumor immunotherapy is the fourth type of tumor treatment after radiotherapy, chemotherapy and surgery. Its mechanism of action is to stimulate and mobilize the body's immune system to achieve the purpose of controlling and killing tumor cells. The methods of tumor immunotherapy mainly include tumor vaccines, adoptive immunotherapy, immune checkpoint blockers, etc. Most of them play anti-tumor effects through T cells. Among them, blocking immune checkpoints is one of the effective ways to enhance T cell activation and is also a hot target for the development of anti-tumor drugs in recent years. Common immune checkpoints such as CTLA-4, PD-1, PD-L1, BTLA, TIM-3, LAG-3, etc. are co-inhibitory molecules that play a role similar to "brakes" in the immune system. When the immune system is overactive, the body will use these immune checkpoints to control the intensity and duration of the immune response, thereby minimizing damage to surrounding normal tissues and avoiding the occurrence of autoimmune diseases. Tumor cells use this mechanism to inhibit the activation of T cells, leading to the occurrence of tumor immune escape.

[0003] At present, monoclonal antibody drugs targeting CTLA-4, PD-1, and PD-L1 have been used clinically for the treatment of tumors. For example, Yervoy, a monoclonal antibody targeting CTLA-4, Opdivo and Keytruda, a monoclonal antibody targeting PD-1, were approved by the FDA as new drugs for the treatment of melanoma in 2011 and 2014, respectively. Tecentrq, a monoclonal antibody targeting PD-L1, was approved by the FDA as a new drug for the treatment of bladder cancer in 2016. The treatment range of these immune checkpoint blockers has gradually expanded to non-small cell lung cancer, renal cell carcinoma, colorectal cancer, head and neck squamous cell carcinoma, and Hodgkin's lymphoma. However, only a small number of these tumor patients respond to CTLA-4, PD-1, and PD-L1 blockers. Therefore, finding other effective immune checkpoints and developing blockers targeting immune checkpoints are of great significance for tumor treatment.

[0004] Butyrophilin-2 (BTNL2) is a member of the butyrophilin family and has a high degree of structural homology with B7 family molecules such as PD-L1, B7-1, and B7-H3. BTNL2 is mainly involved in the activation process of T cells. Its receptor is expressed in activated T cells. BTNL2 inhibits the proliferation of activated T cells and the secretion of cytokines by binding to its receptor, and is a co-inhibitory molecule for T cell activation. Similar to PD-L1, BTNL2 is also associated with a variety of autoimmune diseases such as enteritis, sarcoidosis, and arthritis. In addition, missense mutations at two sites on the BTNL2 exon and a site mutation on the fourth intron are associated with susceptibility to prostate cancer and lung adenocarcinoma, respectively. This suggests that BTNL2 may be a target for tumor treatment.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-human BTNL2 antibody, a biological detection product and the application thereof in anti-tumor drugs to solve the above technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an anti-human BTNL2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain complementary determining region in a heavy chain variable region as shown in SEQ ID NO: 1, and a light chain complementary determining region in a light chain variable region as shown in SEQ ID NO: 2.

[0009] In a second aspect, the present invention further provides a biological product, which includes: the above-mentioned anti-human BTNL2 antibody or its antigen-binding fragment, the biological product is selected from: a reagent, a kit, a test strip, an antibody chip, an antibody probe, a conjugate of an antibody and a chemical drug, an affinity chromatography column or a detector.

[0010] In a third aspect, the present invention further provides the use of an anti-human BTNL2 antibody or an antigen-binding fragment thereof in the preparation of at least one of the following products:

[0011] (1) BTNL2 testing products;

[0012] (2) Tumor diagnosis or auxiliary diagnosis products;

[0013] (3) Autoimmune disease diagnosis or auxiliary diagnosis products;

[0014] (4) BTNL2 enriched products;

[0015] (5) Tumor prevention or treatment products;

[0016] (6) Products for the prevention or treatment of autoimmune diseases;

[0017] (7) Use of combined drugs in the preparation of tumor prevention or treatment products;

[0018] (8) Use of the combination drug in the preparation of products for the prevention or treatment of autoimmune diseases.

[0019] In a fourth aspect, the present invention also provides an isolated nucleic acid molecule encoding the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof for preparing a BTNL2 detection product.

[0020] In a fifth aspect, the present invention also provides a recombinant cell, which comprises: the isolated nucleic acid molecule described above.

[0021] In a sixth aspect, the present invention further provides a composition, which comprises the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof, and the composition is a pharmaceutical composition or a vaccine composition.

[0022] The present invention has the following beneficial effects:

[0023] The present invention uses human BTNL2 extracellular segment protein (hBTNL2-his protein) to immunize mice to prepare specific antibodies targeting human BTNL2, and it is found that the anti-hBTNL2 antibody can specifically recognize and bind to BTNL2, thereby blocking the cell surface ligand BTNL2, and can reverse (or release) the inhibitory effect of hBTNL2 on T cell activation in vitro, and enhance the killing effect of T cells on tumor cells. Therefore, the anti-human BTNL2 specific antibody provided by the present invention has good application prospects in the prevention or treatment of tumor treatment or autoimmune diseases related to BTNL2.

[0024] In addition, this antibody can also be used for the diagnosis or auxiliary diagnosis of diseases related to the BTNL2 marker, thereby achieving early detection and early treatment of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 This is the result diagram of the specific binding of the monoclonal antibody against human BTNL2 to hBTNL2-his protein;

[0027] Figure 2 This is a statistical result diagram of the release of the inhibitory effect of anti-human BTNL2 monoclonal antibody on T cells;

[0028] Figure 3This is a diagram showing the results of the monoclonal antibody against human BTNL2 promoting the killing effect of T cells on human lung cancer cells. DETAILED DESCRIPTION

[0029] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0030] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0031] Definition

[0032] The term "antigen binding fragment" refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen binding fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0033] The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the desired biological activity, such as specific binding to BTNL2 protein or a fragment thereof.

[0034] In the present invention, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment with its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0035] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0036] Typically, the variable region VH of the heavy chain of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 is synonymous with CDR-H1.

[0037] The variable region VL of the antibody light chain can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0038] In a first aspect, the present invention provides an anti-human BTNL2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain complementary determining region in a heavy chain variable region as shown in SEQ ID NO: 1, and a light chain complementary determining region in a light chain variable region as shown in SEQ ID NO: 2.

[0039] The sequence of the heavy chain variable region (VH), SEQ ID NO: 1 is:

[0040] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTVYLQLSSLTSEDTAVYYCPRYYGNYLYAMDYWGQGTSVTVSS

[0041] The sequence of the light chain variable region (VL), SEQ ID NO: 2 is:

[0042] DIVMTQSQKVMSTSVGDRVSITCKASQNVRSAVAWFQQKPGQSPKVLIY LASNRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGAG TKLELK.

[0043] By inputting the above SEQ ID NO: 1 and SEQ ID NO: 2 sequences into the CDR labeling system, the corresponding CDR sequences can be obtained.

[0044] The amino acid sequence of the complementary determining region is discovered and disclosed for the first time in the present invention and is a novel sequence that can endow the antibody or its antigen-binding fragment with the ability to specifically recognize and bind to human BTNL2 protein.

[0045] In a preferred embodiment of the present invention, the heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs: 3-5, and the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs: 6-8.

[0046] The sequence of the heavy chain complementarity determining region, CDR-H1, SEQ ID NO: 3 is: DTYMH;

[0047] The sequence of CDR-H2, SEQ ID NO: 4 is: RIDPANGNTKYDPKFQG;

[0048] The sequence of CDR-H3, SEQ ID NO: 5 is: YYGNYLYAMDY;

[0049] The sequence of CDR-L1, SEQ ID NO: 6 is: KASQNVRSAVA;

[0050] The sequence of CDR-L2, SEQ ID NO: 7 is: LASNRHT;

[0051] The sequence of CDR-L3, SEQ ID NO: 8 is: LQHWNYPLT.

[0052] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a heavy chain framework region, and / or a light chain framework region;

[0053] In a preferred embodiment of the present invention, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs:9-12; for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs:9-12.

[0054] The light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 13-16, for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs: 13-16.

[0055] SEQ ID NO:9: EVQLQQSGAELVKPGASVKLSCTASGFNIK;

[0056] SEQ ID NO:10: WVKQRPEQGLEWIG;

[0057] SEQ ID NO:11: KATITADTSSNTVYLQLSSLTSEDTAVYYCPR;

[0058] SEQ ID NO: 12: WGQGTSVTVSS;

[0059] SEQ ID NO:13: DIVMTQSQKVMSTSVGDRVSITC;

[0060] SEQ ID NO:14: WFQQKPGQSPKVLIY;

[0061] SEQ ID NO:15:

[0062] GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC;

[0063] SEQ ID NO: 16: FGAGTKLELK.

[0064] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region;

[0065] In a preferred embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region;

[0066] In a preferred embodiment of the present invention, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human;

[0067] In a preferred embodiment of the present invention, the species of the constant region is human;

[0068] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of an antibody, wherein Fab'-SH refers to Fab' with a free thiol group on the cysteine ​​residue in the constant region.

[0069] The antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds.

[0070] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0071] In a preferred embodiment of the present invention, the antibody is a chimeric antibody.

[0072] In a second aspect, the present invention further provides a biological product, which includes: the above-mentioned anti-human BTNL2 antibody or its antigen-binding fragment, the biological product is selected from: a reagent, a kit, a test strip, an antibody chip, an antibody probe, a conjugate of an antibody and a chemical drug, an affinity chromatography column or a detector.

[0073] In one embodiment, the above reagent is a product of an anti-human BTNL2 antibody or an antigen-binding fragment thereof, and may also include functional components such as a protein stabilizer and a protective agent. The protein stabilizer is selected from: sucrose, trehalose, BSA, glycerol, mannitol, TritonX-100 and Tween-20. The protective agent is selected from a cryoprotectant, such as a polyol and a sugar. The polyol is selected from, for example, sorbitol, mannitol or a mixture thereof.

[0074] In one embodiment, the form of the reagent includes but is not limited to solid, liquid, and semi-solid.

[0075] The antibody chip refers to a chip formed by immobilizing the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof on a carrier.

[0076] In a preferred embodiment of the present invention, when the biological product is a product with detection function, a detectable marker is marked on the anti-human BTNL2 antibody or its antigen-binding fragment. A detectable marker refers to a class of substances that have characteristics that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which the qualitative or quantitative detection of the corresponding target can be achieved.

[0077] In a preferred embodiment of the present invention, the detectable marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent agent, and a nanoparticle marker.

[0078] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0079] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0080] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0081] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177 Lu, 172 Lu and 18 F.

[0082] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0083] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0084] In a preferred embodiment of the present invention, the kit includes a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; for example, the antibody or antigen-binding fragment thereof is connected to the solid phase by chemical coupling.

[0085] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0086] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0087] In a third aspect, the present invention further provides the use of an anti-human BTNL2 antibody or an antigen-binding fragment thereof in the preparation of at least one of the following products:

[0088] (1) BTNL2 testing products;

[0089] (2) Tumor diagnosis or auxiliary diagnosis products;

[0090] (3) Autoimmune disease diagnosis or auxiliary diagnosis products;

[0091] (4) BTNL2 enriched products;

[0092] (5) Tumor prevention or treatment products;

[0093] (6) Products for the prevention or treatment of autoimmune diseases;

[0094] (7) Use of combined drugs in the preparation of tumor prevention or treatment products;

[0095] (8) Use of combined drugs in the preparation of products for the prevention or treatment of autoimmune diseases;

[0096] In a preferred embodiment of the present invention, the detection product, diagnostic product or auxiliary diagnosis is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector;

[0097] In a preferred embodiment of the present invention, the tumor or autoimmune disease is marked by BTNL2;

[0098] In the above applications (5) and (7), the tumor prevention or treatment product is used to treat all tumors that accelerate tumor progression by inhibiting T cell function through BTNL2. The present invention uses a mouse monoclonal antibody (clone number H2T-3-1-28) to verify that it can relieve the inhibition of BTNL2 on T cells and enhance the tumor killing function of T cells. It also has a corresponding preventive or therapeutic effect on other tumors related to the co-inhibitory molecule (BTNL2) of T cell activation.

[0099] The combination drugs in the above application (7) include, for example, PD-1 monoclonal antibody, PD-L1 monoclonal antibody and CTLA-4 monoclonal antibody.

[0100] In a preferred embodiment of the present invention, the tumor is selected from human melanoma, lung cancer, colon cancer, pancreatic cancer, neuroblastoma, ovarian cancer, breast cancer, liver cancer, non-small cell lung cancer, gastric cancer, adrenocortical carcinoma, cervical squamous cell carcinoma, bile duct cancer, esophageal cancer, head and neck squamous cell carcinoma, thyroid cancer, bladder cancer, thymoma or endometrial cancer.

[0101] In a preferred embodiment of the present invention, the autoimmune disease is selected from at least one of enteritis, Stevens-Johnson syndrome, systemic lupus erythematosus, aplastic anemia, myositis, polyarthritis, idiopathic thrombocytopenia, sarcoidosis, Sjögren's syndrome, uveitis, pulmonary fibrosis, rheumatoid arthritis, myasthenia gravis, psoriasis, cirrhosis, aplastic anemia, encephalopathy, multiple sclerosis, scleroderma and chronic hepatitis;

[0102] In a preferred embodiment of the present invention, the enteritis is Crohn's disease or ulcerative colitis.

[0103] In a preferred embodiment of the present invention, the enteritis is iatrogenic autoimmune colitis;

[0104] In a preferred embodiment of the present invention, the iatrogenic autoimmune colitis is selected from colitis induced by one or more chemotherapeutic agents, colitis induced by adoptive cell therapy, and colitis associated with one or more alloimmune diseases;

[0105] In a preferred embodiment of the present invention, the tumor prevention or treatment product or the autoimmune disease prevention or treatment product is selected from: a pharmaceutical composition or a vaccine composition;

[0106] In a preferred embodiment of the present invention, the tumor prevention or treatment product or the autoimmune disease prevention or treatment product achieves the prevention or treatment of tumors or the prevention or treatment of autoimmune diseases by at least one of the following methods:

[0107] (1) Anti-human BTNL2 antibodies or antigen-binding fragments thereof specifically recognize and bind to BTNL2, block the cell surface ligand BTNL2, and relieve BTNL2's inhibition of T cells;

[0108] (2) Proliferation of activated T cells and secretion of cytokines.

[0109] In a fourth aspect, the present invention also provides an isolated nucleic acid molecule encoding the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof for preparing a BTNL2 detection product.

[0110] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0111] In a fifth aspect, the present invention also provides a recombinant cell, which comprises: the isolated nucleic acid molecule described above.

[0112] In a preferred embodiment of the present invention, the recombinant cell is a bacterium, a fungus or a 293 cell, a 293T cell, a 293FT cell, a CHO cell, a COS cell, or a Per6 cell. 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those skilled in the art.

[0113] In a preferred embodiment of the present invention, the bacteria is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis.

[0114] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0115] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parental cells in nucleic acid content, but may contain mutations.

[0116] The above-mentioned recombinant cells are prepared by transforming the recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and the foreign gene carried can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0117] In a sixth aspect, the present invention further provides a composition, which comprises the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof, and the composition is a pharmaceutical composition or a vaccine composition.

[0118] In an alternative embodiment, the pharmaceutical composition comprises at least one of a pharmaceutically acceptable excipient, a carrier and a diluent.

[0119] The above-mentioned pharmaceutically acceptable carriers include, but are not limited to, fillers, lubricants, disintegrants, binders, glidants and the like.

[0120] In the preferred technical scheme of the present invention, the pharmaceutically acceptable carrier includes but is not limited to polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0121] In an optional embodiment, the vaccine composition includes, in addition to the anti-human BTNL2 antibody or antigen-binding fragment thereof, an adjuvant. The types of adjuvants include but are not limited to: oil-in-water emulsion, water-in-oil, and water-in-oil-in-water emulsion.

[0122] In a preferred embodiment of the present invention, the adjuvant is selected from at least one of Toll-like receptor agonists, RIG-I-like receptor agonists, NOD-like receptor agonists, C-type lectin receptors, STING agonists, bacterial toxins and their derivatives, saponins, cytokines and other adjuvants; other adjuvants are selected from at least one of heat shock protein, A151, GTP-GDP, sodium fluoride, alkyl polypropylene polymers, and dimethyl dioctadecyl quaternary ammonium bromide (DDA).

[0123] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0124] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0125] Example 1

[0126] This example is used to prepare human BTNL2-his protein and mouse anti-human BTNL2 monoclonal antibody.

[0127] 1. Preparation of human BTNL2-his protein

[0128] Genetic engineering technology was used to construct a human BTNL2 gene expression vector. We constructed human BTNL2 cDNA (such as SEQ ID NO: 17) into the pATX2 (AtaGenix, restriction site EcoRI / NotI) expression vector, transformed, shaken, and extracted the endotoxin-free plasmid. The plasmid (500 μg) was transfected into 500 ml 293F cells. When the cell viability dropped to about 30%, the cell culture supernatant was collected, centrifuged at 1500 rpm for 5 minutes to remove cells and cell debris, and then centrifuged at 10000 rpm for 5 minutes to filter the cell culture supernatant with a 0.45 uM filter membrane. The cell culture supernatant was passed through a nickel column to purify the human BTNL2-his protein.

[0129] 2. Preparation of Mouse Anti-human BTNL2 Monoclonal Antibody

[0130] (1) Immunization of mice with human BTNL2-his protein

[0131] Four Balb / c mice were immunized with the prepared human BTNL2-his recombinant protein, and 1 mg / 0.025 ml antigen was injected subcutaneously in the armpits, soles and groins of the mice for immunization. The primary immunization was performed first, and then 2-4 booster immunizations were performed, with an interval of 14 days between each immunization. The mouse serum was taken on the seventh day after the last booster immunization to measure the titer. If the titer met the standard, 0.5 ml of 200 ug / ml antigen solution was injected intraperitoneally for shock immunization. Three days later, spleen cells were taken for cell fusion with myeloma cells.

[0132] (2) Cell fusion

[0133] Mice that had undergone shock immunization and whose serum titers met the criteria were selected, and their eyeballs were removed to collect blood for positive control of hybridoma screening. The mice were then killed by cervical dislocation and disinfected in 75% alcohol for at least 30 seconds. The spleens of the mice were taken to prepare a spleen cell suspension and counted. SP2 / 0 myeloma cells were also taken and counted. The spleen cells and myeloma cells were mixed in a 5:1 ratio in a 50 ml centrifuge tube, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the wall of the centrifuge tube was flicked to loosen the cell pellet. The centrifuge tube was placed in a 37°C water bath, and preheated PEG (1 ml was added within 1 min) was uniformly added to the cell pellet. During the addition of PEG, the centrifuge tube was rotated while gently stirring with the tip of the gun head, and it was left to stand for 90 s. Add preheated 1640 basic medium (first time) at a uniform speed, add 1ml within 1min, and stir gently while adding; add preheated 1640 basic medium (second time) at a uniform speed, add 2ml within 1min, and stir gently while adding; add preheated 1640 basic medium (third time) at a uniform speed, add 9ml within 3min, and stir gently while adding; add preheated 1640 basic medium at a uniform speed, stir gently while adding until 40ml, and place the centrifuge tube in a 37℃ water bath for 3min. The fused cell suspension was centrifuged at 800rpm for 5min, the supernatant was removed, the wall of the centrifuge tube was flicked to loosen the cell sediment, and an appropriate amount of HAT medium was added according to the number of spleen cells, and the mixture was mixed by blowing, and inoculated into a 96-well cell culture plate. After maintaining the HAT selection medium for 7 to 10 days, HT culture medium was used. During the selection culture period, when the hybridoma cells covered 1 / 10 of the bottom of the well, the cell culture supernatant was collected to start detecting specific antibodies and screen out the wells containing positive antibodies.

[0134] (3) ELISA detection of antibodies

[0135] Coat 96-well ELISA plates with 100 μl (2 ug / ml) of BTNL2-his protein antigen solution and incubate overnight at 4°C. Remove the coating solution the next day, add 5% skim milk 300 ul / well to block at room temperature for 1 hour, remove the blocking solution, wash 3 times with PBST, and pat dry on absorbent paper towels. Add primary antibody 100 ul / well and incubate at 37°C for 60 minutes, remove the primary antibody, wash 3 times with PBST, and pat dry on absorbent paper towels. Add secondary antibody 100 ul / well and incubate at 37°C for 30 minutes, remove the secondary antibody, wash 3 times with PBST, and pat dry on absorbent paper towels. Add TMB colorimetric solution 100 ul / well and incubate at 37°C for 5-20 minutes, observe the color development, add stop solution 2M hydrochloric acid 50 ul / well, select 450-620 nm wavelength, and read the absorbance value with an ELISA reader.

[0136] (4) Hybridoma subcloning

[0137] Subclone the hybridoma cells in the positive wells by limiting dilution. Resuspend the hybridoma cells to be cloned from the culture wells, count them, adjust to 10 cells / ml, add 100 μl of diluted cells to each well, and place them at 37°C with 8% CO 2 Incubator, culture for 8-9 days, collect the culture supernatant to detect antibody activity, select the positive wells with good growth of monoclonal hybridoma, transfer to 24-well plate for subcloning or expansion culture.

[0138] (5) Production of mouse anti-human BTNL2 monoclonal antibody

[0139] 7-21 days in advance, mice were intraperitoneally injected with 0.5 ml of Freund's incomplete adjuvant, and hybridoma cells were collected and the density was adjusted to 2×10 6 Mice injected with incomplete adjuvant were selected and intraperitoneally injected with 2×10 hybridoma cells. 6 / 0.5ml / mouse, collect mouse ascites 7-12 days after cell injection, pass the mouse ascites through Protein G affinity chromatography purification column, elute with 0.1M glycine solution (pH 2.5), neutralize with 1M Tris-HCl (pH 9.0) solution, test whether the eluate is neutral with pH test paper, then pass through 50KD ultrafiltration column, replace the antibody in PBS, measure the antibody concentration, take 2ug antibody for gel running, and verify the antibody purity by Coomassie Brilliant Blue staining.

[0140] (6) Verification of mouse anti-human BTNL2 monoclonal antibodies capable of restoring BTNL2's inhibitory function on T cells

[0141] CD3 and CD28 antibodies (anti-CD3 / CD28) were used to induce the activation of primary CD4+ and CD8+ T cells, and the level of IL-2 in the cell culture supernatant was measured by ELISA as a marker of T cell activation and proliferation. To evaluate the effect of hBTNL2-his on T cell activation, anti-CD3 / CD28-coated plates were plated with PBMC cells after hBTNL2-his coating. The obtained research results show that hBTNL2-his has a significant inhibitory effect on the IL-2 level in the PBMC supernatant induced by anti-CD3 / CD28, indicating that BTNL2 can inhibit the activation of T cells. In order to verify whether the H2T-3-1-28 clone we screened can restore the inhibition of BTNL2 on T cells, hBTNL2-his was premixed with the monoclonal supernatant on the anti-CD3 / CD28 coated plate and then co-coated, and then PBMC cells were plated. The IL-2 level was detected by ELISA, and it was found that the monoclonal supernatant of H2T-3-1-28 could significantly relieve the inhibition of BTNL2 on primary T cells. Figure 2 shown.

[0142] Through cell function experiments, we verified a mouse anti-human BTNL2 monoclonal antibody that can restore BTNL2's inhibition of T cells: clone number H2T-3-1-28.

[0143] 3. Hybridoma cell sequencing / variable region gene sequencing

[0144] When the hybridoma cells grow to the logarithmic growth phase, the cells are collected (clone number H2T-3-1-28), and the total RNA of the hybridoma cells is extracted. The first-strand cDNA complementary to the full-length mRNA is obtained by RT-PCR using the 3'RACE technology and the 5'RACE technology. The synthesized cDNA is used as a template, and the antibody heavy chain and light chain variable region genes are amplified by PCR. The antibody variable region genes are connected to the T vector, and the positive clones are transformed and selected for sequencing. The sequencing results are analyzed by bioinformatics to obtain the antibody variable region gene sequence.

[0145] ELISA assay to detect the binding ability of human BTNL2-His recombinant protein and antibody H2T-3-1-28:

[0146] Add 2 μg / mL concentration of human BTNL2-His recombinant protein (ACROBiosystems, BT2-H81Q5) to the ELISA plate (Corning, 3590), 24 wells in total, 100 μL per well, seal the plate and incubate overnight at 4°C; remove the antigen solution, wash the plate three times with 0.05% PBST (PBS, Xi'an Hete Biotechnology, BF001; Tween-20, Sangon Biotechnology, A100777) buffer, 200 μL / well each time, aspirate or invert the plate to remove all the washing solution; wash with 1% The plate was blocked with PBS buffer containing BSA (Sangon Biotechnology, A500023-0100), 300 μL per well, and incubated at room temperature for 1 hour after sealing; the blocking solution was removed, and the plate was washed 5 times with 0.05% PBST buffer, 200 μL / well each time, and all the washing solution was removed by aspirating or inverting the plate; the experimental group antibody H2T-3-1-28 or the control group antibody Blank was added, 10 μg / mL in the first well of each group, and diluted to 11 wells in a 4-fold gradient, and the last well was blank buffer, a total of 12 wells, 100 μL per well, and incubated at room temperature for 1 hour after sealing; the antibody solution was removed, and the plate was washed 5 times with 0.05% PBST buffer, 200 μL / well each time, and all the washing solution was removed by aspirating or inverting the plate; the secondary antibody GoatAnti-Human IgG-HRP (Sino Biologica, SSA001), diluted 1:10000, 100uL per well, sealed and incubated at room temperature for 1 hour; removed the secondary antibody solution, washed the plate 5 times with 0.05% PBST buffer, 200μL / well each time, aspirated or inverted the plate to remove all the washing solution; added TMB (Beijing Meikewande, 1001) substrate solution, 100uL per well, protected from light at room temperature for color development; after reaching the desired intensity, added 1M H 2 SO 4 The solution was used to terminate the reaction, 50 μL per well; the absorbance at 450 nm was measured using an enzyme-linked analyzer; a curve was drawn using the correlation between the sample readings and the concentrations of the two groups of antibodies. The ELISA results showed ( Figure 1 ), antibody H2T-3-1-28 has a higher affinity with human BTNL2-His recombinant protein.

[0147] Example 2

[0148] In this example, the antibody characteristics of the mouse antibodies against human BTNL2 obtained by screening in Example 1 were identified.

[0149] 1. Anti-human BTNL2 antibody H2T-3-1-28 can promote the killing effect of T lymphocytes on human lung cancer cells.

[0150] T cell activators were coated in high-affinity 96-well plates with anti-CD3 antibody at a concentration of 2 μg / mL and anti-CD28 antibody at a concentration of 1 μg / mL, 100 μL per well, and coated overnight at 4°C. Mouse spleen CD8+ T cells were isolated using a kit (Miltenyi Biotec, 130-104-075). Antibody H2T-3-1-28 was added to a 2×10 5 / mL, 100μL H460 cells (human large cell lung cancer cells), incubated at 37°C for 30min; seeded the cells in well plates pre-coated with T cell activators at a ratio of 1:1, 1:2 and 1:10 for H460:T cells; cultured at 37°C for 18-24h; detected the proportion of apoptotic cells by flow cytometry using FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, 556547). The results are shown in Figure 2. Figure 3 shown. Figure 3 The control IgG in the assay was Mouse IgG (Biyuntian, A7050).

[0151] The results showed that the mouse antibody of human BTNL2 had a good killing effect on human lung cancer cells and was dose-dependent.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anti-human BTNL2 antibody or an antigen-binding fragment thereof, characterized in that: It includes a heavy chain complementarity determining region in the heavy chain variable region as shown in SEQ ID NO:1, and a light chain complementarity determining region in the light chain variable region as shown in SEQ ID NO:

2.

2. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The heavy chain complementary determining region includes: CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs: 3-5, and the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs: 6-8.

3. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region, and / or a light chain framework region; Preferably, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 which have at least 80% homology to the amino acid sequences shown in SEQ ID NOs: 9-12, in sequence; The light chain framework region includes LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology to the amino acid sequences shown in SEQ ID NOs: 13-16, in sequence; Preferably, the antibody or antigen-binding fragment thereof further comprises a constant region, wherein the constant region comprises a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region; Preferably, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human; Preferably, the species origin of the constant region is human; Preferably, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of the antibody; Preferably, the antibody is a chimeric antibody.

4. A biological product, characterized in that It includes: The anti-human BTNL2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the biological product is selected from the group consisting of: a reagent, a kit, a test strip, an antibody chip, an antibody probe, a conjugate of an antibody and a chemical drug, an affinity chromatography column or a detector.

5. The biological product according to claim 4, characterized in that When the biological product is a product with detection function, the anti-human BTNL2 antibody or the antigen-binding fragment thereof is labeled with a detectable marker; Preferably, the detectable marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes the color development of a substrate, a radioactive isotope, a chemiluminescent agent and a nanoparticle marker.

6. The biological product according to claim 5, characterized in that The kit comprises a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; Preferably, the solid phase is selected from microspheres, plates and membranes; Preferably, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon and nitrocellulose membranes.

7. Use of the anti-human BTNL2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of at least one of the following products: (1) BTNL2 testing products; (2) Tumor diagnosis or auxiliary diagnosis products; (3) Autoimmune disease diagnosis or auxiliary diagnosis products; (4) BTNL2 enriched products; (5) Tumor prevention or treatment products; (6) Products for the prevention or treatment of autoimmune diseases; (7) Use of combined drugs in the preparation of tumor prevention or treatment products; (8) Use of combined drugs in the preparation of products for the prevention or treatment of autoimmune diseases; Preferably, the detection product, diagnostic product or auxiliary diagnosis is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector; Preferably, the tumor or autoimmune disease is marked by BTNL2; Preferably, the tumor is selected from human melanoma, lung cancer, colon cancer, pancreatic cancer, neuroblastoma, ovarian cancer, breast cancer, liver cancer, non-small cell lung cancer, gastric cancer, adrenocortical carcinoma, cervical squamous cell carcinoma, bile duct cancer, esophageal cancer, head and neck squamous cell carcinoma, thyroid cancer, bladder cancer, thymoma or endometrial cancer; Preferably, the autoimmune disease is selected from at least one of enteritis, Stevens-Johnson syndrome, systemic lupus erythematosus, aplastic anemia, myositis, polyarthritis, idiopathic thrombocytopenia, sarcoidosis, Sjögren's syndrome, uveitis, pulmonary fibrosis, rheumatoid arthritis, myasthenia gravis, psoriasis, cirrhosis, aplastic anemia, encephalopathy, multiple sclerosis, scleroderma and chronic hepatitis; Preferably, the enteritis is Crohn's disease or ulcerative colitis; Preferably, the enteritis is iatrogenic autoimmune colitis; Preferably, the iatrogenic autoimmune colitis is selected from the group consisting of colitis induced by one or more chemotherapeutic agents, colitis induced by adoptive cell therapy treatment, and colitis associated with one or more alloimmune diseases; Preferably, the tumor prevention or treatment product or autoimmune disease prevention or treatment product is selected from: a pharmaceutical composition or a vaccine composition; Preferably, the tumor prevention or treatment product or the autoimmune disease prevention or treatment product achieves the prevention or treatment of tumors or the prevention or treatment of autoimmune diseases by at least one of the following methods: (1) The anti-human BTNL2 antibody or antigen-binding fragment thereof specifically recognizes and binds to BTNL2, blocks the cell surface ligand BTNL2, and relieves the inhibition of BTNL2 on T cells; (2) Proliferation of activated T cells and secretion of cytokines.

8. An isolated nucleic acid molecule, characterized in that The anti-human BTNL2 antibody or antigen-binding fragment thereof encoding any one of claims 1 to 3 is used to prepare a BTNL2 detection product.

9. A recombinant cell, characterized in that It includes: The isolated nucleic acid molecule of claim 8.

10. A composition, characterized in that The invention comprises the anti-human BTNL2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and the composition is a pharmaceutical composition or a vaccine composition.

Images