A rat anti-mouse CD137 antibody or its functional fragment, a tool antibody and their applications

Abstract

The present invention discloses a rat anti-mouse CD137 antibody or its functional fragment, a tool antibody and its application, which relates to the field of biotechnology. This antibody has high specificity, high affinity, and the binding epitope of the antibody to the mouse CD137 molecule is clear, and it can be used for the detection of mouse CD137 molecule. In particular, the rat anti-mouse CD137 antibody provided by the present invention has excellent T cell activation effect and shows good in vivo tumor inhibition effect. Accordingly, the rat anti-mouse CD137 antibody provided by the present invention is not only of great significance for the development and research of CD137 monoclonal antibodies and bispecific antibodies, but also provides a unique research tool for exploring the mechanism of action of specific types of CD137 antibodies in mouse tumor models and new combinations of combination therapies based on CD137 antibodies, potentially guiding the research and development of a new generation of CD137 antibody drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a rat anti-mouse CD137 antibody or a functional fragment thereof, a tool antibody and its application. Background Art

[0002] Immune checkpoint therapy represented by PD-1 antibodies has truly opened a new era of cancer immunotherapy. At present, some domestic PD-1 antibody drugs have been included in the medical insurance, benefiting more patients. However, the effective rate of PD-1 antibody drugs is generally low, with an average of 12%, and less than 20% in non-small cell lung cancer, facing the practical problem of improving the curative effect. Scientists have found that new immune regulation targets other than PD-1 are an important direction for deepening the research of immunotherapy. In addition to negative immune checkpoint molecules such as PD-1, CTLA-4, and LAG3, another class of activating immune checkpoint molecules, such as CD40, OX40, CD137 (4-1BB) targets, etc., have also been used in the research of tumor regulation therapy of T cells, which is an important research direction for improving the curative effect of cancer immunotherapy and expanding the population of beneficiaries, and has attracted much attention internationally.

[0003] CD137 is one of the most concerned molecules. CD137 is a member of the TNFRSF family, which is related to the activation of immune cells and is antigen-inducible. Although CD137 is widely expressed, it is mainly expressed in T cells, especially in CD8+ T cells and NK cells, and these two cell populations are the core cells of cancer immunity. Preclinical models have confirmed that the regulation of anti-tumor activity by CD137 signaling is mainly mediated by CD8+ T cells, and NK cells can compensate for the functional defects of CD8+ T cells, so that the two cell populations jointly eradicate mouse tumors through functional complementarity. In addition, CAR-T cells modified with CD137 signaling are more effective and have been approved by the US Food and Drug Administration (FDA) and the Drug Administration for the treatment of B-cell leukemia and lymphoma. Accumulated research has shown that CD137 signaling prolongs the survival of CD8+ T cells and forms memory T cells.

[0004] In the early stage, two CD137 antibodies were undergoing clinical trials abroad: Urelumab (BMS-663513, by Bristol-Myers Squibb), which showed a clear immune activation effect in vivo, but there was an excessive immune stimulation phenomenon, which manifested as nonspecific immune activation, especially acting on the liver and hematopoietic tissue, leading to a certain degree of liver damage, and low-dose application may hinder its clinical anti-tumor efficacy; Utomilumab (PF-05082566, by Pfizer) is an antibody with good systemic toxicity control, showing weak in vivo anti-tumor activity. Although these two early-developed antibodies were found to have certain defects, they are still in clinical trials, using the strategy of reducing the dosage and combining PD-1 / PD-L1 antibodies to improve efficacy. There are currently 16 CD137 antibody research projects underway worldwide. In recent years, my country has begun to develop a new generation of CD137 antibodies that are safer and more effective. A total of three domestic companies are involved in the development of new CD137 target drugs, all of which are anti-CD137 monoclonal antibodies, one of which is in Phase I clinical trials, and two are in preclinical research stages, and are still in the early clinical stages.

[0005] Due to the limitations of human experiments, preclinical experiments, especially preclinical mouse in vivo experiments, are an important platform for developing a new generation of anti-tumor antibodies with low side effects and high efficiency. At present, rat CD137 antibodies are limited to a group of antibodies produced early in the world. There is no report on the production of rat anti-mouse CD137 in China. The overall resources of rat anti-mouse CD137 antibodies are small, which limits the development and research. Different CD137 antibodies have different characteristics, including binding epitopes, affinity, categories, and then determine their different immunomodulatory activities. In order to further promote the development of CD137 antibody drugs, exert stronger anti-tumor effects, and limit CD137 antibody-related adverse reactions, it is urgent to prepare diverse and different characteristics of rat anti-mouse CD37 antibodies, provide screening development and test evaluation basis for drug experiments in mice, and deepen the study of anti-tumor activity. This can not only conduct in-depth discussions on the mechanism and potential mechanism of disease occurrence, but also effectively promote the research and development of CD137 antibody drugs and the clinical trial process, and reduce the time cost of preclinical testing, but also reduce the risk of clinical experiments, and accelerate the development of a new generation of anti-human CD137 antibodies.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a rat anti-mouse CD137 antibody or its functional fragment, a tool antibody and its application, so as to provide a basis for screening, development, testing and evaluation of drug experiments carried out in mice, deepen the research on anti-tumor activity, which can not only deeply explore the pathogenesis and potential mechanisms of diseases, but also effectively promote the research, development and clinical trial process of CD137 antibody drugs, reduce the time cost of pre-clinical testing, and at the same time reduce the clinical trial risk, and accelerate the research and development of a new generation of anti-human CD137 antibodies.

[0008] Definition of terms

[0009] The term "binding protein" generally refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antibody functional fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, Fab', F(ab')2, Fd, Fv, scFv, bispecific antibodies and antibody minimal recognition units, as well as single-chain derivatives of these antibodies and fragments. The types of antibodies can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD. 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".

[0010] The term "antibody" in this article is used in the broadest sense, which can include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, as long as they exhibit the required biological activities, such as specifically binding to an antigen or its fragment. "Antibody fragments" include parts of full-length antibodies, preferably their antigen-binding regions or variable regions. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), bivalent antibodies or domain antibodies.

[0011] Under normal circumstances, the variable regions VH / VL of the heavy and light chains of an antibody can be obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0012] The present invention is implemented as follows:

[0013] In the first aspect, the present invention provides a rat anti-mouse CD137 antibody or its functional fragment, and the antibody or its functional fragment includes the following complementarity-determining regions:

[0014] CDR-VH1: SDGVH;

[0015] CDR-VH2: IIYYDGDTDYDSAIKS;

[0016] CDR-VH3: IDFRY;

[0017] CDR-VL1: RASSSLSYMY;

[0018] CDR-VL2: ETSKLSS;

[0019] CDR-VL3: QQWNSTPLT.

[0020] Rat anti-mouse antibody is rare and has high technical requirements. The inventor screened and identified a rat anti-mouse CD137 antibody or its functional fragment with high affinity and strong specificity for the CD137 molecule. The binding epitope of this antibody to the mouse CD137 molecule is clear. It can be used for the research and development of CD137 antibody drugs, so as to exert stronger anti-tumor effects and at the same time limit the adverse reactions related to CD137 antibodies.

[0021] After identification, this antibody has a certain blocking ability for the natural ligand receptor of the CD137 molecule. This antibody also has a clear ability to co-activate T cells and anti-tumor activity, especially anti-tumor activity in mouse tumors. This information provides a new tool for studying the role of specific CD137 antibodies in mouse tumor models and the role and new mechanism in the combination therapy of CD137 antibodies.

[0022] In a preferred embodiment of the application of the present invention, the above antibody includes a light chain framework region FR1-L, FR2-L, FR3-L and FR4-L shown in SEQ ID NO: 1-4 in sequence, and / or, a heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NO: 5-8 in sequence.

[0023] The sequences of SEQ ID NO: 1-8 are shown in the following table:

[0024]

[0025] In a preferred embodiment of the application of the present invention, the above antibody further includes a constant region.

[0026] In an alternative embodiment, the constant region is selected from the constant regions of any one of IgM, IgD, IgG, IgA and IgE.

[0027] In an alternative embodiment, the species origin of the constant region is mouse; the sequence of the light chain constant region of the constant region is as shown in SEQ ID NO.9, and the sequence of the heavy chain constant region of the constant region is as shown in SEQ ID NO.10;

[0028] In an alternative embodiment, the functional fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, bispecific antibody and scFv of an antibody.

[0029] The functional fragments of the above antibodies generally have the same binding specificity as their source antibodies. It is easy for those skilled in the art to understand from the content recorded 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 cleave disulfide bonds.

[0030] The functional fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0031] In a second aspect, the present invention also provides the use of a rat anti-mouse CD137 antibody or its functional fragment in any one of the following:

[0032] (a) Preparing a product for detecting the level of mouse CD137;

[0033] (b) Preparing an activation product for activating immune cells;

[0034] (c) Screening a mouse transplanted tumor efficacy model;

[0035] (d) Screening a mouse primary tumor efficacy model;

[0036] (e) Screening CD137 antibody drugs and combination therapy drugs;

[0037] (f) Preparing a CD137 ligand blocker.

[0038] Experiments have proved that the rat anti-mouse CD137 antibody provided by the present invention has a good T cell activation effect and shows a good in vivo tumor suppression effect. Therefore, the rat anti-mouse CD137 antibody provided by the present invention not only has important significance and application potential for the development and research of CD137 monoclonal antibodies and bispecific antibodies, but also provides a new tool for studying the mechanism of action of specific CD137 antibodies in mouse tumor efficacy models and the role and new mechanism in the combination therapy of CD137 antibodies.

[0039] The efficacy model is any tumor model of mice that is sensitive or insensitive to CD137 antibody treatment, including spontaneously occurring tumors, induced tumor models, and transplanted tumor models.

[0040] The above immune cells are immune cells expressing CD137, and the immune cells are selected from lymphocytes, dendritic cells, macrophages, granulocytes, mast cells, or natural killer cells.

[0041] The lymphocytes are T lymphocytes or B lymphocytes.

[0042] The above products include, but are not limited to, reagents, kits, chips, or microplates. For example, an enzyme-labeled rat anti-mouse secondary antibody complex for detecting mouse CD137 is prepared.

[0043] In an alternative embodiment, the above tumors are mouse melanoma, mouse breast cancer, mouse lung cancer, mouse colon cancer, mouse mammary tumor, mouse kidney cancer, mouse lymphoma, mouse lymphoid tumor, mouse mastocytoma, mouse liver cancer, mouse pituitary tumor, mouse myeloma, mouse cranial nerve tumor, mouse Leydig cell tumor, mouse gastric cancer, RM-1 - mouse prostate cancer cells, or mouse pancreatic cancer.

[0044] For example, the above tumors are CT26 mouse colon cancer, MC38 - mouse intestinal cancer, B16 - mouse melanoma, LLC - mouse lung cancer, CMT-93 mouse colon cancer, Colon26 - mouse colon cancer, RENCA - mouse kidney cancer, C127 - mouse mammary tumor, 4T1 - mouse breast cancer, NF639 - mouse breast cancer, YAC-1 - mouse lymphoma, P388D1 - mouse lymphoid tumor, P815 - mouse mastocytoma, BpRc1 - mouse liver cancer, GT1-1 - mouse pituitary tumor, H22 - mouse liver cancer, P3 / ag - mouse myeloma, Neuro-2a - mouse cranial nerve tumor, MLTC-1 - mouse Leydig cell tumor, MFC - mouse gastric cancer, RM-1 - mouse prostate cancer cells, AtT-20 - mouse pituitary tumor, or LTPA - mouse pancreatic cancer.

[0045] Generally, the mouse transplanted tumor efficacy model in the foregoing (c) is an animal model in which a mouse immortalized tumor cell line or mouse primary cancer tissue or cells are transplanted onto a mouse to grow into a tumor. For example, the mouse breast cancer transplanted tumor model is an animal model in which a mouse immortalized mammary tumor cell line or mouse primary breast cancer tissue or cells are transplanted onto a mouse to grow into a tumor, and the mouse breast cancer transplanted tumor model is selected from luminal A breast cancer, luminal B breast cancer, HER-2 overexpressing breast cancer, or basal-like breast cancer.

[0046] The above-mentioned mouse transplanted tumor efficacy model is applicable to drug screening, efficacy evaluation or clinical prediction. In particular, it is suitable for the research of immunomodulatory therapeutic drugs including the CD137 antibody of the present invention (or in combination with this antibody and other drugs or therapies).

[0047] The mouse primary tumor efficacy model of application (d) can be selected from mouse breast cancer primary tumor models, and the mouse breast cancer primary tumor models are selected from spontaneous breast cancer models, induced breast cancer models or genetically engineered mouse breast cancer models; the induced breast cancer model is, for example, applied to experimental animals through chemical induction, using inducers such as N-methyl-N-nitrosourea (MNU) or DMBA, etc., through intragastric administration, topical application or intravenous injection and other routes.

[0048] The genetically engineered mouse breast cancer model is selected from transgenic models or gene knockout models. The transgenic model is, for example, to direct the expression of oncogenes using specific promoters such as mammary gland, colorectal, etc. The gene knockout model is, for example, to establish a tumor model by knocking out tumor suppressor genes in mice, such as knocking out the tumor suppressor gene p53 to simulate a naturally occurring tumor model.

[0049] The mouse primary tumor efficacy model can be used to study the internal mechanism of tumor occurrence or metastasis.

[0050] In application (e), the combined therapeutic drugs for mice include rat anti-mouse CD137 antibody and at least one of the following drugs: chemotherapeutic drugs, small molecule targeted drugs, anti-tumor immunomodulatory drugs, oncolytic viruses and physical therapies.

[0051] The chemotherapeutic drugs are selected from platinum-based, docetaxel, paclitaxel, taxol, vinorelbine, vinca alkaloids, 5-fluorouracil-related drugs, capecitabine, gemcitabine, anthracyclines or irinotecan.

[0052] The small molecule targeted drugs are selected from at least one of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKI), echinoderm microtubule-associated protein-like 4-anaplastic large cell lymphoma kinase fusion gene (EML4-ALK) inhibitors, KIF5B-RET fusion gene inhibitors, BCL-2 inhibitors, HDAC inhibitors, vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors (TKI), osteopontin (OPN) inhibitors, Hedgehog (Hh) signaling pathway inhibitors, WNT / β-catenin signaling pathway inhibitors, PI3K / AKT / mTOR signaling pathway inhibitors, Ras / Raf / MAPK signaling pathway inhibitors, Notch signaling pathway inhibitors and transforming growth factor β (TGF-β) inhibitors.

[0053] The anti-tumor immunomodulatory drug is selected from at least one of anti-LAG3 antibody, anti-Tim-3 antibody, Tigit antibody, anti-CD20 antibody, anti-CD25 antibody, anti-OX40 antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-BTLA antibody, anti-GITR antibody, anti-PD-L1 antibody, LAG3 inhibitor, CTLA-4 inhibitor, TIM3 inhibitor, BTLA inhibitor, TIGIT inhibitor, CD38 inhibitor, CD47 inhibitor, IDO inhibitor, Ang2 inhibitor, EGFR inhibitor, VISTA inhibitor, CSF1R inhibitor, CCR2 inhibitor, CXCR4 inhibitor, CXCR2 inhibitor, CCR4 inhibitor, CXCL12 inhibitor, IL-6R inhibitor and IL-10 inhibitor.

[0054] The above-mentioned platinum-based drugs include, but are not limited to, anti-tumor platinum complexes, such as those selected from nedaplatin, carboplatin, cisplatin or oxaliplatin.

[0055] The above-mentioned physical therapies are selected from radiotherapy, thermotherapy, electrotherapy, microwave, ultrasound, radiofrequency, acupuncture or cryotherapy.

[0056] The above-mentioned oncolytic viruses are selected from alphavirus, measles virus, vesicular stomatitis virus, human enteric cytopathic orphan virus, adenovirus, herpes simplex virus, vaccinia virus, reovirus or coxsackievirus; the above-mentioned alphavirus is selected from at least one of M1 virus and Getah virus.

[0057] In application (f), the antibody provided by the present invention has partial blocking ability for CD137 ligand at high concentration, so it can be used to prepare the corresponding blocking agent.

[0058] In addition, the inventors found that the antibody provided by the present invention can synergistically activate mouse T lymphocytes with an anti-mouse CD3 antibody, and has stronger co-activation ability than other anti-CD137 antibodies. Under the action of the anti-CD137 antibody and the anti-CD3 antibody, CD8+ T cell proliferation is the strongest.

[0059] In the third aspect, the present invention also provides a tool antibody, which includes the above-mentioned rat anti-mouse CD137 antibody or its functional fragment. This antibody may be further engineered, such as potentially preparing single-chain antibodies and bispecific antibodies of various structures. This tool antibody or its further engineered fragment can be used in mouse immune efficacy model research, including but not limited to screening of mouse model immune efficacy, providing a basis for screening, development and test evaluation of drug experiments conducted in mice, research on anti-tumor activity and mechanism, etc.

[0060] Fourthly, the present invention also provides a reagent or kit for immunodetection of mouse CD137, which contains the above-mentioned rat anti-mouse CD137 antibody or its functional fragment; or contains the above-mentioned tool antibody.

[0061] The rat anti-mouse CD137 antibody provided by the present invention has high binding affinity and specificity for the CD137 molecule and can be used to detect the mouse CD137 molecule.

[0062] The reagent or kit further includes a label that can be detected.

[0063] The label that can be detected refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument. Through these characteristics, qualitative or quantitative detection of the corresponding target can be achieved.

[0064] In an alternative embodiment, the label that can be detected includes, but is not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0065] In actual use, those skilled in the art can select a suitable label according to the detection conditions or actual needs. No matter which label is used, it falls within the protection scope of the present invention.

[0066] Fifthly, the present invention also provides a nucleic acid encoding the rat anti-mouse CD137 antibody or its functional fragment, which includes: the nucleic acid encoding the heavy chain as shown in SEQ ID NO.11 and the nucleic acid encoding the light chain as shown in SEQ ID NO.12.

[0067] Sixthly, the present invention also provides a vector that contains the nucleic acid encoding the above-mentioned rat anti-mouse CD137 antibody or its functional fragment.

[0068] Seventhly, the present invention also provides a host cell that contains the above-mentioned vector.

[0069] The present invention has the following beneficial effects:

[0070] The present invention provides a rat anti-mouse CD137 antibody, which has high specificity and high affinity. Moreover, the binding epitope of the antibody to the mouse CD137 molecule is clear. After identification, the antibody provided by the present invention has the ability to block the natural ligand-receptor at high concentrations, and the antibody also has a clear ability to co-activate T cells, has a good T cell activation effect, and shows a good in vivo tumor suppression effect in mice. The present invention not only has important significance and application potential for the development and research of CD137 monoclonal antibodies and bispecific antibodies, but also provides a brand-new tool for studying the role of specific CD137 antibodies in mouse tumor models and the role and new mechanism in the combination therapy of CD137 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0072] Figure 1 It is a diagram showing the purity identification result of the DB-68 antibody; Figure 1 It is a diagram showing the SDS-PAGE electrophoresis result of the DB-68 antibody after affinity chromatography purification. The left lane is the protein marker, and the right lane is DB-68. It can be seen that two bands appear in the antibody lane, one is about 55KD and the other is about 25KD, and the antibody purity > 90%;

[0073] Figure 2 It is a diagram showing the specificity identification result of the DB-68 antibody. The DB-68 antibody only specifically binds to the mouse CD137 molecule, does not cross-react with the human CD137 molecule, and does not bind to other molecules with the same label as the mouse CD137 molecule, showing high specificity;

[0074] Figure 3 It is the binding and dissociation curve of the DB-68 antibody and mouse CD137 analyzed by Fortebio Octet; different lines represent different concentrations of mCD137-his, from top to bottom are 250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.81 nM, 3.9 nM;

[0075] Figure 4Schematic diagram of mCD137 protein fragment; mE3 (mE3-hFc), a fusion protein of amino acids 1-85 of the extracellular region of mouse CD137 molecule and human Fc; mE4 (mE4-hFc), a fusion protein of amino acids 1-117 of the extracellular region of mouse CD137 molecule and human Fc; mE400 (mE400-mFc), a fusion protein of amino acids 118-185 of the extracellular region of mouse CD137 molecule and mouse Fc;

[0076] Figure 5 Figure showing the identification results of the expression of mCD137 protein fragment; The expressed protein was determined to be the target protein by comparing with protein marker and by identifying the tag protein in the fusion protein with specific antibody.

[0077] Figure 6 Figure showing the analysis results of the binding of DB-68 antibody to the mCD137 region; DB-68 does not bind to mE3 (mE3-hFc), that is, it does not bind to the region of amino acids 1-85 of the extracellular region of mouse CD137 molecule; DB-68 antibody binds to mE4 (mE4-hFc), that is, it binds to the region of amino acids 1-117 of the extracellular region of mouse CD137 molecule; DB-68 antibody does not bind to mE400 (mE400-mFc), that is, it does not bind to the region of amino acids 118-185 of the extracellular region of mouse CD137 molecule. Therefore, the binding site of DB-68 antibody to mouse CD137 is only limited to the region of amino acids 86-117, that is, the third CDR region.

[0078] Figure 7 Figure showing the results of the blocking of the binding of DB-68 antibody to mouse CD137 molecule and its ligand; The figures show the results of the binding of 1 μg / ml of mCD137-hF, 5 μg / ml and 25 μg / ml of DB-68 antibody mixed with 1 μg / ml of mCD137-hFc overnight and then with mouse CD137 ligand molecule.

[0079] Figure 8 Figure showing the experimental results of the co-activation of mouse CD4+ T lymphocytes by DB-68 antibody; In the left figure, only αCD3e (2 ng / ml) was added to the culture medium, in the middle figure, αCD3e (2 ng / ml) + αCD137-1 (control antibody, 2 ng / ml) was added, and in the right figure, αCD3e (2 ng / ml) + DB-68 (2 ng / ml) was added.

[0080] Figure 9Experimental result graph of the co - activation of mouse CD8+ T lymphocytes by DB - 68 antibody; in the left figure, only αCD3e (2 ng / ml) was added to the culture medium, in the middle figure, αCD3e (2 ng / ml) + αCD137 - 1 (control antibody, 2 ng / ml) was added, and in the right figure, αCD3e (2 ng / ml) + DB - 68 (2 ng / ml) was added;

[0081] Figure 10 Antitumor activity result graph of DB - 68 antibody in a mouse intestinal cancer model. CT26 (mouse intestinal cancer cell line, purchased from ATCC) was inoculated subcutaneously into BALB / c mice at a dose of 5×10 5 / mouse. When the tumors reached 6 - 7 mm in size, the treatment group started intratumoral injection of DB - 68 antibody at a dose of 5 μg / mouse, administered every other day for three consecutive times. The control group received an intratumoral injection of the same volume of PBS solution. There was a significant difference in the tumor growth rate between the DB - 68 antibody treatment group and the control group. NS, P > 0.05; *, P < 0.05; ****, P < 0.0001. It shows that although the DB - 68 antibody cannot completely eliminate the subcutaneous transplanted tumors in mice, it can inhibit tumor growth. Detailed implementation methods

[0082] Reference to the embodiments of the present invention will now be provided in detail, with one or more examples described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a further embodiment.

[0083] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby incorporated by reference in its entirety.

[0084] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0085] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0086] Example 1

[0087] This example provides a method for preparing a rat anti-mouse CD137 monoclonal antibody, which specifically includes the following steps carried out in sequence:

[0088] 1. Immunization of animals.

[0089] The immunogen is the fusion protein mCD137-hFc, that is, mouse CD137 molecule plus human Fc tag (purchased from Beijing Sino Biological Inc.). Six- to eight-week-old purebred Lewis rats were selected. On day 0, primary immunization was carried out. The mCD137-hFc fusion protein and Freund's complete adjuvant (purchased from sigma) were made into a water-in-oil mixture by high-frequency oscillation. Each rat was immunized with 100 μg of protein, and the volume was 0.3 mL. Immunization was carried out by intraperitoneal and subcutaneous injection, and a total of 2 rats were immunized. On day 14, booster immunization was carried out. The mCD137-hFc fusion protein and Freund's incomplete adjuvant (purchased from sigma) water-in-oil mixture. Each rat was immunized with 100 μg of protein, and the volume was 0.3 mL. Immunization was carried out by intraperitoneal and subcutaneous injection. On days 28 and 42, booster immunization and fourth immunization were carried out respectively. The mCD137-hFc fusion protein and Freund's incomplete adjuvant (sigma) water-in-oil mixture, 100 μg of protein per rat, and the volume was 0.3 mL. Immunization was carried out by intraperitoneal and subcutaneous injection.

[0090] On day 45, blood was collected from the tail vein, and rat serum was collected by centrifugation. The serum titer was detected by ELISA. The titers of the immunized rats all reached or exceeded 1:10000. One rat was selected and on day 56, final immunization was carried out. 100 μg of antigen was dissolved in 0.3 mL of 1×PBS and immunized by intraperitoneal injection for boosting. Cell fusion was carried out 4 days later.

[0091] 2. Cell fusion.

[0092] (1) Preparation of immunized spleen cells: Three days after the last booster immunization, the rats were sacrificed by cervical dislocation. The spleens were aseptically removed and washed once with culture medium. The spleens were ground and passed through a 400-mesh stainless steel sieve to make a single-cell suspension. The cells were washed 2 times with serum-free DMEM culture medium, counted, and 10 8 spleen lymphocyte suspensions were reserved for use.

[0093] (2) Take the mouse myeloma cell P3-X63-Ag8.653 (purchased from ATCC) in the logarithmic growth phase. The cells were centrifuged, washed 2 times with serum-free DMEM culture medium, counted, and 1×10 7 cells were reserved for use.

[0094] (3) Fusion:

[0095] ①Mix myeloma cells and spleen cells in a ratio of 1:10 or 1:5, wash them once with serum-free DMEM in a 50 mL centrifuge tube, and centrifuge at 1200 rpm / min for 8 min; discard the supernatant, and use a pipette to suck out the residual liquid to avoid affecting the concentration of polyethylene glycol (PEG, purchased from Sigma). Gently tap the bottom of the centrifuge tube to slightly loosen the cell pellet.

[0096] ②Add 1 mL of 45% PEG (molecular weight 4000) solution pre-warmed at 37°C within 90 s, and gently shake while adding. Incubate in a 37°C water bath for 90 s.

[0097] ③Add serum-free DMEM culture medium pre-warmed at 37°C to terminate the action of PEG, and add 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL respectively every 2 min.

[0098] ④Centrifuge at 800 rpm / min for 6 min.

[0099] ⑤Discard the supernatant, and resuspend with 20% fetal bovine serum DMEM culture medium containing HAT (purchased from Sigma).

[0100] ⑥Add the above cells to a 96-well plate with a feeder cell layer, add 100 μL to each well, and inoculate 20 96-well plates with one immunized spleen.

[0101] ⑦Place the culture plate in an incubator at 37°C and 5% CO 2 for culture.

[0102] 3. Primary screening of hybridomas and detection of secreted antibodies.

[0103] (1) HAT screening: After spleen cells and myeloma cells are treated with PEG, a mixture of various cells is formed. Only the fusion bodies of spleen cells and myeloma cells can survive in the HAT selection culture medium, and other fusion bodies or non-fused cells cannot survive. After maintaining the culture for 10 days, perform a half-volume medium change.

[0104] (2) Antibody screening: Use the enzyme-linked immunosorbent assay (ELISA) method to detect whether the antibodies secreted in the hybridoma supernatant can bind to the target protein. The method is as follows: Coat with 1 μg / mL of mCD137-his (i.e., the fusion protein of mouse CD137 molecule and his tag, purchased from Beijing Sino Biological Inc.). After blocking, take out 50 μL of the supernatant from each well of each plate after fusion (half volume to prevent sucking out hybridoma cells), and add them to the blocked ELISA plate respectively. Color development is carried out through HRP-goat anti-rat secondary antibody and substrate, and the OD value is detected. In this experiment, at least 40 positive clones are initially screened out.

[0105] 4. Hybridoma cloning.

[0106] The positive hybrid clones detected are cloned at least three times to ensure the stability of the hybridomas. The cloned hybridoma cells also need to be recloned regularly to prevent the mutation or chromosome loss of the hybridoma cells, thereby losing the ability to produce antibodies. In this experiment, the limited dilution method was used for cloning, and the antibodies screened in the experiment were subcloned more than 3 times.

[0107] One day before cloning, prepare the feeder cell layer. For 40 positive clones, the 10 clones with the strongest positivity need to be subcloned into one 96-well plate, and the remaining 30 clones are each subcloned into half a 96-well plate, for a total of 25 96-well plates. Gently blow the hybridoma cells to be cloned out of the culture wells, count them. If subcloning one plate, take out about 100 hybridoma cells (for subcloning half a plate, take 50 hybridoma cells), add them to 10 ml (for subcloning half a plate, 5 ml) of complete medium containing HT (purchased from Sigma), mix well, and pipette into the 96-well plate, 100 μl / well, trying to make each well contain 1 cell, and incubate in a 37 °C, 5% CO 2 incubator. Cell clones can be seen forming in 8 - 9 days. Around 10 days, for all 25 96-well plates where hybridomas have grown to form clones, perform ELISA to detect whether the antibodies in the supernatant bind to the target protein. Transfer the cells in the positive wells to a 24-well plate for expansion culture and cryopreservation.

[0108] 5. Cryopreservation and resuscitation of hybridoma cells.

[0109] (1) Cryopreservation of hybridoma cells: Each ampoule for cryopreserving hybridoma cells contains 1×10 7 or more cells. The cell cryopreservation solution: 50% calf serum; 40% DMEM culture medium; 10% DMSO (dimethyl sulfoxide, purchased from Sigma). During cryopreservation, use a programmable freezer to gradually cool the cells from room temperature to -80 °C, and then place them in liquid nitrogen. The cells can be stored in liquid nitrogen for several years or longer.

[0110] (2) Resuscitation of hybridoma cells: Carefully take out the glass ampoule from liquid nitrogen, place it in a 37 °C water bath, and thaw the cryopreserved cells within 1 minute. Wash the cells twice with 20% DMEM culture medium, and then transfer them into a culture flask with feeder layer cells prepared the previous day, and place it in a 37 °C, 5% CO 2 incubator for culture. When cell colonies form, perform ELISA to detect the antibody activity in the supernatant. The present invention relates to hybridomas that have experienced repeated freeze-thaw processes, and their antibody secretion activity and yield are stable.

[0111] Example 2

[0112] In this example, the rat anti-mouse CD137 monoclonal antibody (DB68) prepared in the above example was assayed for antibody purity, specificity, and affinity, and the antigen-binding epitope was analyzed, and its blocking effect on the binding of CD137 molecule to CD137 ligand was verified.

[0113] 1. Purity of DB-68 antibody

[0114] (1) Production of DB-68 antibody

[0115] Select DB-68 hybridoma and resuscitate it in a T75 culture flask. Wait until the cells cover the flask, about 5×10 7 cells. Transfer the cells into a special roller bottle for hybridoma, add 100 ml of 5% DMEM and culture for 2 days. After collecting the supernatant, replace it with 100 ml of serum-free DMEM and culture for another 5 days. Then collect the supernatant again. Mix the two supernatants, centrifuge to remove cell debris, and filter through a 0.2 μm filter membrane to further remove impurities to prevent clogging of the affinity chromatography column during the affinity chromatography process.

[0116] (2) Purification of DB-68 antibody

[0117] Use an AKTA protein purification system and a Protein G affinity chromatography column (GE Healthcare, USA) to purify the DB-68 antibody in the hybridoma culture supernatant according to the set program. Dialyze to replace the purified antibody buffer with 1×PBS, and ultrafiltrate and centrifuge the antibody to the predetermined concentration through an ultrafiltration centrifugal tube (Millipore, USA), and aliquot and freeze-dry for storage at -80 °C.

[0118] (3) Identification of antibody purity

[0119] Mix 2 μg of the purified antibody with protein loading buffer and boil in boiling water for 5 minutes. After cooling to room temperature, perform electrophoresis using a 4-12% gradient SDS-PAGE gel at a constant voltage of 140 V for 70 minutes. Stain the gel with Coomassie Brilliant Blue overnight and then decolorize with decolorizing solution. The results are shown in Figure 1 . It can be seen that there are two bands in the antibody lane, one about 55 KD and one about 25 KD. In the DB-68 antibody lane, the 55 KD and 25 KD proteins account for more than 90% of the protein bands in this lane, that is, the antibody purity is above 90%, and the proportion of impurity proteins is less than 10%. After the antibody purity is identified, subsequent experiments can be carried out.

[0120] 2. Identification of the specificity of DB-68 antibody by ELISA

[0121] Coat mCD137-his, HCD137-his, PD-L1-his, EGFR-his, PD-1-his, CD137L-his, and EpCam-his fusion proteins at 1 μg / mL respectively. After blocking with 5% milk (BD), add 1 μg / mL of the antibody against DB-68 and incubate at 37 °C for 1 hour. Then add the goat anti-rat secondary antibody labeled with HPR and incubate at 37 °C for 1 hour. Finally, add the substrate for color development and measure the OD450 value.

[0122] The experimental results are shown in Figure 2 , mCD137-his is a fusion protein of mouse CD137 molecule with his tag, hCD137-his is a fusion protein of human CD137 molecule and his tag, and the other several proteins are all fusion proteins with his tags of different proteins (all purchased from Beijing Sino Biological Inc.). The results show that the antibody in Example 1 of the present invention has strong specificity for only recognizing and binding to mouse CD137 molecule, has no cross-reaction with human CD137, and does not bind to other proteins with his tags, indicating that the DB-68 antibody has high specificity.

[0123] 3. Detection of the affinity of the DB-68 antibody

[0124] The specific method is as follows:

[0125] (1) The AMC sensor (PALL Corporation, USA) is pre-wetted in the equilibration solution (PBS solution containing 0.1% BSA + 0.02% TWEEN20) for 10 minutes. Dilute the DB-68 antibody to 20 μg / mL with the equilibration solution and add it to the second column of a 96-well plate in the dark, 200 μL / well.

[0126] (2) Dilute mCD137-his serially from 250 nM to 3.9 nM and add it to the fourth column of the 96-well plate in the dark, 200 μL / well. Well H4 is used as a blank control and 200 μL of the equilibration solution is added.

[0127] (3) Add the equilibration solution to the first and third columns, 200 μL / well.

[0128] (4) Use the Fortebio octet96 instrument for detection. Equilibrate the sensor in the first column for 120 seconds to obtain the baseline equilibrium curve, then perform antibody immobilization in the second column for 300 seconds. Perform re-equilibration in the third column for 120 seconds, bind the antigen in the fourth column for 180 seconds to obtain the binding curve, and return to the third column to dissociate for 300 seconds to obtain the dissociation curve.

[0129] (5) Use the Fortebio Octet96 analysis software to fit and analyze the curve to obtain the affinity value.

[0130] Table 1 Results of the binding and dissociation of the PD-L1 antibody to PD-L1 analyzed by Fortebio Octet

[0131]

[0132] The experimental results are shown in Figure 3 and Table 1. The affinity of the antibody in Example 1 for binding to the mCD137-his protein measured by the Fortebio Octet molecular interaction analyzer was 1.02×10 -10 mol / L, that is, it can reach 10 -10 mol / L. The smaller the affinity, the stronger the binding ability.

[0133] Figure 3 The different lines in it represent different concentrations of mCD137-his. From top to bottom, they are 250 nM, 125 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.81 nM, and 3.9 nM respectively.

[0134] 4. Analysis of the binding epitope of the DB-68 antibody to the mCD137 molecule

[0135] (1) Regional expression of the mCD137 molecule

[0136] To analyze the specific binding site of the DB-68 antibody to the mCD137 molecule, the mCD137 molecule was regionally expressed.

[0137] First, specific primers were designed, and different regions of mCD137 were amplified by PCR. Then the amplified fragments were inserted into the expression vector PcDNA3.1, and the sequence of the inserted target gene fragment was determined to be completely correct by sequencing. When the 293 cells covered the bottom of the 6-well plate, the expression plasmid was transfected into the 293 cells by transfection. After the transfection was completed, 2 ml of serum-free medium was added and cultured for 72 h. The expressed target protein entered the culture supernatant in a secreted form. Usually, the protein in the supernatant could reach 0.5 - 1 μg / ml.

[0138] (2) Identification of the regional protein of the mCD137 molecule

[0139] Collect the culture supernatant in a 6-well plate. After centrifuging about 2 ml to remove cell debris, add 15 μl of Protein A or Protein G agarose gel suspension (GE Healthcare, USA). Mix overnight at 4°C so that the mCD137 fusion protein fragments with human or mouse Fc tags in the supernatant can fully bind to Protein A or Protein G agarose gel. Subsequently, by centrifugation, Protein A or Protein G agarose gel will deposit at the bottom of the centrifuge tube, and the fusion protein can be enriched at the same time. Mix the enriched protein with protein loading buffer and boil in boiling water for 5 minutes. The target protein can dissociate from Protein A or Protein G agarose gel. After centrifugation, the target protein is in the supernatant, while Protein A or Protein G agarose gel sediments at the bottom of the tube.

[0140] Perform SDS-PAGE electrophoresis on the proteins in the supernatant and transfer them to a nitrocellulose membrane. After blocking with 5% milk, use HRP-labeled goat anti-human or goat anti-mouse secondary antibody to recognize the human Fc tag of mE3-hFc and mE4-hFc and the mouse Fc tag of mE400-mFc. At the same time, determine the correctness of the expressed protein by comparing the expressed protein with the protein marker in two aspects.

[0141] Refer to Figure 4 As shown, mE3, mE4, and mE400 represent the first 85 amino acids, the first 117 amino acids, and the last 68 amino acids of the mCD137 molecule, respectively. Specifically, mE3 (mE3-hFc) represents: the fusion protein of amino acids 1-85 in the extracellular region of mouse CD137 molecule and human Fc; mE4 (mE4-hFc) represents: the fusion protein of amino acids 1-117 in the extracellular region of mouse CD137 molecule and human Fc; mE400 (mE400-mFc) represents: the fusion protein of amino acids 118-185 in the extracellular region of mouse CD137 molecule and mouse Fc.

[0142] The SDS-PAGE-ECL test identifies that the expression sizes of the protein fragments are all correct, as shown in Figure 5 shown.

[0143] (3) Analysis of the binding epitope of DB-68 antibody to mCD137 molecule

[0144] Mix 50 μl of the correctly identified mE3, mE4, mE400 and control supernatant (empty vector) + 50 μl of coating buffer, and add them to the ELISA plate respectively for overnight coating at 4°C. The next day, after blocking, add DB-68 antibody, 1 μg / ml, incubate at 37°C for 1 hour, then add HRP-labeled goat anti-rat secondary antibody, incubate at 37°C for 1 hour, add substrate for color development, and measure the OD450 value.

[0145] The experimental results are shown in Figure 6 The antibody in Example 1 of the present invention has the ability to recognize and bind to the region between amino acids 85-117 of the mCD137 molecule, i.e., the third CDR region.

[0146] 5. Blocking effect of DB-68 antibody on the binding of mouse CD137 to its ligand.

[0147] The blocking effect was detected by ELISA.

[0148] Coat mCD137L-mFc (mouse CD137 ligand molecule plus mouse Fc tag, constructed, expressed and identified in this laboratory), 1 μg / ml. At the same time, 5 μg / ml and 25 μg / ml of DB-68 antibody were respectively mixed with 1 μg / ml of mCD137-hFc and placed overnight at 4°C. The next day, the mixture was added to the ELISA plate coated with mouse CD137 ligand and incubated at 37°C for 2 h. HRP-labeled goat anti-human secondary antibody was used to detect mCD137-hFc, and the OD450 value was read.

[0149] The results are shown in reference to Figure 7 As shown, the experiment shows that when the DB-68 antibody is 5 μg / ml, only a very small part of the mCD137 molecules can be blocked from binding to their ligand, while when the DB-68 antibody is 25 μg / ml, 48% of the mCD137 molecules can be blocked from binding to their ligand.

[0150] 6. Full-length nucleic acid sequencing of hybridoma antibodies

[0151] According to the technical manual of the reagent (Thermo Fisher Scientific, USA), total RNA was isolated from hybridoma cells. According to the technical manual of the PrimeSeCpT™ First Strand cDNA Synthesis Kit (Takara, CAT, number: 610A), the total RNA was reverse transcribed into cDNA using isotype-specific antisense primers (or universal primers). According to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GESTcript, the antibody fragments of VH, VL, CH, and CL were amplified. The amplified antibody fragments were respectively cloned into standard cloning vectors and sequenced. Sequence analysis tools for variable regions: (i) NCBI NucleotideBLAST; (ii) IMGT / V Quest program; (iii) NCBI Ig BLAST. Colony PCR was used to screen for inserts of the correct size. At least five inserts of the correct size for each fragment were sequenced, and the different clone sequences were aligned to obtain a consensus sequencing result.

[0152] The heavy chain DNA sequencing result is shown in reference to SEQ ID NO.13, including the signal peptide sequence and the termination sequence.

[0153] The light chain DNA sequencing result is shown in reference to SEQ ID NO.14, including the signal peptide sequence and the termination sequence.

[0154] Experimental Example 1

[0155] This experimental example verified that the DB-68 antibody has the function of co-activating mouse T lymphocytes.

[0156] (1) Preparation of mouse lymphocyte suspension

[0157] The mouse was sacrificed by cervical dislocation, and the spleen was taken out in a laminar flow hood. 3 ml of mouse lymphocyte separation solution was placed in a 35 mm culture dish. The spleen was placed on a nylon mesh above the culture dish, and the spleen was ground into single cells with the piston in the syringe and entered the mouse lymphocyte separation solution below. The separation solution containing spleen cells was immediately transferred to a 15 ml centrifuge tube, and 500 ul of serum-free 1640 was gently and slowly overlaid. Centrifuge at 800 g for 30 min. The lymphocytes in the mouse spleen entered the upper layer of serum-free DMEM by gradient centrifugation. The lymphocyte layer was aspirated into a new tube, washed once with 5% 1×PBS, and the cells were resuspended in 1 ml of 5% PBS. The cells were counted, and the total number was 3 - 4×10 7 cells.

[0158] (2) Analysis of mouse T lymphocyte proliferation by CFSE method

[0159] 3 - 4×10 7 mouse spleen cells were resuspended in 1 ml of 5% PBS. 1.1 ul of 5 mM CFSE stock solution was suspended in 110 ul of 1×PBS, and then added to the spleen cell suspension. After thorough mixing, the cells were incubated at room temperature in the dark for 5 minutes, and the cells were washed twice with 10-fold volume of 5% 1×PBS. Count before the last centrifugation, a total of 1×10 7 cells. After centrifugation, the supernatant was discarded, and the cells were resuspended in 10 ml of 1640 complete medium. The cell concentration was about 1×10 6 / ml. 1 ml / well was added to a 24-well plate, a total of 10 wells. Among them, three wells were only added with αCD3e (145-2C11 from BD) at a concentration of 2 ng / ml; three wells were added with αCD3e (2 ng / ml) + αCD137-1 (control antibody, 2 ng / ml); three wells were added with αCD3e (2 ng / ml) + DB-68 (2 ng / ml), and cultured at 37 °C in 5% CO 2 2.

[0160] (3) Flow cytometry analysis

[0161] At 0 h, 24 h, 48 h, and 72 h after CFSE staining, one well of cells under the above three culture conditions was taken (for 0 h, it was one well of cells without adding any reagents) for flow cytometry analysis once: The cells were first washed once with 1 ml of 1×PBS and 2 ml, and the supernatant was discarded by centrifugation. 0.5 μl of live / dead dye (BV605) was added to 500 μl of 1×PBS, mixed well, and then added to the cell pellet, mixed well, protected from light, and left at room temperature for 15 min. Then, the cells were washed twice with 2 ml of 1×PBS by centrifugation, and the supernatant was discarded; 2 μl / tube of the blocking solution Purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block) was added, mixed well, and protected from light for 15 min; then 5 μl of PE-Rat Anti-Mouse CD4 and 5 μl of APC-Rat Anti-Mouse-CD8a were added, mixed well, protected from light for 30 min. After washing twice with 1 ml of 1×PBS, the pellet was resuspended in 200 μl / tube of 1×PBS, and the co-activation ability of different rat anti-mouse CD137 antibodies on mouse CD4+ and CD8+ T lymphocytes was analyzed using a flow cytometer.

[0162] Figure 8 Among them, the different proliferation conditions of CD4+ T lymphocytes after being stimulated and cultured under three conditions for 72 hours were analyzed by flow cytometry. The left figure shows that only αCD3e (2 ng / ml) was added to the culture medium, the middle figure shows the addition of αCD3e (2 ng / ml) + αCD137-1 (control antibody, 2 ng / ml), and the right figure shows the addition of αCD3e (2 ng / ml) + DB-68 (2 ng / ml). Among them, the proliferation of CD4+ T cells was the strongest under the condition of αCD3e + DB-68, indicating that DB-68 has a stronger co-activation ability than the control CD137 antibody.

[0163] Figure 9 Among them, the different proliferation conditions of CD8+ T lymphocytes after being stimulated and cultured under three conditions for 72 hours were analyzed by flow cytometry: The left figure shows that only αCD3e (2 ng / ml) was added to the culture medium, the middle figure shows the addition of αCD3e (2 ng / ml) + αCD137-1 (control antibody, 2 ng / ml), and the right figure shows the addition of αCD3e (2 ng / ml) + DB-68 (2 ng / ml). Among them, the proliferation of CD8+ T cells was the strongest under the condition of αCD3e + DB-68, and at the same concentration, the co-activation ability of DB-68 was stronger than that of the control CD137 antibody, and the proliferation of CD8+ T cells was stronger than that of CD4+ T cells under the same conditions.

[0164] From Figures 8 - 9 the results, it can be seen that the DB-68 antibody has a strong co-activation ability, especially for mouse CD8+ T lymphocytes.

[0165] Experimental Example 2

[0166] This experimental example verifies the anti-tumor activity of the DB-68 antibody in a mouse xenograft tumor model.

[0167] (1) Establishment of a mouse intestinal cancer model

[0168] Female BALB / c mice about 6 - 8 weeks old were selected. CT26 (a mouse intestinal cancer cell line, purchased from ATCC) was inoculated subcutaneously into the mice at 5×10 5 / mouse, about 100 μl, and the inoculated skin bumps were made to be as hemispherical as possible. 15 BALB / c mice were inoculated. Around the 7th day after inoculation, the inoculated tumor cells formed tumors with a size of 6 - 7 mm under the skin of the mice. The long and short diameters of the tumors were measured with a caliper. 10 BALB / c mice with basically the same tumor size were selected and divided into a control group and a treatment group, with 5 mice in each group. The mice were housed separately in cages and marked.

[0169] (2) Anti-tumor treatment with DB-68 antibody

[0170] On the 6th - 7th day after inoculation, when the diameter of the subcutaneous xenograft tumors was about 6 - 7 mm, the treatment group started intratumoral injection of DB-68 antibody at 5 μg / mouse, with a volume of 50 μl. The drug was administered every other day for three consecutive times. The control group was injected with the same volume of PBS solution intratumorally. And at the beginning of the treatment, the tumor size was measured with a caliper every other day, and the tumor area was calculated until the end of the animal experiment, that is, when the tumor area exceeded 225 mm 2 .

[0171] The results are as shown in Figure 10 . There were significant differences in the growth rate of tumors between the DB-68 antibody treatment group and the control group. NS, P > 0.05; *, P < 0.05; ****, P < 0.0001. Although the DB-68 antibody could not completely eliminate the subcutaneous xenograft tumors in mice, it could inhibit the growth of tumors.

[0172] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A rat anti-mouse CD137 antibody or a functional fragment thereof, characterized in that, the antibody or the functional fragment thereof comprises the following complementarity-determining regions: CDR-VH1: SDGVH; CDR-VH2: IIYYDGDTDYDSAIKS; CDR-VH3: IDFRY; CDR-VL1: RASSSLSYMY; CDR-VL2: ETSKLSS; and CDR-VL3: QQWNSTPLT; the functional fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv and scFv of the antibody.

2. The rat anti-mouse CD137 antibody or a functional fragment thereof according to claim 1, characterized in that, the antibody comprises a light chain framework region FR1-L, FR2-L, FR3-L and FR4-L shown in SEQ ID NO:1-4 in sequence, and / or, a heavy chain framework region FR1-H, FR2-H, FR3-H and FR4-H shown in SEQ ID NO:5-8 in sequence.

3. The rat anti-mouse CD137 antibody or a functional fragment thereof according to claim 2, characterized in that, the antibody further comprises a constant region.

4. The rat anti-mouse CD137 antibody or a functional fragment thereof according to claim 3, characterized in that, the constant region is selected from the constant region of any one of IgM, IgD, IgG, IgA and IgE.

5. The rat anti-mouse CD137 antibody or a functional fragment thereof according to claim 3, characterized in that, the species origin of the constant region is mouse; the sequence of the light chain constant region of the constant region is as shown in SEQ ID NO.9, and the sequence of the heavy chain constant region of the constant region is as shown in SEQ ID NO.

10.

6. Use of the rat anti-mouse CD137 antibody or a functional fragment thereof according to any one of claims 1-5 in any one of the following: (a) Preparing a product for detecting the level of mouse CD137; (b) Preparing an activation product for activating immune cells in vitro; (c) Screening a mouse transplanted tumor efficacy model; (d) Screening a mouse primary tumor efficacy model; (e) Screening a CD137 antibody drug and a combined therapeutic drug; (f) Preparing a drug for treating mouse colon cancer.

7. The use according to claim 6, characterized in that, the product is a reagent or a kit; the tumor is mouse melanoma, mouse lung cancer, mouse colon cancer, mouse breast cancer, mouse kidney cancer, mouse lymphoid tumor, mouse mastocytoma, mouse liver cancer, mouse pituitary tumor, mouse myeloma, mouse cranial nerve tumor, mouse testicular interstitial cell tumor, mouse gastric cancer, RM-1-mouse prostate or mouse pancreatic cancer.

8. The use according to claim 7, characterized in that, The tumor is CT26 murine colon cancer, MC38 murine intestinal cancer, B16 murine melanoma, LLC murine lung cancer, CMT-93 murine colon cancer, Colon26 murine colon cancer, RENCA murine renal cancer, C127 murine mammary tumor, 4T1 murine breast cancer, NF639 murine breast cancer, YAC-1 murine lymphoma, P388D1 murine lymphoid tumor, P815 murine mastocytoma, BpRc1 murine liver cancer, GT1-1 murine pituitary tumor, H22 murine liver cancer, P3 / ag murine myeloma, Neuro-2a murine cranial nerve tumor, MLTC-1 murine testicular interstitial cell tumor, MFC murine gastric cancer, RM-1 murine prostate cancer, AtT-20 murine pituitary tumor or LTPA murine pancreatic cancer.

9. The application according to claim 6, characterized in that, the tumor is murine breast cancer or murine lymphoma.

10. The application according to claim 6, characterized in that, the combined therapeutic drug comprises the rat anti-mouse CD137 antibody and at least one drug selected from the following: chemotherapeutic drugs, small molecule targeted drugs, anti-tumor immunomodulatory drugs and oncolytic viruses; the chemotherapeutic drugs are selected from platinum-based, docetaxel, paclitaxel, taxol, vinorelbine, vinca alkaloids, 5-fluorouracil-related drugs, capecitabine, gemcitabine, anthracyclines or irinotecan; the small molecule targeted drugs are selected from at least one of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKI), echinoderm microtubule-associated protein-like 4-anaplastic large cell lymphoma kinase fusion gene (EML4-ALK) inhibitors, KIF5B-RET fusion gene inhibitors, BCL-2 inhibitors, HDAC inhibitors, vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGF-TKI), osteopontin (OPN) inhibitors, Hedgehog (Hh) signaling pathway inhibitors, WNT / β-catenin signaling pathway inhibitors, PI3K / AKT / mTOR signaling pathway inhibitors, Ras / Raf / MAPK signaling pathway inhibitors, Notch signaling pathway inhibitors and transforming growth factor β (TGF-β) inhibitors; The anti-tumor immunomodulatory drug is selected from at least one of an anti-LAG3 antibody, an anti-Tim-3 antibody, a Tigit antibody, an anti-CD20 antibody, an anti-CD25 antibody, an anti-OX40 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-BTLA antibody, an anti-GITR antibody, an anti-PD-L1 antibody, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD38 inhibitor, a CD47 inhibitor, an IDO inhibitor, an Ang2 inhibitor, an EGFR inhibitor, a VISTA inhibitor, a CSF1R inhibitor, a CCR2 inhibitor, a CXCR4 inhibitor, a CXCR2 inhibitor, a CCR4 inhibitor, a CXCL12 inhibitor, an IL-6R inhibitor, and an IL-10 inhibitor; The oncolytic virus is selected from alphavirus, measles virus, vesicular stomatitis virus, human enteric cytopathic orphan virus, adenovirus, herpes simplex virus, vaccinia virus, reovirus, or coxsackievirus; the alphavirus is selected from at least one of M1 virus and Getah virus.

11. A tool antibody, Characterized in that, It comprises the rat anti-mouse CD137 antibody or its functional fragment according to any one of claims 1-5.

12. A reagent or kit for immunodetection of mouse CD137, Characterized in that, It contains the rat anti-mouse CD137 antibody or its functional fragment according to any one of claims 1-5; or contains the tool antibody according to claim 11.

13. A vector, Characterized in that, It contains a nucleic acid encoding the rat anti-mouse CD137 antibody or its functional fragment according to any one of claims 1-5.

14. A host cell, Characterized in that, It contains the vector according to claim 13.

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