An anti-human CD137 antibody and its application

Abstract

The present invention discloses an anti-human CD137 antibody and its application, and relates to the field of biomedical technology. The present invention provides a novel anti-human CD137 antibody, which can cross-bind to monkey CD137, has strong human T cell activation characteristics, and is named clone BXMD12. The monoclonal antibody has a unique variable region sequence, binds to the CD137-CRDI+CRDII+CRDIII region, and has blocking activity for the natural ligand receptor binding of the CD137 molecule. It is a "blocking" antibody, and the whole antibody realizes eukaryotic expression and purification batch preparation, which can be used to prepare reagents or kits for detecting human or monkey CD137. The anti-human CD137 antibody provided by the present invention is functionally efficient and synergistically stimulates T cell activation and proliferation, especially for activating the expansion of CD8+T cells, and is potentially used for immunomodulatory treatment research and application of immune-related diseases such as tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-human CD137 antibody and an application thereof. Background Art

[0002] The breakthrough brought by PD-1 antibody in treating tumors is that PD-1 antibody is based on immune checkpoint blockade, thereby reducing the inhibitory signals of T cell proliferation and activity, and promoting CD8+T lymphocytes (Cytotoxic T Lymphocytes, CTLs) to directly kill tumor cells. The successful use of immune checkpoint inhibitors in tumor treatment has confirmed the feasibility of killing and treating tumors by regulating the immune system, opening up new horizons for tumor treatment. The main problem with PD-1 antibody treatment is that only a small number of patients are effective in this drug. Improving the effect of tumor immunotherapy and expanding the number of patients who benefit are the focus of widespread attention and research hotspots after PD-1 antibody treatment. Discovering new immune regulatory targets and combined immunotherapy have become internationally recognized directions for improving tumor immunotherapy, which is expected to benefit tumor patients and may eventually cure tumors.

[0003] In 1993, Kwon first cloned 4-1BB (CD137) from CD8+T cells, which has the function of co-stimulating T cells. He then reported that CD137 antibodies have the ability to regulate immunity in vivo and eradicate mouse tumor transplantation models. Subsequently, the preclinical series of anti-tumor treatment studies showed the excellent anti-tumor activity of this target antibody, including low immunogenic tumors. Therefore, CD137 molecular regulation has become one of the most concerned anti-tumor immune regulation targets. CD137 is a classic T cell co-activation molecule, which is antigen-induced, especially biased towards CD8+T cell expression. Its regulation helps CD8+T cell survival and promotes memory T cell differentiation. CD8+T cells are the core effector cells of anti-tumor. The CD137 molecule is introduced into chimeric antigen receptor T cells (CAR-T cells, Chimeric Antigen Receptor T-Cells) and is considered to be the best regulatory molecule for CAR-T cells to date. It also shows that this molecule is of great significance to tumor immunoregulatory treatment and has always been a highly concerned immune regulation. Its antibody regulatory preparation is the main application form. In short, the antibody against this target is mainly used in tumor treatment and secondly has therapeutic potential in certain autoimmune diseases, all of which have been verified in preclinical studies.

[0004] Urelumab is the first CD137 antibody to enter clinical trials. It fully demonstrates its efficient ability to activate T cells in vivo. The super-strong immune activation even leads to a certain immune overreaction. Subsequently, another different antibody, Utomilumab, was admitted to clinical trials, but its clinical effect was not as good as Urelumab. These two antibodies are still undergoing clinical verification [1,2]. Based on this, the international community is focusing on developing more characteristic CD137 antibodies. Recently, it has been reported that new CD137 antibodies have entered preclinical research and are considering the possibility of entering clinical trials, including CD137 alone and PD-1 combined treatment, which shows the desire for new antibodies and the development trend of their research and development [3].

[0005] Based on the analysis of basic research in recent years, there are obvious differences in the specific binding sites of Urelumab and Utomilumab produced in the early stage. One is non-blocking to the natural ligand receptor, while the other is blocking to the natural ligand receptor. This may lead to different immunotherapy effects and toxic reactions. Balancing the co-stimulatory efficacy and toxic reactions is a problem that needs to be weighed in clinical practice. Given that the systemic application (i.e., intravenous administration) of the above-mentioned super-strong co-stimulatory CD137 antibody may cause immune over-activation and systemic inflammatory side effects, the co-stimulatory CD137 antibody with moderate stimulation intensity is suitable for tumor targeting, reducing dosage, and effectively performing local immune regulation [4], which may avoid the accompanying toxicity during systemic application. As for the antibody itself, it is urgent to understand the mechanism and molecular basis of the production of "strong" co-stimulatory T cells and "weak" co-stimulatory T cells of personalized CD137 antibodies, so that it may be suitable for clinical testing and treatment applications with different needs. Based on these new understandings, it is necessary to produce a new generation of anti-human CD137 antibody clones with distinct characteristics. This process usually involves "finely" screening out CD137 antibody clones with differences and distinct characteristics from a large number of CD137 antibodies. The activity characteristics are fundamentally related to the unique sequence of the antibody clone and the individuality of the unique spatial structure it forms.

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

[0007] The purpose of the present invention is to explore unique antibodies with different synergistic activity characteristics through comparative studies of a group of anti-human CD137 antibodies, especially to map the antibody binding to the CD137 extracellular region CRDs (cysteine ​​rich domains), clarify the structural partitioning of antibody binding to CD137-CRD, confirm that the synergistic stimulatory activity of different antibody clones is unrelated to the binding blocking property of the ligand receptor, and the proximity of the binding CD137-CRDI antibody is not the main reason for the high synergistic stimulatory activity, showing that the production of antibodies with different synergistic antibody immune synergistic stimulatory activity antibodies has a high degree of spatial structure dependence. Here, one of the CD137 antibodies (named: BXMD12) with significantly stronger synergistic T cell stimulating activity is provided, which does not bind to the CD137-CRDIV+STP region, that is, it binds to the CD137 extracellular region away from the cell membrane, which is another clear feature of the antibody. Different binding regions potentially affect the functional activity of the antibody, and further obtain the unique variable region sequence of the antibody. As follows, the present invention will systematically introduce the characteristics and basic features of the antibody strain, production and preparation, and recommend its scope of application.

[0008] Unless otherwise defined, all technical terms, notes and other scientific terms or scientific terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art to which the present invention relates. In some cases, for clarity and / or for ease of reference, terms with commonly understood meanings are defined herein, and including such definitions herein need not be interpreted as representing significant differences commonly understood in the art. Many techniques and steps described or quoted herein are well understood by those of ordinary skill in the art and are generally used using conventional methods. Unless otherwise stated, steps involving the use of commercially available test kits and reagents are generally performed according to the experimental schemes and / or parameters defined by the manufacturer.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides an anti-human CD137 antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises the following complementary determining regions:

[0011] CDR-VH1:SYGVH;

[0012] CDR-VH2:VIWSGGSTDYNAAFIS;

[0013] CDR-VH3:NSITTVVADY;

[0014] CDR-VL1: KASQNVGINVD;

[0015] CDR-VL2: SASYRYS;

[0016] CDR-VL3:QQYNSYPLT.

[0017] The present invention provides a novel anti-human CD137 (also known as anti-human 4-1BB) antibody, which can cross-bind to monkey (Rhesus) natural CD137 membrane expression protein, has strong human T cell activation characteristics, and is named clone BXMD12. The clone has a unique variable region sequence and has blocking activity against the natural ligand receptor binding of the molecule, and is a "blocking" antibody. The whole antibody has achieved eukaryotic expression, purification and batch preparation, and can be used for human and monkey 4-1BB detection. It is functionally efficient and synergistically stimulates T cell activation and proliferation, especially for activating the expansion of CD8+T cells. The full-length sequence of the antibody can be further engineered for expression.

[0018] The BXMD12 antibody provided by the present invention has high affinity and provides a new antibody selection for the treatment of cancers related to the CD137 target.

[0019] The term "antibody" refers in particular to a protein comprising at least two heavy chains and two light chains connected by disulfide bonds. The term "antibody" includes naturally occurring antibodies as well as recombinant forms of antibodies, for example, antibodies expressed in prokaryotes, unglycosylated antibodies, humanized antibodies and chimeric antibodies. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy chain constant region comprises three or - in the case of IgM- or IgE-type antibodies - four heavy chain constant domains (CH1, CH2, CH3 and CH4), wherein the first constant domain CH1 is adjacent to the variable region and the second constant domain CH2 can be connected by a hinge region. The light chain constant region consists of only one constant domain. The variable region can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs), wherein each variable region comprises three CDRs and four FRs. However, the term "antibody" according to the present invention also includes antibodies like heavy chain antibodies (ie antibodies consisting of only one or more (especially two) heavy chains) and nanobodies (ie antibodies consisting of only a single monomeric variable domain).

[0020] In a preferred embodiment of the present invention, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L whose sequences are sequentially shown in SEQ ID NOs: 1-4, and / or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are sequentially shown in SEQ ID NOs: 5-8.

[0021] In an alternative embodiment, the antibody further comprises a constant region.

[0022] In an alternative embodiment, the constant region is selected from the constant region of any one of IgM, IgD, IgG, IgA and IgE.

[0023] For example, IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0024] The light chain constant region of the antibody is selected from the constant region of a kappa light chain or a lambda light chain.

[0025] The constant region of the antibody can be derived from any human antibody or human antibody consensus sequence, or can be selected from constant regions of other species. In particular, the heavy chain constant region can be any type, such as γ, δ, α, μ or ε type heavy chain.

[0026] In an optional embodiment, the species of origin of the constant region is mouse; the sequence of the light chain constant region of the constant region is shown in SEQ ID NO.9, and the sequence of the heavy chain constant region of the constant region is shown in SEQ ID NO.10.

[0027] The heavy chain containing the signal peptide of the BXMD12 antibody has the amino acid sequence shown in SEQ ID NO. 11. The light chain containing the signal peptide has the amino acid sequence shown in SEQ ID NO.12.

[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] In a second aspect, the present invention further provides a reagent or kit for detecting CD137 protein, which comprises the above-mentioned anti-human CD137 antibody or a functional fragment thereof.

[0030] The BXMD12 antibody provided by the present invention has a unique variable region sequence, has good binding activity to the CD137 protein expressed on the natural cell membrane of humans and monkeys, and can be used for the detection of human and monkey CD137.

[0031] In an optional embodiment, the antibody or its functional fragment in the above reagent or kit is labeled with a detectable marker.

[0032] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0033] In an alternative embodiment, detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents, and nanoparticle labels.

[0034] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0035] In an optional embodiment, the 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, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59 ... 5, 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), pre-chlorophyll protein (preCP), etc.).

[0036] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0037] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 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.

[0038] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0039] In an optional embodiment, the nanoparticle marker includes but is not limited to nanoparticles and colloids. The nanoparticles are, for example, selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0040] In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0041] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0042] In a third aspect, the present invention further provides an anti-human CD137 antibody or a functional fragment thereof for use in any of the following:

[0043] (1) Expand T cell subsets;

[0044] (2) Preparation of products that promote IFN-γ secretion by T lymphocytes;

[0045] (3) Preparation of products that promote IL-10 secretion by T lymphocytes;

[0046] (4) Preparation of products that promote T lymphocyte activation and / or proliferation;

[0047] (5) Preparation and detection of CD137 products;

[0048] (6) Preparation of combination therapeutic drugs;

[0049] In an optional embodiment, the T cell subset is selected from: at least one of CD4+ T cells and CD8+ T cells;

[0050] In an alternative embodiment, the T cell subset is selected from CD8+ T cells;

[0051] In an optional embodiment, in application (4), an anti-human CD137 antibody or a functional fragment thereof, a co-antigen stimulation signal agent or a co-stimulatory signal agent is used in the preparation of a product for promoting T lymphocyte activation and / or proliferation;

[0052] In an optional embodiment, the antigen stimulation signal reagent is selected from anti-T cell antigen receptor (T cellreceptor, TCR) antibody, anti-CD3γ antibody, anti-CD3δ antibody, anti-CD3ε antibody, anti-CD3ζ antibody; the co-stimulation signal reagent is selected from at least one of anti-CD28 antibody, anti-CD27 antibody, anti-CD134 (OX40), anti-CD26 antibody, anti-CD40 antibody, anti-GITR antibody, anti-CD270 antibody, anti-CD5 antibody, anti-CD154 antibody, anti-CD79a antibody, anti-CD22 antibody, anti-CD66d antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD79b antibody, and anti-ICOS antibody.

[0053] In a fourth aspect, the present invention also provides a use of an anti-human CD137 antibody or a functional fragment thereof in combination with an antigen stimulation signal reagent or a stimulation signal reagent in the preparation of an anti-tumor drug, wherein the antigen stimulation signal reagent is selected from anti-T cell antigen receptor T cell antigen receptor (TCR) antibody, anti-CD3γ antibody, anti-CD3δ antibody, anti-CD3ε antibody, and anti-CD3ζ antibody; the co-stimulatory signal reagent is selected from at least one of anti-CD28 antibody, anti-CD27 antibody, anti-CD134 (OX40), anti-CD26 antibody, anti-CD40 antibody, anti-GITR antibody, anti-CD270 antibody, anti-CD5 antibody, anti-CD154 antibody, anti-CD79a antibody, anti-CD22 antibody, anti-CD66d antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD79b antibody, and anti-ICOS antibody.

[0054] In an alternative embodiment, the tumor is a solid tumor.

[0055] In an optional embodiment, the tumor is one or more of a digestive system tumor, a respiratory system tumor, a reproductive system tumor, a musculoskeletal system tumor, a nervous system tumor, an endocrine system tumor, a circulatory system tumor, a urinary system tumor, and a reproductive system tumor.

[0056] In an alternative embodiment, the tumor is a digestive system tumor.

[0057] In a further optional embodiment, the digestive system tumor is one or more of an esophageal tumor, a gastric tumor, a cardia tumor, an intestinal tumor, a liver tumor, and a gallbladder tumor.

[0058] In an optional embodiment, the tumor is colon cancer, rectal cancer, colorectal cancer, and the colorectal tumor includes one or more of polypoid type, stricture type and ulcerative type.

[0059] In a preferred embodiment of the present invention, the above-mentioned tumor is an epithelial tumor, including but not limited to papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma or squamous cell carcinoma.

[0060] The gastrointestinal cancer is selected from the group consisting of esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, pancreatic cancer, bile duct cancer, small intestine cancer, colorectal cancer and anal cancer.

[0061] In an alternative embodiment, the adenocarcinoma includes but is not limited to lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0062] In an alternative embodiment, the adenoma includes but is not limited to a cystadenoma, a fibroadenoma, a pleomorphic adenoma or a polypoid adenoma.

[0063] In an alternative embodiment, breast cancer includes but is not limited to breast ductal carcinoma, breast epithelial carcinoma or breast lobular carcinoma. Breast cancer, preferably stage II to IV and / or poorly differentiated invasive ductal carcinoma, comedo carcinoma and medullary carcinoma (preferably grade 2).

[0064] Ovarian cancer, serous and mucinous carcinomas (preferably stages Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0065] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0066] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0067] Lung cancer, preferably includes small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma) and large cell lung cancer.

[0068] The squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma (eg esophageal squamous cell carcinoma), lip squamous cell carcinoma, uterine squamous cell carcinoma, vaginal squamous cell carcinoma and skin squamous cell carcinoma.

[0069] Oral squamous cell carcinoma (OSCC) is also called oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include but are not limited to tongue squamous cell carcinoma.

[0070] In other embodiments, the squamous cell carcinoma may also be squamous cell carcinoma formed by squamous metaplasia in the bronchus, bladder, renal pelvis and other parts.

[0071] In a fifth aspect, the present invention also provides an antibody conjugate comprising the above-mentioned anti-human CD137 antibody or a functional fragment thereof.

[0072] The antibody conjugates of the present invention can be detected using various labeling coupling technologies, such as enzyme, fluorescent and isotope labels.

[0073] The molecule coupled to the humanized antibody or antigen-binding fragment thereof can be an imaging agent, or other molecule.

[0074] Imaging agents can be, for example, radioactive elements, enzymes, chemicals and other substances that emit fluorescence when exposed to a certain spectrum of radiation such as ultraviolet light. Many fluorescent materials are known and can be used as imaging agents. These imaging agents include, for example, fluorescein, rhodamine, auramine, Texas red, AMCA blue and fluorescent yellow.

[0075] Detectable imaging agents include, but are not limited to, radioactive labels such as isotopes 3 H. 14 C. 32 P. 35 S. 36 Cl, 51 Cr, 57 Co. 58 Co. 59 Fe, 90 Y. 121 I. 124 I. 125 I. 131 I. 111 In, 211 At 198 Au, 67 Cu, 225 Ac, 213 Bi, 99 Tc and 186 Re, can be linked to the antibody of the present invention or its humanized antibody using conventional chemistry known in the field of antibody imaging. Imaging agents also include fluorescent labels and labels routinely used in the art for MRI-CT imaging. They also include enzyme labels, such as horseradish peroxidase. Labels also include chemical moieties such as biotin, which can be detected by binding to a specific cognate detectable moiety such as labeled avidin.

[0076] In a sixth aspect, the present invention further provides a nucleic acid molecule encoding the above-mentioned anti-human CD137 antibody or a functional fragment thereof.

[0077] The DNA sequence of the heavy chain of the BXMD12 antibody containing the signal peptide is shown in SEQ ID NO.13, and the DNA sequence of the light chain of the BXMD12 antibody containing the signal peptide is shown in SEQ ID NO.14.

[0078] The term "nucleic acid molecule" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and intersugar (backbone) bonds. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and can contain natural bases, including adenine, guanine, cytosine, thymine and uracil. Modified bases may also be contained. Examples of these modified bases include nitrogenous and denitrogenous adenine, guanine, cytosine, thymine and uracil; as well as xanthine and hypoxanthine.

[0079] In a seventh aspect, the present invention also provides a vector comprising the above-mentioned nucleic acid molecule.

[0080] The term "vector" is used herein in its most general sense and includes any intermediate for nucleic acid, which enables the nucleic acid to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed in the cell. The vector comprises a plasmid, a phagemid, a phage or a viral genome. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, typically a circular DNA double strand, which can replicate independently of chromosomal DNA.

[0081] In an eighth aspect, the present invention also provides a recombinant cell comprising the above-mentioned vector.

[0082] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. The term "recombinant cell" according to the present invention comprises prokaryotes (e.g., E. coli) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats or primates. Cells can be derived from multiple tissue types and include primary cells and cell lines. Nucleic acids can be present in host cells in single copy form or in two or more copy forms, and in one embodiment, are expressed in recombinant cells.

[0083] In an alternative embodiment, the recombinant cell is a eukaryotic cell;

[0084] In an alternative embodiment, the recombinant cell is a mammalian cell;

[0085] In an alternative embodiment, the recombinant cell is HEK293.

[0086] In a ninth aspect, the present invention further provides a method for preparing the above-mentioned anti-human CD137 antibody or a functional fragment thereof, comprising: (a) culturing the above-mentioned recombinant cell; (b) recovering the anti-human CD137 antibody or a functional fragment thereof from the culture product of step (a).

[0087] The present invention has the following beneficial effects:

[0088] The anti-human CD137 antibody provided by the present invention has the activity of binding to human soluble CD137 (sCD137) protein, and can bind to CD137 protein expressed by natural cell membrane of human or monkey with high affinity, thereby being used to prepare a reagent or kit for detecting the expression of corresponding human or monkey CD137 protein molecule.

[0089] The inventors mapped the antibody binding to the CD137 extracellular region CRDs (cysteine ​​rich domains), and clarified the structural partitioning of the antibody binding to CD137-CRD. The antibody binds to the CD137 extracellular CRDI+CRDII+CRDIII region. This confirms that the synergistic stimulatory activity of different antibody clones is not related to the binding blocking property of the ligand receptor, and that the proximity of the CD137-CRDI antibody is not the main reason for the high synergistic stimulatory activity, indicating that the production of antibodies with different synergistic antibody immune synergistic stimulatory activity is highly spatially structure-dependent.

[0090] It has blocking activity on the natural ligand receptor binding of CD137 molecules and is a "blocking" antibody. The whole antibody has achieved eukaryotic expression, purification and batch production.

[0091] The anti-human CD137 antibody provided by the present invention is functionally stronger in co-stimulating T cell activation and proliferation, and is particularly suitable for activating the expansion of CD8+T cells. The full-length sequence of the antibody can be further engineered for expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0093] Figure 1The SDS-PAGE electrophoresis identification diagram of two soluble CD137 proteins, (A) CD137-His protein; (B) CD137-Fc protein; His and Fc fusion proteins expressed by CD137 extracellular domain DNA sequence (NP-001552.2), expressed in HEK293 cells, purity 95%;

[0094] Figure 2 The ELISA results of the antibody provided by the present invention and the natural CD137L binding to the human CD137-His protein are shown; specifically, the OD450 value of the ELISA of the antibody provided by the present invention and the natural ligand protein (CD137L-Fc) binding to the CD137-His protein, and the OD value of the control without adding the antibody and CD137L is <0.1;

[0095] Figure 3 The figure is a scatter plot of cells transfected with 293-T cells expressing CD137 by flow cytometry. As shown in the figure, the detection rate of GFP protein transfected by plasmid is about 18%, and the effective transfection cell membrane CD137 expression rate detected by the antibody BXMD12 provided by the present invention is 16.9%, indicating that the detection rate of cells transfected with exogenous human CD137 molecules after staining CD137 protein with the antibody provided by the present invention is nearly 100%;

[0096] Figure 4 The results of the test of binding of the antibody provided by the present invention to mouse CD137 are statistically shown. The ELISA test of binding of the antibody provided by the present invention to mouse CD137 protein shows that the antibody provided by the present invention does not bind to mouse CD137 molecules, while human CD137 ligand (CD137L) obviously binds to mouse CD137-His protein. The results show that the antibody provided by the present invention does not have the activity of cross-binding to mouse CD137 molecules;

[0097] Figure 5 The ELISA detection of the binding of the antibody provided by the present invention to monkey soluble CD137-His and its membrane expressed protein and the flow cytometry detection scatter plot; A, ELISA detection of the binding of the antibody provided by the present invention to monkey soluble CD137 protein shows that the antibody provided by the present invention does not bind to the monkey CD137-His protein, while CD137L obviously binds to the monkey CD137-His protein. The results show that the antibody BXMD12 provided by the present invention does not cross-bind to the soluble monkey CD137 molecule (OD value is less than 0.1); B, the flow cytometry detection scatter plot shows that the antibody provided by the present invention can effectively bind to the membrane expressed monkey CD137;

[0098] Figure 6 Schematic diagram of the extracellular region CRD of human CD137 and its expressed fragment protein; Figure 6The entire extracellular region of hCD137 is shown and covers different CD137-CRD fragments; hE, hE3, and hE4 represent different hCD137 expression fragments; the STP region (serine, threonine, and proline-rich region) is located at amino acids 160-186;

[0099] Figure 7 This is the result of the ELISA test of the blocking effect of antibodies on ligands. Figure 7 The horizontal axis is the unit of antibody serial dilution. The OD value reflects the degree of antibody interference with CD137-CD137L. The lower the OD value, the more interference with ligand-receptor binding. The results show that the BXMD12 antibody can block CD137-CD137L binding. BXD12 is BXMD12, and the other antibodies are BXF12, BXA10, BXE8, BXG12, BXD10, BXD3 and BXC11.

[0100] Figure 8 The binding and dissociation curves of CD137-His fusion protein at different dilution concentration gradients (0-0.08 nM) were measured by BLI, and the affinity of BXMD12 antibody was analyzed by biolayer interferometry (BLI);

[0101] Fig. 9 This is a diagram showing the experimental results of using BXMD12 antibody for western blotting; Fig. 9 Western Blot analysis of BXMD12 antibody in a non-reduced (without 2-mercaptoethanol) state showed that it had binding activity to CD137-His protein (with Fig. 9 A), does not bind to CD137-His fusion protein in the reduced state (with 2-mercaptoethanol) Fig. 9 B);

[0102] Fig.10 The experimental results of antibody-co-stimulation amplification of lymphocytes; 5×10 5 PBMCs (cell concentration 1×10 6 / ml) for 12 hours, then add anti-CD28, BXD12, BXE8, BXD3 2μg / ml monoclonal antibodies, and add IL-2 50IU / ml to each well. Continue to culture until the 10th day, and count the total number of cells by placenta blue staining. Asterisks represent statistical differences in the number of cells between the marked groups;

[0103] Fig.11The figure shows the experimental results of BXMD12 synergizing with OKT3 to stimulate lymphocytes to produce cytokines; after 72 hours of OKT3+CD137 stimulation of PBMC, the culture supernatant was collected to determine the concentrations of IFNγ (A) and IL-10 (B), and the concentrations of the two cytokines in the supernatant were detected by ELISA. The antibodies provided by the present invention significantly assisted OKT3 in activating lymphocytes and promoting the production of IFNγ and IL-10. The concentrations of IFNγ and IL-10 in the control culture with OKT3 alone were less than 500pg / ml and 5pg / ml, respectively. BXMD12 showed a higher ability to promote the production of IFNγ and IL-10. DETAILED DESCRIPTION

[0104] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0105] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); 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.

[0106] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0107] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0108] Example 1 Preparation process of the antibody of the present invention

[0109] 1. Animal Immunization

[0110] The immunogen used was a soluble eukaryotic expressed human CD137 tag (Fc) protein (Sino-Bio), which is a human IgG1-Fc fusion protein. CD137 contains extracellular region amino acids (Met-Gln186) and is expressed in HEK293 cells. After purification by Protein A affinity chromatography, the protein purity was >90%. Purebred BALB / c mice aged 4-6 weeks were selected, and the primary immunization began 2 months before fusion. To increase the immunogenicity of the soluble protein, the adjuvants used in this experiment included Freund's complete adjuvant and Freund's incomplete adjuvant (Sigma).

[0111] The immunization procedure is as follows: on day 0, primary immunization, 0.25 mL of complete adjuvant emulsified antigen, 10-100 μg per mouse, intraperitoneal injection, for a total of 5 mice; on day 14, secondary immunization, 0.25 mL of incomplete adjuvant emulsified antigen, 10-50 μg per mouse, intraperitoneal injection; on day 24, blood was collected from the tail vein, incubated at 37°C for 1 hour, and then centrifuged at 4°C for 2 hours, and the supernatant was collected for the initial serum titer detection; on day 35, third immunization, 0.25 mL of incomplete adjuvant emulsified antigen, 10-50 μg per mouse, intraperitoneal injection; on day 45, blood was collected from the tail vein, incubated at 37°C for 1 hour, and then centrifuged at 4°C for 2 hours, and the supernatant was collected, and the serum titer was detected by ELISA. The titers of the five immunized mice all reached or exceeded 1:512000. Two mice with titers exceeding 1:512000 were selected for final immunization on day 56. The antigen was dissolved in PBS, 0.25 mL per mouse, 10-50 μg, and injected into the tail vein and peritoneum. One week later (day 60), subsequent cell fusion was performed.

[0112] 2. Cell Fusion

[0113] (1) Preparation of feeder cells: Mouse peritoneal macrophages were selected. Mice of the same strain as the immunized mice, BALB / c mice aged 6 to 10 weeks, were killed by pulling the neck, and immersed in 75% alcohol for 3 to 5 minutes. The skin was cut open with sterile scissors to expose the peritoneum. 5 to 6 mL of pre-cooled culture medium was injected with a sterile syringe, and the cells were rinsed repeatedly. The rinsing fluid was aspirated and placed in a 10 mL centrifuge tube. The cells were separated at 1200 rpm / min for 5 to 6 minutes, and suspended with culture medium containing 20% ​​calf serum (NCS) or fetal calf serum (FCS). The cell number was adjusted to 1×10 5 / mL, add to 96-well plate, 100μL / well, and culture in a 37℃ CO2 incubator.

[0114] (2) Preparation of immune spleen cells: Three days after the last booster immunization, the mice were killed by cervical dislocation, and the spleens were removed aseptically and washed once with culture medium.

[0115] The spleen was ground and passed through a stainless steel mesh. After centrifugation, the cells were washed twice with culture medium, counted, and 108 The spleen lymphocyte suspension was prepared for later use.

[0116] (3) Preparation of myeloma cells: Centrifuge the logarithmically growing myeloma cells, wash twice with serum-free culture medium, count, and take 1×10 7 Cells to spare.

[0117] (4) Integration:

[0118] ① Mix myeloma cells and spleen cells at a ratio of 1:10 or 1:5, wash once with serum-free incomplete culture medium in a 50mL centrifuge tube, and centrifuge at 1200rpm / min for 8min; discard the supernatant and use a pipette to suck out the remaining liquid to avoid affecting the concentration of polyethylene glycol (PEG). Gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly.

[0119] ② Add 1 mL of 45% PEG (molecular weight 4000) solution pre-warmed at 37°C within 90 seconds, shaking gently while adding. Incubate in a 37°C water bath for 90 seconds.

[0120] ③ Add incomplete culture medium preheated at 37°C to terminate the action of PEG, and add 1mL, 2mL, 3mL, 4mL, 5mL and 6mL every 2min.

[0121] ④ Centrifuge at 800 rpm / min for 6 min.

[0122] ⑤ Remove the supernatant and resuspend in HAT selection medium containing 20% ​​calf serum.

[0123] ⑥ Add the above cells to a 96-well plate with a feeder cell layer, add 100 μL to each well, and inoculate 10 96-well plates for one immune spleen, for a total of 20 plates.

[0124] ⑦ Place the culture plate in a 37°C, 5% CO2 incubator.

[0125] 3. Hybridoma screening and secreted antibody detection

[0126] (1) HAT selection of hybridomas. After being treated with PEG, spleen cells and myeloma cells form a mixture of various cells. After being maintained in HAT selection culture medium for 7 to 10 days, HT culture medium is used, maintained for 2 weeks, and then switched to general culture medium. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom of the well, specific antibodies are detected to screen out the required hybridoma cell lines. During the selection culture period, half of the culture medium is generally replaced every 2 to 3 days.

[0127] (2) Antibody detection was performed using an enzyme-linked immunosorbent assay (ELISA). The hybridoma culture supernatant was coated with 2 μg / mL concentration of human CD137-Fc and paired CD137-his (Met-Gln186, His fusion protein, Sino-Qiao) for double screening and detection. The color was developed using enzyme-labeled anti-mouse secondary antibodies and substrates, and the OD value was detected. In this experiment, 40 positive wells (CD137-Fc negative and CD137-his positive) were initially screened and further cloned.

[0128] 4. Hybridoma cloning

[0129] The positive hybrid clones detected were cloned to avoid being inhibited by cells that did not secrete antibodies, resulting in loss of antibody-secreting cells. The cloned hybridoma cells also needed to be re-cloned regularly to prevent hybridoma cell mutation or chromosome loss, thereby losing the ability to produce antibodies, and the hybridoma was stable after freeze-thaw experiments. In this experiment, the limiting dilution method was used for cloning, and the antibodies screened in the experiment were subcloned more than 4 times.

[0130] (1) Prepare the feeder cell layer (for cell fusion) one day before cloning.

[0131] (2) Gently blow up the hybridoma cells to be cloned from the culture well and count them.

[0132] (3) Adjust the cell population to 3–10 cells / mL.

[0133] (4) Take the cell culture plate with the feeder cell layer prepared the day before, add 100 μL of diluted cells to each well, and incubate in a 37°C, 5% CO2 incubator.

[0134] (5) Change the medium on the 7th day, and then every 2 to 3 days.

[0135] (6) Cell clone formation can be seen in 8 to 9 days, and antibody activity can be detected by ELISA in time.

[0136] (7) Transfer the cells from the positive wells to a 24-well plate for expansion.

[0137] (8) Detect antibodies, expand the culture, and repeat the process of re-cloning if necessary, thereby obtaining the clone BXMD12 in this example.

[0138] 5. Hybridoma Cell Cryopreservation and Thawing

[0139] (1) The ability to secrete antibodies may be lost during cell culture. Hybridoma cryopreservation can verify the stability of hybridomas and preserve hybridoma strains for a long time. Each ampoule of hybridoma cell cryopreservation contains 1×10 7The above, cell freezing solution: 50% calf serum; 40% incomplete culture medium; 10% DMSO (dimethyl sulfoxide). When freezing, the temperature can be immediately lowered from room temperature to 0℃ and placed in a -70℃ ultra-low temperature refrigerator, and then transferred to liquid nitrogen the next day. It can also be frozen using a cell freezing device. Cryopreserved cells should be revived regularly to check the activity of the cells and the stability of secreted antibodies. Cells can be stored in liquid nitrogen for several years or longer.

[0140] (2) Cell recovery: Carefully remove the glass ampoule from the liquid nitrogen and place it in a 37°C water bath to thaw the frozen cells within 1 minute. Wash the cells twice with complete culture medium and then transfer them to a culture bottle of feeder layer cells prepared the day before and culture them in an incubator at 37°C and 5% CO2. When the cells form colonies, the supernatant antibody activity is detected by ELISA. The present invention relates to hybridomas that undergo repeated freezing and thawing processes and have stable antibody secretion activity and yield.

[0141] 6. Full-length nucleic acid sequencing of monoclonal antibodies

[0142] According to the technical manual of Trizol® reagent (AMBION, CAT, No. 15596-026), total RNA was isolated from hybridoma BXMD12 cells. According to the technical manual of PrimeSeCpTTM first-strand cDNA synthesis kit (Takara, CAT, No. 610A), 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, antibody fragments of VH, VL, CH and CL were amplified. The amplified antibody fragments were cloned into standard cloning vectors and sequenced. The sequence analysis tools of variable regions were: (i) NCBI NucleotideBLAST; (ii) IMGT / V Quest program; (iii) NCBI Ig BLAST. Colony PCR was used to screen for inserts with the correct size. No less than five fragments with inserts of the correct size were sequenced. The sequences of different clones were compared to obtain consistent sequencing results (the heavy chain of BXMD12 antibody containing the signal peptide has the sequence of SEQ ID The amino acid sequence of the light chain containing the signal peptide is shown in SEQ ID NO.11. The light chain containing the signal peptide has the amino acid sequence shown in SEQ ID NO.12. The DNA sequence of the heavy chain containing the signal peptide of the BXMD12 antibody is shown in SEQ ID NO.13, and the DNA sequence of the light chain containing the signal peptide of the BXMD12 antibody is shown in SEQ ID NO.14).

[0143] The biological characteristics of the antibodies are detected in Examples 2-8 as follows.

[0144] Example 2 BXMD12 antibody binds to human soluble CD137 (sCD137) protein

[0145] The BXMD12 antibody provided by the present invention confirms the binding to the soluble form of the fusion protein of the extracellular region of human CD137.

[0146] ① The test materials used two CD137 fusion proteins: human CD137-Fc protein and CD137-His fusion protein. The two CD137 fusion proteins were expressed in eukaryotic expression system 293 cells and purified by affinity chromatography to obtain purified proteins with a purity greater than 90% (see attached Figure 1 : Electrophoresis identification of human CD137 extracellular region CD137-Fc and CD137-His fusion expression proteins), both of which exist in dimer and monomer forms respectively;

[0147] ② The specific binding of the antibody to the above two sCD137 proteins was verified as follows: In a 96-well ELISA test plate, 1 μg / ml human CD137-His protein was coated, 100 μl per well, overnight at 4 degrees, washed three times with 0.05% Tween-20 washing solution, blocked with 5% milk powder at 4 degrees overnight, and washed three times again. Add the purified antibody or human CD137 ligand (Arg71-Glu254, CD137L-Fc) 1 μg / ml, 100 μl per well, 37 degrees for 2 hours. Wash five times with 0.05% Tween-20 washing solution, add HRP-labeled goat anti-human IgG (1:5000) 100 μl / well, 37 degrees for 1 hour. Wash five times with 0.05% Tween-20, add TMB colorimetric solution to react in the dark for 5 minutes, terminate the reaction with sulfuric acid stop solution, and detect the OD value at 450nm with an enzyme marker.

[0148] Attached Figure 2 The reactivity of the BXMD12 antibody and CD137L provided by the present invention in binding to human CD137-His protein was determined by ELISA, and the results showed that the binding force of the antibody was significantly higher than that of natural CD137L on CD137-His protein.

[0149] Example 3 BXMD12 antibody binds to native cell membrane expressed CD137 protein

[0150] The pCMV3-CD137-GFPSpark expression plasmid was used to transfect the cultured 293T cells using Invitrogen Lipofectamine 2000 reagent. After 48 hours, the cells were collected and 1×10 cells were transfected with the expression plasmid. 6Place in flow tube for immunofluorescence staining: first add 1 μg mouse anti-human CD137 monoclonal antibody, add 1 μg mouse IgG1 isotype as control in the control tube, stain for 25 minutes at room temperature in the dark, wash twice with 2 ml 1% FBS PBS buffer, then add APC-labeled rabbit anti-mouse IgG secondary antibody, stain for 25 minutes at room temperature in the dark, wash twice, resuspend the cells in 200 μl buffer, and use flow cytometry to detect the expression of CD137 molecules on the membrane of transfected 293-T cells.

[0151] The results show (see Appendix Figure 3 ), antibody and 293T-CD137 transfected cells were combined with flow cytometry detection, wherein the control antibody used was anti-mIgG1 isotype instead of the mouse anti-human CD137 monoclonal antibody of the present invention, and the results showed that the detection rate of plasmid transfection GFP protein was about 18%, and the expression rate of effective transfection cell membrane CD137 detected by antibody BXMD12 was 16.9%, indicating that cells transfected with exogenous human CD137 molecules, stained with the BXMD12 antibody provided by the present invention for effective expression of CD137 protein, had a detection rate of nearly 100%, and the antibody had the activity of binding to the natural structure cell membrane to express CD137 protein.

[0152] Example 4 BXMD12 antibody does not cross-bind to mouse CD137 protein

[0153] Mouse CD137-His protein also uses eukaryotic expression protein (recombinant HEK 293 expresses ECD-His-Tag fusion protein, CD137 is Met 1-Leu187). The expressed mouse CD137 fusion protein is used for ELISA test, diluted with coating buffer to a concentration of 1μg / ml, 100μl / well added to 96-well plate, coated overnight at 4 degrees, washed three times with washing buffer, added 200μl 5% milk powder blocking solution at 4 degrees overnight, washed three times again; added 100μl diluted antibody BXMD12 100μl (1μg / ml), 37 degrees for 2h. Then washed five times, added enzyme-labeled rat anti-mouse IgG (1:5000) 100μl / well, and added color development solution A+B after the reaction.

[0154] The results showed that antibody BXMD12 did not bind to the mouse CD137 molecule, but bound to the human CD137 ligand, while CD137L clearly bound to the mouse CD137-His protein, see Appendix Figure 4 ELISA was used to detect the binding of antibody BXMD12 to mouse CD137 protein. The results showed that the BXMD12 antibody provided by the present invention did not have the activity of cross-binding to mouse CD137 molecules.

[0155] Example 5 BXMD12 antibody cross-binds to monkey (Rhesus) natural CD137 membrane expression protein

[0156] The monkey CD137-His protein adopts eukaryotic expression protein (recombinant HEK 293 expresses ECD-His-Tag fusion protein, CD137 is Met 1-Leu186). In addition, the pCMV3-RhesusCD137-His plasmid is used to transfect 293F cells. The monkey CD137-His protein is used for ELISA test, diluted with coating buffer to a concentration of 1μg / ml, 100μl / well is added to a 96-well plate, coated overnight at 4 degrees, washed three times with washing solution, 200μl 5% milk powder blocking solution is added at 4 degrees overnight, washed three times again, and 100μl of 1μg / ml diluted by blocking solution is added. 100μl (1μg / ml) of the BXMD12 antibody provided by the present invention is added, and 37 degrees for 2h. Then wash five times, add 100μl / well of enzyme-labeled rat anti-monkey IgG (1:5000), and add color development solution A+B after the effect.

[0157] The results showed that the BXMD12 antibody provided by the present invention did not bind to the soluble monkey CD137-His protein, while the human CD137 ligand (hCD137L) clearly bound to the monkey CD137-His protein. Figure 5 AELISA was used to detect the binding between the BXMD12 antibody provided by the present invention and the monkey soluble CD137-His protein. The results showed that the BXMD12 antibody provided by the present invention had the activity of BXMD12 not cross-binding to the monkey soluble CD137 protein, and correspondingly, the human CD137 ligand had cross-binding activity.

[0158] At the same time, the plasmid-transfected cells were collected for flow cytometry analysis of the Rhesus CD137 protein on the cell membrane, and the APC-labeled rabbit anti-mouse secondary antibody was used to detect the mouse anti-human CD137 monoclonal antibody, which was combined with the transfected 293F to express CD137. The plasmid-transfected cells were used for flow cytometric analysis of the expression of monkey CD137 protein on the cell membrane, and the APC-labeled anti-mouse secondary antibody was used to detect the anti-human CD137 monoclonal antibody, which was combined with the transfected 293F to express CD137 protein.

[0159] The results showed that BXMD12 has the activity of cross-binding to the natural state of monkey CD137 expression protein on the cell membrane, and the detection rate is 1.8% (the transfection rate of plasmid transfection of 293F cells is about 1.8%), indicating that the transfected monkey CD137 expression cells have a 100% detection rate, while the non-binding antibody control staining has only 0.1% positive cells. See attached Figure 5B: Flow cytometry was used to detect the binding of the BXMD12 antibody BXD12 provided by the present invention to the monkey CD137-293F cell membrane expressed protein. The cross-binding reactivity of BXMD12 with human soluble and cell membrane expressed CD137, as well as with monkey CD137, is summarized in Table 1.

[0160] Table 1. Binding of the present antibody to different forms of human and monkey CD137 protein

[0161]

[0162] Note: hCD137-His, human CD137-His; rCD137, monkey CD137 protein; ++strong binding; NO, no binding, YES, binding

[0163] Example 6 Localization of BXMD12 antibody binding to extracellular cysteine-rich regions (CRDs) of human CD137

[0164] ① Expression of human CD137 extracellular cysteine-rich region (CRDs) fragment

[0165] The human CD137 extracellular region includes 186 amino acids and has four cysteine-rich regions (CRDI, CRDII, CRDIII and CRDIV). A disulfide bond is formed between two cysteines in CRD to produce a local spatial structure, in which natural CD137L mainly binds to CD317-CRDIII. Based on the expression of the extracellular region protein hE, we further expressed the hE3 and hE4 fragments, expressing the cut CD137 extracellular region CRD1+CRDII and CRDI+CRDII+CRDIII, respectively, including the extracellular region N-terminal guide sequence, CRD1+CRDII including 1-86 amino acids, CRDI+CRDII+CRDIII including 1-118 amino acids, see the attached Figure 6 Schematic diagram of the human CD137 extracellular region CRD and its expression fragment protein. The protein was transfected into COS-7 cells in a eukaryotic expression system, and the culture supernatant of the transfected cells was identified to be effectively expressed (same as the above CD137-Fc extracellular region protein expression and identification), and the expression product was used for ELISA detection to analyze the antibody binding to the CRDs region.

[0166] ② The BXMD12 antibody provided by the present invention binds and is localized in the CD137 extracellular domain CRDI+CRDII+CRDⅢ.

[0167] The direct ELISA detection OD value of antibody BXMD12 binding to the full-length fragment of the extracellular region (hE) was >2.0, and the ELISA detection OD values ​​were 0.066 and 0.27, respectively. The mapping analysis of the binding area showed that the BXMD12 antibody BXMD12 provided by the present invention binds to the CD137 extracellular CRDI+CRDII+CRDIII region (Table 2).

[0168] Table 2. OD values ​​and binding region locations of BXMD12 and human CD137-CRDs fragments measured by ELISA

[0169]

[0170] Example 7 Blocking activity of BXMD12 antibody on human CD137-CD137L binding

[0171] The antibody blocking CD137-CD137L binding assay uses two assay methods:

[0172] ① Based on biofilm layer interferometry (BLI) technology

[0173] Using ForteBio Octet biomolecular interaction technology, the AMC sensor was pre-wetted in the balancing solution for 10 minutes, the balancing solution was added to the first column of the light-proof 96-well plate, and the 8 antibodies were added to the second column of the light-proof 96-well plate. The third column was the balancing solution, the fourth column was the antigen hCD137-his, and the fifth column was hCD137L-hFc. The antigen and ligand were diluted to 5μg / ml, and a volume of 200μl was added to each well. The fortebio octet96 instrument was used for detection. The sensor was balanced in the first column for 60s to obtain the basic equilibrium curve, the second column was solidified with antibodies for 300s, the third column was re-balanced for 60s, the fourth column was bound to antigen for 180s, and the fifth column was bound to ligand for 180s. The method is the same as above, the ligand is fixed to bind to the antigen, and whether the antibody can bind to the antigen is detected. The sensor was balanced for 60s, the ligand was solidified for 300s, re-balanced for 60s, the antigen was bound for 180s, and the antibody was bound for 180s.

[0174] ②ELISA detection of antibody blocking test of ligand. Coat CD137-his protein (2μg / ml) with 100μL per well, overnight at 4 degrees, wash three times. Add 5% milk powder blocking solution to block 200μl / well, 37 degrees for 2h, wash 3 times. Add 100μL premixed solution (CD137 ligand / CD137 antibody: 1 / 0, 1 / 1, 1 / 2.5, 1 / 12.5) to each well, 37 degrees for 2h, wash five times. Add 100μL goat anti-human horseradish enzyme marker (1:6000) to each well, react at 37 degrees for 2h, wash five times. Mix the color development solution A+B in equal proportions, add 100μl to each well, color for 10min, add 50μl stop solution, and measure the absorbance with an enzyme marker.

[0175] Given that natural CD137L mainly binds to CD317-CRDIII, and CRDIV+STP also involves the formation of a CD137L-CD137 protein complex, ForteBio Octet biomolecular interaction technology and ELISA detection methods both show that the BXMD12 antibody or BXMD12 provided by the present invention clearly has the characteristics of blocking CD137L-CD137 binding, and the blocking activity is compared with other antibodies as shown in the attached Figure 7 It has a certain blocking activity, which is consistent with the expected result of BXMD12 binding to the CD137 extracellular region and mapping the CRDI+CRDII+CRDIII region.

[0176] Example 8 Types and affinities of the BXMD12 antibodies provided by the present invention

[0177] ① Methods for determining antibody affinity and determining antibody categories

[0178] Using ForteBio Octet biomolecular interaction technology, the AMC sensor was pre-wetted in PBS equilibration solution with 0.1% BSA and 0.02% TWEEN20 for 10 minutes, and then the mouse anti-human CD137 monoclonal antibody was diluted to 20μg / ml with the equilibration solution and added to the second column of a light-proof 96-well plate, 200μl / well. The CD137-His protein was diluted from 500nM to 31.3nM and added to the fourth column of a light-proof 96-well plate, 200ul / well. The equilibration solution was added to the first and third columns, 200μl / well. Using the Fortebio octet96 instrument for detection, the sensor was equilibrated in the first column for 120s to obtain the basic equilibrium curve, and then the antibody was immobilized in the second column for 300s. Re-equilibration was performed in the third column for 120s, and the antigen was bound in the fourth column for 180s to obtain the binding curve, and the dissociation curve was obtained by returning to the third column for 300s. The curve was fitted and analyzed using Fortebio Octet96 analysis software to obtain affinity values. Antibody subclass determination was performed using the classic ELISA method, and the kit was a product of Bo'ao Biology Co., Ltd (Cat no. BF06004), which can classify heavy chains IgA, IgM, IgG (IgG1, IgG2, IgG3 and IgG4), and identify light chain typing of κ light chain or λ light chain.

[0179] ②BXMD12 antibody high affinity and subclass

[0180] Biomembrane optical interference technology (ForteBio Octet) was used to determine the binding constant and dissociation constant of the antigen-antibody interaction and the antibody affinity. The binding and dissociation curves of the BXMD12 antibody provided by the present invention combined with CD137-His protein at different dilution concentration gradients (500nM-31nM) are shown in the attached Figure 8 The affinity KD value of the BXMD12 antibody provided by the present invention is calculated to be 6.9×10 -9 (3.9×10 -8 ), and the various parameter values ​​in the affinity determination are shown in Table 3. The BXMD12 antibody provided by the present invention was determined to be IgG1 by ELISA, and the light chain was a κ light chain.

[0181] Table 3. BXMD12 affinity parameter values ​​analyzed by biolayer interferometry (BLI)

[0182]

[0183] Note: The antigen bound by the antibody in the assay is human CD137-His fusion protein

[0184] Experimental Example 1 This experimental example is used to produce and purify antibodies.

[0185] The expression and affinity chromatography purification strategies of the eukaryotic system are adopted. The production and purification of the antibody can be carried out according to the antibody sequence provided. The full-length light chain DNA (shown in SEQ ID NO.14) and the full-length heavy chain DNA (shown in SEQ ID NO.13) are cloned into pcDNA3.1 or pcDNA3.4 respectively, transformed into competent Escherichia coli, amplified plasmids, and the sequence correctness is verified by restriction digestion and sequencing, and then quantified by spectrophotometer. The following plasmid dosage is the DNA mass (µg) after the light chain vector and the heavy chain expression vector are mixed in a ratio of 2:1, which is used for transient transfection expression in mammalian cells ExpiCHO-S™ (ThemoFisher).

[0186] Day 1: Resuscitate and expand freshly thawed ExpiCHO-S™ cells to a cell density of approximately 4-6×10 6 viable cells / mL.

[0187] Day 2: Split ExpiCHO-S™ culture and adjust cell density to 3-4×10 6 viable cells / mL, and cells were grown overnight.

[0188] Day 3: Determine viable cell density and percent viability. The cell density should reach approximately 7-10 × 10 6 The viability should be 95-99% before transfection can proceed.

[0189] Dilute the cells to a final density of 6 × 10 cells / mL using fresh ExpiCHO™ Expression Medium pre-warmed to 37°C. 6 Gently shake the culture flask to mix the cells.

[0190] Day 4: Transfection. Use cold reagents (4°C), ExpiFectamine™ CHO and cold plasmid solution to mix thoroughly to prepare ExpiFectamine™ CHO / plasmid. The transfection culture volume should be 0.5-1.0µg DNA / mL of total light chain variable region plasmid (for example, a 1L culture flask system, the culture volume is 280mL, the volume of plasmid DNA is 8mL, and the total amount of plasmid needs to be 4.0-8.0 µg).

[0191] Follow the steps below:

[0192] (i) Gently invert the bottle of ExpiFectamine™ CHO reagent 4-5 times to mix thoroughly. Dilute the plasmid DNA with cold OptiPRO™ medium (ThemoFisher) and mix with the DNA by shaking or inverting the tube at room temperature to make the total plasmid DNA 0.5-1.0 µg / mL culture volume and mix thoroughly.

[0193] (ii) Dilute 640 µL of ExpiFectamine™ CHO Reagent (ThemoFisher) with 7.4 mL of OptiPRO™ Medium and mix by shaking or inverting the tube.

[0194] (iii) Add the diluted ExpiFectamine™ CHO Reagent to the diluted DNA and mix by shaking or inverting the tube.

[0195] (iv) Incubate the ExpiFectamine™ CHO / plasmid DNA complex at room temperature for 1-5 minutes (immediately add the ExpiFectamine™ CHO / DNA complex to the cells, mix for no longer than 5 minutes, and then slowly transfer the solution to the culture flask in step 4, gently shaking the culture flask during the addition process. Incubate the cells on an orbital shaker at 120 ± 5 rpm in a humidified atmosphere of 8% CO2 at 37°C in an incubator (25-mm orbit).

[0196] Day 5: The day after transfection (Day 1, 18-22 hours after transfection), add 1.2 mL of ExpiFectamine™ CHO Enhancer and 48 mL of ExpiCHO™ Supplement according to the selected experimental protocol: Transfer the culture flask to a 32°C incubator with 5% CO2 in humidified air and culture with shaking.

[0197] Day 12-14: Collect culture supernatant. The best time to collect antibody protein is usually 8-10 days after transfection.

[0198] Antibody purification: centrifuge at 4000g for 20min to remove cell particles or debris, filter with 0.22μm or 0.45μm membrane filter, measure the pH of the supernatant, adjust the pH to 7.0-7.4 with 0.2M Na2HPO4 or 0.2M NaH2PO4 (1M NaOH or 1M HCl), fully equilibrate the protein A purification column (Sigma) with buffer (PBS, pH7-7.4), load the cell culture supernatant onto the fully equilibrated column at 4°C using an AKTA system or peristaltic pump, equilibrate the column with 3 CV-5 CV binding buffer; elute the antibody / protein from the column with elution buffer using an AKTA system. Collect the eluate at 1-3 mL / min according to UV280 absorbance, desalt with a desalting column, quantify with a spectrophotometer, aliquot, and store at -80 degrees or freeze-dried.

[0199] Experimental Example 2 The antibody provided by the present invention is used for Western Blot detection of CD137 analysis

[0200] 1. Sample preparation

[0201] Mix 20 μl of 2 μg CD137-His protein with 5 μl of 5* loading buffer containing 2-mercaptoethanol, boil in boiling water for 5 min, cool and centrifuge for later use. Mix 20 μl of 2 μg CD137-His protein with 5 μl of 5* loading buffer without 2-mercaptoethanol and centrifuge for later use.

[0202] 2. Electrophoresis

[0203] Perform SDS-PAGE and load the samples on reducing / non-reducing PAGE gels, respectively. The loading order from left to right is reducing / non-reducing CD137-His protein and protein marker. The voltage is 140V and the electrophoresis is performed in an ice bath for 60 minutes.

[0204] 3. Transfer

[0205] Take nitrocellulose membranes of appropriate size for transfer, voltage 70V, ice bath electrotransfer for 70 minutes.

[0206] 4. Immunological testing

[0207] Ponceau staining for 5 minutes, rinse with water until the bands are clear. Then block with 5% skim milk / PBST at room temperature for 1 hour. Dissolve BXMD12 antibody in 2ml 5% skim milk / PBST blocking solution. Put the membrane in a hybridization bag and incubate with the prepared BXMD12 antibody at 4°C overnight. The next day, rinse the membrane with PBS + 0.05% Tween 20 for 3 times, 10 minutes each time. Then incubate with anti-mouse IgG antibody (1:5000 dilution) at room temperature for 1 hour. Rinse the membrane with PBS + 0.05% Tween 20 for 3 times, 10 minutes each time. In the dark room, mix 0.5ml of each of the A and B solutions in the ECL kit and drop them evenly on two nitrocellulose membranes, absorb the surface liquid, and use a chemiluminescence imager to take pictures of the results.

[0208] The results of immunoblotting and Western Blot analysis of the antibody binding to CD137-His protein showed that the BXMD12 antibody had binding activity to CD137-His protein in a non-reduced (without 2-mercaptoethanol) state (with Fig. 9 A), does not bind to CD137-His (attached) in the reduced state (with 2-mercaptoethanol) Fig. 9 B), indicating that the antibody provided by the present invention binds to the natural structure CD137, but does not bind to the non-natural structure CD137 after the disulfide bond is destroyed. The antibody provided by the present invention can be used to identify the existence of the CD137 protein spatial structure related thereto.

[0209] Experimental Example 3 Antibodies cooperate with antigen stimulation signals to expand CD8+ T cells

[0210] T cells need to be activated under the action of antigen TCR / CD3 stimulation signals and co-stimulatory signals. If there is only an antigen stimulation signal, the T cells lack co-stimulatory signals and go to exhaustion. For this reason, the above two signals are required for T cell activation and expansion. The most important T cell co-stimulatory signals include CD28 and CD137, but the co-stimulatory effects of the two are different. CD28 mainly co-stimulates CD4+T cells, while CD137 co-stimulatory signals tend to activate CD8+T cells, the latter cell subset is the core effector cell of anti-tumor. The antibodies provided by the present invention have been shown to effectively activate T cells, have characteristics that are biased towards the CD8+T subset, can be used to activate and amplify CD8+T cells in vitro, and also show the potential to activate CD8+T cells in vivo to exert anti-tumor activity. The antibodies provided by the present invention have applications in activating CD8+T cell amplification, as shown in the following method:

[0211] (1) Density gradient centrifugation method for isolating PBMCs

[0212] Peripheral Blood Mononuclear Cell (PBMC), mainly including lymphocytes and monocytes, refers to a cell population with a single nucleus in peripheral blood. Human PBMC is obtained by Ficoll density gradient centrifugation. Ficoll is a polymer of sucrose that maintains normal physiological osmotic pressure, does not destroy the biological membrane, and maintains cell activity. The red blood cells and granulocytes in the blood have a high specific gravity and sink to the bottom of the tube after centrifugation. The specific gravity of lymphocytes and monocytes is less than or equal to the specific gravity of the layering fluid. After centrifugation, they are located at the top of the cell components of the layering fluid, and above them is the invisible component plasma. Therefore, cells on the surface of the layering fluid can be absorbed to separate PBMC from peripheral blood. The specific operation process is as follows:

[0213] ① Add 4 ml of Ficoll, a lymphocyte separation medium, into a 10 ml centrifuge tube.

[0214] ② Take 2 ml of anticoagulated peripheral blood (whole blood) and mix thoroughly with sterile PBS at a ratio of 1:1. Use a pipette to slowly superimpose it on the layered liquid surface along the tube wall. Be sure to move gently and pay attention to maintaining a clear interface. The final volume of lymphocyte separation solution and diluted blood is in a ratio of 1:1.

[0215] ③ Horizontal centrifugation at 400 g for 20 minutes. The specific speed should be adjusted according to the requirements of the lymphocyte separation medium Ficoll or the centrifuge rotor used, usually 1800-2000rpm.

[0216] ④ After centrifugation, the tube is divided into three layers: the upper layer is the plasma liquid layer, the middle layer is Ficoll, and the lower layer is the visible cell layer such as red blood cells and granulocytes. At the interface between the upper and middle layers, there is a thin layer of white cells mainly composed of mononuclear cells, that is, mononuclear cells PBMC.

[0217] ⑤ Remove part of the upper layer of liquid, leaving about 1 mL, and gently aspirate the mononuclear cells with a pipette. Place them in another centrifuge tube, add 5-7 ml of PBS buffer containing 1% fetal bovine serum (FBS), centrifuge at 300 g for 10 minutes, and wash the cells twice.

[0218] ⑥After the second centrifugation, discard the supernatant, add RPMI1640 containing 10% FBS, calculate cell viability and cell count by trypan blue staining, and adjust and resuspend the cells in RPMI1640 containing 10% FBS to a concentration of 1x10 6 / ml, for future use.

[0219] (2) PBMC stimulation culture method and detection

[0220] ① Remove the above concentration 1x10 6 / ml of cell suspension was placed in a 24-well plate, 1ml per well, for a total of 5 wells, and placed in a carbon dioxide cell culture incubator with a certain humidity and incubated at 37°C.

[0221] ② Prepare anti-CD3 antibody as antigen stimulation signal reagent, select clone OKT3, and prepare it with 10% 1640 at a concentration of 0.2 ng / ml; prepare BXMD12 antibody of costimulatory signal with 10% 1640 at 1 mg / ml for standby use; similarly prepare another reference costimulatory signal CD28 antibody with 10% 1640.

[0222] ③ Set up OKT3, OKT3+anti-CD28, OKT3+BXMD12 and no stimulation control respectively. During the operation, add 1 ml of OKT3 (0.2 ng / ml) reagent to 5 wells of cells respectively.

[0223] ④At this point, the cell culture concentration in each well is 0.5x10 6 / ml, the concentration of various stimulation reagents is OKT3 is 0.1 ng / ml, which is a suboptimal antigen stimulation concentration. The above conditions are cultured in a cell culture incubator for 12 hours, and then different antibodies are added to the corresponding experimental wells to form BXME8 (or OKT3+BXMD12) and CD28 antibodies (take 1mg / ml, add 4μl / well), and the co-stimulation culture conditions are established, so that the final concentration is 2μg / ml of co-stimulatory antibodies. And IL-2 50IU / ml is added.

[0224] ⑤ Under the above culture conditions, culture the cells in a tissue culture incubator for 10 days.

[0225] ⑥After the cell culture is completed, remove the cells, centrifuge gently (800rpm, 5min), collect the cells, reselect the cells with 10% PBS, and count the number of live cells with placenta blue (the results of lymphocyte expansion are shown in the attached Fig.10 ).

[0226] Flow cytometry staining and CD8+ T cell expansion detection and analysis methods

[0227] The 6-color lymphocyte subset analysis kit (BD Multitest 6-Color TBNK Reagent) from BD Company of the United States was used. The product is stored in 1 mL of buffered saline solution containing 0.1% sodium azide. It contains FITC-labeled CD3 cells, clone SK7; PE-labeled CD16, clone B73.1 and CD56, clone NCAM16.2; PerCP-Cy5.5-labeled CD45 cells, clone 2D1 (Hle-1); PE-Cy7-labeled CD4, clone SK3; APC-labeled CD19, clone SJ25C1; and APC-Cy7-labeled CD8, clone SK1. Lymphocyte staining was performed according to the instructions to analyze the main T cell subsets. The results are shown in Table 4. After 10 days of culture with different costimulatory signals, compared with simple OKT3 stimulation, the costimulatory signal CD28 antibody mainly promoted the expansion of CD4+T cells, and the absolute number increased from 5.88x10 4 Expanded to 2.37x10 6 , amplified 1393 times. Compared with the amplification of CD4+T cells, CD137 synergistic signal tends to amplify CD8+T cells more, BXMD12 synergistically amplified 21 times. The stronger the CD137 synergistic stimulatory activity, the higher the amplification of CD8+T cells. Therefore, the antibodies provided by the present invention can cooperate with antigen stimulation signals to promote the amplification of CD8+T cells.

[0228] Table 4. Changes in expanded T cell subsets under stimulation by different co-stimulatory signals

[0229]

[0230] Note: The values ​​in the table are flow cytometry analysis data of PBMC before culture and cells cultured under different stimulation conditions for 10 days. BXD12 in the table refers to BXMD12

[0231] Experimental Example 4 Antibodies activate T cells to promote cytokine production

[0232] Healthy PBMCs were isolated by Ficoll Paque PLUS (GE Healthcare Life Sciences) and plated at 1 × 10 4 / / Cells were cultured at a density of 0.1 ml / well, and anti-human CD3 antibody (OKT3, Miltenyi Biotec Inc.) and this CD137 monoclonal antibody were added at 0.1 ng / ml and 10 ng / ml, respectively. A single CD137 monoclonal antibody was added at a concentration of 10 μg / ml. After 72 hours, the culture supernatant was collected to determine the concentrations of IFNγ and IL-10. The ELISA detection method was used. The ELISA kit was a product of Thermo Scientific, Rockford, IL, USA. The concentrations of the two cytokines in various supernatants were detected according to the instructions.

[0233] Reference Fig.11 As shown, the antibody provided by the present invention significantly assists OKT3 in activating lymphocytes and promoting the production of IFNγ and IL-10. BXMD12 shows stronger co-stimulatory ability.

[0234] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0235] References:

[0236] 1. Segal NH, et al. Results from an Integrated Safety Analysis ofUrelumab, an Agonist Anti-CD137 Monoclonal Antibody. Clin Cancer Res; 2017;23(8): 1929–36

[0237] 2. Chester C, et al. Immunotherapy targeting 4-1BB: mechanisticrationale, clinical results, and future strategies. Blood 2018; 4;131(1):49-57

[0238] 3. Qiao Y, et al. Cancer immune therapy with PD-1-dependent CD137 co-stimulation provides localized tumour killing without systemic toxicity. NatCommun 2021.12:6360.

[0239] 4. Aznar MA,et al. Intratumoral Delivery of Immunotherapy−−ActLocally, Think Globally. J Immunol 2017; 198:31-39.

Claims

1. An anti-human CD137 antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment comprises the following complementarity determining region: CDR-VH1:SYGVH; CDR-VH2:VIWSGGSTDYNAAFIS; CDR-VH3:NSITTVVADY; CDR-VL1: KASQNVGINVD; CDR-VL2: SASYRYS; CDR-VL3:QQYNSYPLT.

2. The anti-human CD137 antibody or a functional fragment thereof according to claim 1, characterized in that: The antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L, whose sequences are sequentially shown in SEQ ID NOs: 1-4, and / or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H, whose sequences are sequentially shown in SEQ ID NOs: 5-8.

3. The anti-human CD137 antibody or a functional fragment thereof according to claim 2, characterized in that: The antibody also includes a constant region.

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

5. The anti-human 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 shown in SEQ ID NO.9, and the sequence of the heavy chain constant region of the constant region is shown in SEQ ID NO.

10.

6. The anti-human CD137 antibody or a functional fragment thereof according to claim 2, characterized in that: The functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

7. A reagent or kit for detecting CD137 protein, characterized in that: It comprises the anti-human CD137 antibody or a functional fragment thereof according to any one of claims 1 to 6.

8. Use of an anti-human CD137 antibody or a functional fragment thereof as claimed in any one of claims 1 to 6 in any of the following: (1) A co-antigen stimulation signal reagent is used to expand T cell subsets in vitro; the T cell subsets are CD8+ T cells; the antigen stimulation signal reagent is an anti-CD3γ antibody, an anti-CD3δ antibody, an anti-CD3ε antibody or an anti-CD3ζ antibody; (2) Synergistic antigen stimulation signal reagents are used to prepare products that promote IFN-γ secretion by T lymphocytes in vitro; the antigen stimulation signal reagents are anti-CD3γ antibodies, anti-CD3δ antibodies, anti-CD3ε antibodies or anti-CD3ζ antibodies; (3) Synergizing antigen stimulation signal reagents to prepare products that promote IL-10 secretion by T lymphocytes in vitro; the antigen stimulation signal reagents are anti-CD3γ antibodies, anti-CD3δ antibodies, anti-CD3ε antibodies or anti-CD3ζ antibodies; (4) Synergistic antigen stimulation signal reagents are used to prepare products that promote the activation and / or proliferation of T cell subsets in vitro; the antigen stimulation signal reagents are anti-CD3γ antibodies, anti-CD3δ antibodies, anti-CD3ε antibodies or anti-CD3ζ antibodies; the T cell subsets are CD8+ T cells; (5) Preparing products for detecting CD137.

9. An antibody conjugate, characterized in that: It is formed by coupling the anti-human CD137 antibody or its functional fragment according to any one of claims 1 to 6 and a marker; the marker is selected from enzyme, fluorescein, isotope or biotin labeling.

10. A nucleic acid molecule, characterized in that It encodes the anti-human CD137 antibody or its functional fragment according to any one of claims 1 to 6.

11. A carrier, characterized in that It contains the nucleic acid molecule according to claim 10.

12. A recombinant cell, characterized in that It contains the vector according to claim 11.

13. The recombinant cell according to claim 12, characterized in that The recombinant cell is a eukaryotic cell.

14. The recombinant cell according to claim 13, characterized in that The recombinant cell is a mammalian cell.

15. The recombinant cell according to claim 14, characterized in that The recombinant cell is HEK293.

16. A method for preparing the anti-human CD137 antibody or a functional fragment thereof according to any one of claims 1 to 6, characterized in that: It includes: (a) culturing the recombinant cell according to any one of claims 12 to 15; (b) recovering the anti-human CD137 antibody or a functional fragment thereof from the culture product of step (a).

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