An anti-human BTNL2 antibody, a biological detection product, and its application in anti-tumor drugs

Abstract

The present invention discloses an anti-human BTNL2 antibody, a biological detection product, and its application in anti-tumor drugs, and relates to the field of biomedical technology. The antibody comprises a heavy chain complementary determining region in the heavy chain variable region as shown in SEQ ID NO: 1, and a light chain complementary determining region in the light chain variable region as shown in SEQ ID NO: 2. The antibody can specifically recognize and block human BTNL2, making BTNL2 unable to inhibit the function of T cells and enhancing the tumor killing function of T cells. Therefore, the specific anti-human BTNL2 antibody provided by the present invention has good preventive or therapeutic application prospects in tumor treatment or autoimmune diseases related to BTNL2. In addition, the antibody can also be used for the diagnosis or auxiliary diagnosis of diseases related to the BTNL2 marker.

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 applications thereof in anti-tumor drugs. Background Art

[0002] Tumor immunotherapy is the fourth cancer treatment option, following radiotherapy, chemotherapy, and surgery. Its mechanism of action is to stimulate and mobilize the body's immune system to control and kill tumor cells. Tumor immunotherapy approaches primarily include tumor vaccines, adoptive immunotherapy, and immune checkpoint blockade, most of which utilize T cells to exert their anti-tumor effects. Blockade of immune checkpoints is an effective approach to enhance T cell activation and has become a popular target for anti-tumor drug development in recent years. Common immune checkpoints, such as CTLA-4, PD-1, PD-L1, BTLA, TIM-3, and LAG-3, are co-inhibitory molecules that act like "brakes" on the immune system. When the immune system is overactive, the body utilizes these checkpoints to control the intensity and duration of the immune response, thereby minimizing damage to surrounding normal tissues and preventing the development of autoimmune diseases. Tumor cells exploit this mechanism to suppress T cell activation, leading to immune escape.

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

[0004] Butyrophilin-2 (BTNL2) is a member of the butyrophilin family and shares high structural homology with B7 family molecules such as PD-L1, B7-1, and B7-H3. BTNL2 is primarily involved in T cell activation. Its receptor is expressed on activated T cells. BTNL2 inhibits the proliferation of activated T cells and the secretion of cytokines by binding to its receptor, acting as a co-inhibitory molecule for T cell activation. Similar to PD-L1, BTNL2 is also associated with various autoimmune diseases, including enteritis, sarcoidosis, and arthritis. Furthermore, missense mutations at two sites in BTNL2 exons and a mutation at one site in intron 4 are associated with susceptibility to prostate cancer and lung adenocarcinoma, respectively. This suggests that BTNL2 may be a target for cancer therapy.

[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 its application 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, comprising 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.

[0009] In a second aspect, the present invention further provides a biological product comprising: the above-mentioned anti-human BTNL2 antibody or its antigen-binding fragment, the biological product being 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) Cancer 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 in combination with other drugs in the preparation of products for the prevention or treatment of autoimmune diseases.

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

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

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

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

[0023] The present invention uses the human BTNL2 extracellular segment protein (hBTNL2-his protein) to immunize mice to prepare specific antibodies targeting human BTNL2. It was found that the anti-hBTNL2 antibody can specifically recognize and bind to BTNL2, thereby blocking the cell surface ligand BTNL2, and can reverse (or eliminate) the inhibitory effect of hBTNL2 on T cell activation in vitro, thereby enhancing the killing effect of T cells on tumor cells. Therefore, the specific anti-human BTNL2 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 following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

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

[0027] Figure 2 This is a statistical result diagram showing that anti-human BTNL2 monoclonal antibodies relieve the inhibitory effect on T cells;

[0028] Figure 3This figure shows the results of the anti-human BTNL2 monoclonal antibody promoting the killing effect of T cells on human lung cancer cells. DETAILED DESCRIPTION

[0029] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, 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 invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[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 person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (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); and PCR: The Polymerase Chain Reaction. 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 of noun

[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, as well as 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, so 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 terms "complementarity determining region" or "CDR" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and refer to the regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the hypervariable 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 this field 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 CDR regions, 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 an antibody heavy chain can be obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. HCDR1 and CDR-H1 are synonymous.

[0037] The variable region VL of the antibody light chain can be obtained by connecting the following numbered CDRs and FRs 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, comprising 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.

[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. It 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 origin 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 antibody F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv, wherein Fab'-SH refers to Fab' in which the cysteine ​​residues in the constant region have a free thiol group.

[0069] The antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that functional fragments of the above antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds.

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

[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 comprising: the above-mentioned anti-human BTNL2 antibody or its antigen-binding fragment, the biological product being 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 reagent is a preparation of an anti-human BTNL2 antibody or antigen-binding fragment thereof, and may also include functional components such as protein stabilizers and protective agents. Protein stabilizers are selected from sucrose, trehalose, BSA, glycerol, mannitol, Triton X-100, and Tween-20. Protective agents are selected from cryoprotectants, such as polyols and sugars. Polyols, for example, are selected from sorbitol, mannitol, or mixtures 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 a detection function, the anti-human BTNL2 antibody or antigen-binding fragment thereof is labeled with a detectable marker. A detectable marker is a substance that has properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, or radioactivity, and which can be used to achieve qualitative or quantitative detection of the corresponding target.

[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 substrate color development, a radioactive isotope, a chemiluminescent reagent, 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 falls within the scope of protection 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), peridinin-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-phosphate glucose deoxidase.

[0081] In an alternative embodiment, 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, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0083] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; 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) Cancer 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 the drug in combination with other 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 BTNL2's inhibition of 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: 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 at least one selected from 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 the group consisting of 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 means:

[0107] (1) Anti-human BTNL2 antibodies or their antigen-binding fragments specifically recognize and bind to BTNL2, blocking the cell surface ligand BTNL2 and relieving 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 further provides an isolated nucleic acid molecule encoding the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof for use in 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, comprising: the isolated nucleic acid molecule described above.

[0112] In preferred embodiments of the present invention, the recombinant cells are bacteria, fungi, or 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, or Per6 cells. 293 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 include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.

[0116] The recombinant cells are prepared by transforming a recombinant expression vector into a host cell (e.g., a microorganism) using conventional methods in the art. The host microorganism can be any of a variety of conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate and the exogenous gene carried by it 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 comprising the above-mentioned anti-human BTNL2 antibody or antigen-binding fragment thereof, wherein 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, etc.

[0120] In the preferred technical solution of the present invention, the pharmaceutically acceptable carrier includes but is not limited to one or a combination of 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, and indigo.

[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 emulsions, water-in-oil emulsions, and water-in-oil-in-water emulsions.

[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 derivatives thereof, 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] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

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

[0125] Example 1

[0126] This example involves the preparation of human BTNL2-his protein and mouse anti-human BTNL2 monoclonal antibody.

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

[0128] A human BTNL2 gene expression vector was constructed using genetic engineering techniques. Human BTNL2 cDNA (SEQ ID NO: 17) was inserted into the pATX2 (AtaGenix, EcoRI / NotI restriction sites) expression vector, transformed, and cultured to extract the endotoxin-free plasmid. 500 μg of the plasmid was transfected into 500 ml of 293F cells. When cell viability dropped to approximately 30%, the cell culture supernatant was collected and centrifuged at 1500 rpm for 5 minutes to remove cells and cell debris. The supernatant was then filtered through a 0.45 μM filter membrane. The cell culture supernatant was then passed through a nickel column to purify the human BTNL2-his protein.

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

[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. Immunization was performed by subcutaneous injection of 1 mg / 0.025 ml of the antigen into the axilla, paw, and groin. A primary immunization was followed by two to four booster immunizations, each 14 days apart. Seven days after the final booster immunization, serum was collected for titer measurement. Serum titers meeting the recommended titer were then intraperitoneally injected with 0.5 ml of a 200 μg / ml antigen solution for a boost immunization. Three days later, spleen cells were harvested and fused with myeloma cells.

[0132] (2) Cell fusion

[0133] Mice that had undergone shock immunization and whose serum titers met the requirements 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 50ml centrifuge tube, centrifuged at 1000rpm for 5min, the supernatant was discarded, 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 was uniformly added to the cell pellet (1ml was added within 1min). During the addition of PEG, the centrifuge tube was rotated while gently stirring with the tip of the gun head, and the mixture was left to stand for 90s. Add preheated 1640 minimal medium (first volume) at a constant rate, adding 1 ml over 1 minute while stirring gently. Add preheated 1640 minimal medium (second volume) at a constant rate, adding 2 ml over 1 minute while stirring gently. Add preheated 1640 minimal medium (third volume) at a constant rate, adding 9 ml over 3 minutes while stirring gently. Add preheated 1640 minimal medium at a constant rate, adding gently while stirring until the volume reaches 40 ml. Place the centrifuge tube in a 37°C water bath for 3 minutes. Centrifuge the fused cell suspension at 800 rpm for 5 minutes, remove the supernatant, and gently tap the tube to loosen the cell pellet. Add an appropriate amount of HAT medium based on the number of splenocytes, mix thoroughly by pipetting, and plate into 96-well cell culture plates. Maintain in HAT selection medium for 7-10 days before switching to HT medium. During the selection period, when hybridoma cells have covered 1 / 10 of the bottom of the well, collect the cell culture supernatant and begin testing for specific antibodies to screen wells containing positive antibodies.

[0134] (3) ELISA detection of antibodies

[0135] Coat a 96-well microtiter plate 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 and add 300 ul / well of 5% skim milk to block at room temperature for 1 hour. Remove the blocking solution, wash three times with PBST, and pat dry on an absorbent paper towel. Add 100 ul / well of primary antibody and incubate at 37°C for 60 minutes. Remove the primary antibody, wash three times with PBST, and pat dry on an absorbent paper towel. Add 100 ul / well of secondary antibody and incubate at 37°C for 30 minutes. Remove the secondary antibody, wash three times with PBST, and pat dry on an absorbent paper towel. Add 100 ul / well of TMB colorimetric solution and incubate at 37°C for 5-20 minutes. Observe the color development, add 50 ul / well of 2M hydrochloric acid as the stop solution, select a wavelength of 450-620 nm, and read the absorbance value with a microtiter plate reader.

[0136] (4) Hybridoma subcloning

[0137] Subclone hybridoma cells from positive wells by limiting dilution. Resuspend the hybridoma cells from the culture wells and count them. Adjust the concentration to 10 cells / ml. Add 100 μl of the diluted cells to each well and incubate in a 37°C 8% CO2 incubator for 8-9 days. Collect the culture supernatant and test for antibody activity. Select positive wells with good monoclonal hybridoma growth and transfer them to 24-well plates for subcloning or expansion.

[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. 7-12 days after cell injection, the mouse ascites was collected and passed through a Protein G affinity chromatography purification column. The ascites was eluted with 0.1M glycine solution (pH 2.5) and neutralized with 1M Tris-HCl (pH 9.0). The eluate was tested for neutrality using pH test paper. The eluate was then passed through a 50KD ultrafiltration column. The antibody was replaced in PBS and the antibody concentration was determined. 2μg of antibody was run on a gel and the antibody purity was verified 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] Primary CD4+ and CD8+ T cells were activated using CD3 and CD28 antibodies (anti-CD3 / CD28). IL-2 levels in the cell culture supernatant were 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 coated with hBTNL2-his before seeding PBMCs. The obtained research results show that hBTNL2-his has a significant inhibitory effect on the IL-2 level in the supernatant of PBMC induced by anti-CD3 / CD28, suggesting 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 pre-mixed with the supernatant of the monoclonal strain on the anti-CD3 / CD28 coated plate and then co-coated. PBMC cells were then plated and the IL-2 level was detected by ELISA. It was found that the supernatant of the monoclonal 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] After the hybridoma cells reached the logarithmic growth phase, the cells were collected (clone number H2T-3-1-28) and total RNA of the hybridoma cells was extracted. 3'RACE and 5'RACE techniques were used to obtain first-strand cDNA complementary to the full-length mRNA by RT-PCR. The synthesized cDNA was used as a template for PCR amplification to obtain the antibody heavy and light chain variable region genes. The antibody variable region genes were ligated to a T vector, and positive clones were transformed and selected for sequencing. The sequencing results were 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] Human BTNL2-His recombinant protein (ACROBiosystems, BT2-H81Q5) at a concentration of 2 μg / mL was added to 24 wells of an ELISA plate (Corning, 3590). 100 μL per well was added, and the plate was sealed and incubated overnight at 4°C. The antigen solution was removed, and the plate was washed three times with 0.05% PBST (PBS, Xi'an Hitech Biotechnology, BF001; Tween-20, Sangon Biotechnology, A100777) buffer, 200 μL per well each time. All the washing solution was removed by aspiration or inversion of the plate. The plate was then washed 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 per well each time, and all the washing solution was removed by aspiration or inversion. 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. The last well was filled with 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 per well each time, and all the washing solution was removed by aspiration or inversion. The secondary antibody Goat Anti-Human IgG-HRP (Sino Biologica, SSA001), diluted 1:10000, 100 μL 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, 200 μL / well each time, aspirated or inverted the plate to remove all the washing solution; added TMB (Beijing Meikewande, 1001) substrate solution, 100 μL per well, protected from light and room temperature for color development; after reaching the desired intensity, added 1M H2SO4 solution to stop the reaction, 50 μL per well; measured the absorbance at 450 nm using an enzyme-linked analyzer; and plotted a curve using the correlation between the sample readings and the concentrations of the two groups of antibodies. ELISA results showed ( Figure 1 ), the antibody H2T-3-1-28 has a high affinity to the human BTNL2-His recombinant protein.

[0147] Example 2

[0148] In this example, the antibody characteristics of the mouse-derived anti-human BTNL2 antibody 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] High-affinity 96-well plates were coated with 2 μg / mL of anti-CD3 antibody and 1 μg / mL of anti-CD28 antibody as T cell activators, 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 2×10 5 / mL, 100μL of H460 cells (human large cell lung cancer cells) were incubated at 37°C for 30 minutes; the cells were seeded into well plates pre-coated with T cell activator at a ratio of 1:1, 1:2, and 1:10 for H460:T cells; the cells were cultured at 37°C for 18-24 hours; the proportion of apoptotic cells was detected by flow cytometry using the 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 against human BTNL2 had a good killing effect on human lung cancer cells in a dose-dependent manner.

[0152] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection 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 identical to the heavy chain variable region set forth in SEQ ID NO: 1, and a light chain complementarity determining region identical to the light chain variable region set forth in SEQ ID NO: 2, said complementarity determining regions being determined by the Kabat numbering scheme.

2. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 1, characterized in that The heavy chain variable 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 variable 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.

4. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 3, characterized in that The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 9-12; 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.

5. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region, which comprises a heavy chain constant region and / or a light chain constant region.

6. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 5, characterized in that: 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.

7. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

8. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The species origin of the constant region is human.

9. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 1, characterized in that The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, Fab'-SH and scFv of the antibody.

10. The anti-human BTNL2 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody is a chimeric antibody.

11. 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 10, wherein the biological product is selected from the group consisting of: a reagent, a kit, a test strip, an antibody chip, an antibody probe, an affinity chromatography column, or a detector.

12. The biological product according to claim 11, 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 marked with a detectable marker.

13. The biological product according to claim 12, characterized in that The detectable marker is selected from at least one of a fluorescent dye, an enzyme that catalyzes substrate color development, a radioactive isotope, a chemiluminescent reagent, and a nanoparticle marker.

14. The biological product according to claim 11, characterized in that The kit comprises a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase.

15. The biological product according to claim 14, characterized in that The solid phase is selected from the group consisting of microspheres, plates, and membranes.

16. The biological product according to claim 14, characterized in that The solid phase is selected from the group consisting of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

17. Use of the anti-human BTNL2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 in the preparation of at least one of the following products: (1) BTNL2 testing products; (2) BTNL2 enriched products; (3) Products for the prevention or treatment of lung cancer.

18. The use according to claim 17, characterized in that The detection product is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector.

19. The use according to claim 17, characterized in that The lung cancer prevention or treatment product is selected from: a pharmaceutical composition or a vaccine composition.

20. An isolated nucleic acid molecule, characterized in that It encodes the anti-human BTNL2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

21. A recombinant cell, characterized in that It includes: The isolated nucleic acid molecule of claim 20.

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

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