Monoclonal antibody capable of binding to human CD137 or functional fragment thereof and application thereof

Abstract

The invention belongs to the technical field of biomedicine and antibody engineering, and relates to a monoclonal antibody capable of being combined with human CD137 protein or a functional fragment of the monoclonal antibody, and application of the monoclonal antibody capable of being combined with the human CD137 protein or the functional fragment of the monoclonal antibody as an antibody conjugate, a chimeric antigen receptor and / or a composition. The monoclonal antibody is specifically combined with cell membrane surface CD137 protein and soluble CD137 protein through a specific nucleic acid sequence and an encoding antibody of the monoclonal antibody, has clear basic characteristics of CD137 combination specificity, sensitivity, affinity and the like, and can be used for detecting cell membrane surface CD137 and body fluid sCD137. The monoclonal antibody capable of being combined with the human CD137 protein, which is obtained by screening, has high sensitivity, high specificity and high affinity.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and antibody engineering technology, and more specifically relates to the use of human CD137 monoclonal antibodies or their functional fragments as antibody conjugates, chimeric antigen receptors and / or compositions. Background Technology

[0002] Immunotherapy has provided a new avenue for cancer treatment and has become one of the important methods in cancer therapy. Antibody drugs, represented by immune checkpoint inhibitors such as programmed cell death receptor 1 (PD-1), programmed cell death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), and lymphocyte activation gene 3 (LAG-3), have benefited patients with various cancers. However, only 20%-30% of cancer patients benefit from this therapy; most patients show no initial response or partial response, and even those who do experience tumor progression, which is considered to be related to the presence of other immunosuppressive molecules and incomplete activation of the immune system. Combination immunotherapy has become an important development strategy for improving cancer response rates, including immunotherapy combined with chemotherapy, radiotherapy, anti-angiogenic drugs, and immune checkpoint therapy. Screening and combining new immunotherapeutic targets and optimizing treatment strategies are a major trend in current cancer treatment.

[0003] CD137 (4-1BB / TNFRSF9) is a member of the tumor necrosis factor receptor (TNFR) superfamily. Initially discovered to be expressed on activated T cells, it is a promising target for tumor immunotherapy (Melero I. et al. 2023). CD137 is an immune co-stimulatory molecule, primarily expressed on activated T cells, NK cells, and vascular endothelial cells. As a T cell co-stimulatory molecule, CD137 mediates T cell activation, and CD137-stimulating antibodies can induce effective anti-tumor immune responses (Melero I. et al. 1997; Vinay DS. et al. 2016). Furthermore, following stimulation by inflammatory cytokines, CD137-induced expression has been found on the surface of vascular wall cells, such as vascular endothelial cells and vascular smooth muscle cells, in inflamed tissues (Drenkard D. et al. 2007; Olofsson PS. et al. 2008).

[0004] The expression level of CD137 in tumor blood vessels is highly correlated with the malignancy of the tumor (Broll K. et al. 2001). Based on research experience with CD137 agonists and angiogenesis inhibitors in tumor therapy, it has been shown that targeting CD137 with agonist antibodies can eliminate tumor cells and generate durable anti-tumor immunity (Romero P. et al. 1998; Shao Z. et al. 2008). With the development of agonist monoclonal antibodies targeting CD137, CD137 agonist antibodies have shown good therapeutic effects on various tumors, such as colon cancer, lung cancer, melanoma, and ovarian cancer (Claus C. et al. 2019; ChuDT. et al. 2019). The CD137 pathway preferentially activates CD8. + T cells, and CD8 + T cells are the main effector cells in anti-tumor immunity (Gramaglia et al. 2000; Lee et al. 2002; Hamid O. et al. 2022). Research on CD137 agonist antibodies for anti-tumor effects is currently in early clinical trials (Phase I and II); there are also reports that CD137 agonist antibodies can alleviate symptoms of some autoimmune diseases. In chimeric antigen receptors (CARs), the intracellular domain of CD137 delivers a more durable activation signal to chimeric antigen receptor T cells (CAR-T) and delays CAR-T cell depletion.

[0005] CD137 exists in two forms: membrane-bound and soluble (sCD137). Recent studies have discovered that CD137... + T-subsets can serve as biomarkers defining a healthy immune system and are associated with successful anti-tumor immunotherapy. sCD137, produced through differential gene splicing or metalloproteinase cleavage, is upregulated in autoimmune diseases and cancers. sCD137 competitively blocks the binding of CD137 ligand (CD137L) to CD137, antagonizing the immune activation of membrane-bound CD137 and CD137-agonist antibodies. High sCD137 expression is a potential biomarker for poor prognosis in tumor immunotherapy. Therefore, developing CD137 antibodies with diagnostic and therapeutic potential is of great significance for tumor diagnosis and treatment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention discloses a monoclonal antibody capable of binding to human CD137, which has a heavy chain variable region and a light chain variable region, thereby completing this invention.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a human CD137-binding monoclonal antibody or a functional fragment thereof, wherein the human CD137-binding monoclonal antibody comprises complementarity-determining region (CDR) sequences of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the CDR sequences of the heavy chain variable region and the light chain variable region respectively contain CDR1, CDR2, and CDR3.

[0009] The amino acid sequence of the heavy chain variable region CDR region is shown below:

[0010] The amino acid sequence of VHCDR1 is SEQ ID NO.1 or SEQ ID NO.4;

[0011] The amino acid sequence of VHCDR2 is SEQ ID NO.2, SEQ ID NO.5, or SEQ ID NO.7;

[0012] The amino acid sequence of VHCDR3 is SEQ ID NO.3 or SEQ ID NO.6;

[0013] The amino acid sequence of the CDR region of the light chain variable region is shown below:

[0014] The amino acid sequence of VLCDR1 is selected from SEQ ID NO.8;

[0015] The amino acid sequence of VLCDR2 is SEQ ID NO.9;

[0016] The amino acid sequence of VLCDR3 is selected from SEQ ID NO.10 or SEQ ID NO.11;

[0017] Specifically, when the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.2, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.3; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M001, and the VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M001 are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively; the VH and VL nucleotide sequences of CD137-M001 are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively.

[0018] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M006. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M006 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively; and the VH and VL nucleotide sequences of CD137-M006 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively.

[0019] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M009. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M009 are shown in SEQ ID NO.16 and SEQ ID NO.15, respectively; and the VH and VL nucleotide sequences of CD137-M009 are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively.

[0020] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.7, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.3; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M051. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M051 are shown in SEQ ID NO.17 and SEQ ID NO.15, respectively; and the VH and VL nucleotide sequences of CD137-M051 are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively.

[0021] When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.11, the monoclonal antibody that can bind human CD137 protein is CD137-M077. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M077 are shown in SEQ ID NO.16 and SEQ ID NO.18, respectively; and the VH and VL nucleotide sequences of CD137-M077 are shown in SEQ ID NO.29 and SEQ ID NO.30, respectively.

[0022] The five antibody heavy chain constant regions have the same amino acid sequence as shown in SEQ ID NO.19; the five antibody light chain constant regions have the same amino acid sequence as shown in SEQ ID NO.20; the five antibodies encode the heavy chain constant regions with the same nucleotide sequence as shown in SEQ ID NO.31; and the five antibodies encode the light chain constant regions with the same nucleotide sequence as shown in SEQ ID NO.32.

[0023] Furthermore, the sequence of the complementarity-determining region of the heavy chain variable region (VH) and light chain variable region (VL) of the monoclonal antibody capable of binding to human CD137 protein also includes its mutant sequence, the mutant sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity.

[0024] Furthermore, the monoclonal antibody or its antigen-binding fragment capable of binding to human CD137 protein includes an antibody constant region Fc, which comprises a heavy chain constant region and a light chain constant region.

[0025] Furthermore, the light chain constant region of the monoclonal antibody or its functional fragment that can bind to human CD137 protein is a κ-type or λ-type light chain constant region.

[0026] Preferably, the light chain constant region is κ-type, and the amino acid sequence is as shown in SEQ ID NO.20.

[0027] Furthermore, the heavy chain constant region of the monoclonal antibody or its functional fragment that can bind to human CD137 protein is selected from the heavy chain constant region of any one of the antibodies IgD, IgE, IgM, IgA and IgG.

[0028] Furthermore, the heavy chain constant region IgG includes IgG1, IgG2a, IgG2b, IgG4, and IgG4.

[0029] Preferably, the heavy chain constant region is selected from IgG1, and the amino acid sequence is shown in SEQ ID NO.19.

[0030] Furthermore, the functional fragment is selected from one or more of Fab, Fab', F(ab')2, Fv, Fd, scFv, scFv-Fc chimeric fragments and bispecific antibodies.

[0031] Furthermore, the species source of the constant region is mouse, rat, goat, rabbit, or human.

[0032] Secondly, the present invention provides a nucleic acid molecule encoding a monoclonal antibody or a functional fragment thereof that can bind to human CD137 protein.

[0033] Thirdly, the present invention provides a carrier comprising a nucleic acid molecule containing a monoclonal antibody or a functional fragment thereof capable of binding to human CD137 protein as described in the second aspect of the present invention.

[0034] Fourthly, the present invention provides a host cell containing a nucleic acid molecule of a monoclonal antibody or a functional fragment thereof capable of binding to human CD137 protein as described in the second aspect of the present invention.

[0035] Fifthly, embodiments of the present invention provide an antibody conjugate comprising the antibody or a functional fragment thereof described in the first aspect of the present invention.

[0036] In a sixth aspect, the present invention provides a chimeric antigen receptor comprising the monoclonal antibody or a functional fragment thereof described in the first aspect of the present invention.

[0037] In a seventh aspect, the present invention provides a detection composition comprising an antibody or a functional fragment thereof as described in any one of the first to sixth aspects, a nucleic acid molecule, a vector or host cell or an antibody-drug conjugate.

[0038] In an eighth aspect, the present invention provides the use of monoclonal antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors or host cells, antibody conjugates, chimeric antigen receptors or their encoding nucleic acid molecules, constructs or vectors, transformed immune cells, and / or compositions described in the fifth aspect of the present invention in disease diagnosis and in the preparation of medicaments that positively modulate immune cell activity and / or enhance immune responses.

[0039] Furthermore, the diseases mentioned include tumors, autoimmune diseases, and infectious diseases.

[0040] Furthermore, the application may include its use in the preparation of medicaments for the prevention and / or treatment of tumors or infectious diseases.

[0041] In a ninth aspect, the present invention provides a kit for detecting CD137 protein, the kit comprising instructions and detection reagents, wherein the detection reagents are reagents that can bind to human CD137 monoclonal antibodies or their antigen-binding fragments, nucleic acid molecules, vectors or host cells, antibody conjugates, chimeric antigen receptors or their encoded nucleic acid molecules, constructs or vectors, transformed immune cells and / or compositions described in the fifth aspect of the present invention.

[0042] Furthermore, the reagents or kits can be used for the detection of CD137 on the cell membrane surface and sCD137 in body fluids.

[0043] Beneficial effects

[0044] This study utilizes an immune mouse phage antibody library to screen for high-performance monoclonal antibodies that bind to human CD137. Compared to mouse hybridoma antibody screening technology, this method offers a greater chance of obtaining high-affinity antibodies. The human CD137-binding monoclonal antibody in this invention exhibits higher sensitivity and specificity than commercially available CD137 monoclonal antibodies (4B4-1), with significantly higher affinity. It can specifically recognize CD137 and soluble CD137 protein on the cell membrane surface and can be used for the detection of CD137 and sCD137 on the cell membrane surface. Attached Figure Description

[0045] Figure 1 SDS-PAGE can be used to identify the purity of human CD137 monoclonal antibody.

[0046] In the diagram, A: non-reduction condition; B: reduction condition; M: Marker; 1: CD137-M001; 2: CD137-M006;

[0047] 3: CD137-M009; 4: CD137-M051; 5: CD137-M077

[0048] Figure 2 It can be used in conjunction with human CD137 monoclonal antibody to detect CD137 protein-specific ELISA identification.

[0049] Figure 3 It can be combined with human CD137 monoclonal antibody for detection of CD137 protein-specific Western blotting identification.

[0050] In the figure, M: Marker; 1: CD137-His recombinant protein under non-reducing conditions; 2: CD137-His recombinant protein under reducing conditions.

[0051] Figure 4 It can bind to human CD137 monoclonal antibodies to recognize CD137-GFP recombinant plasmids transfected into 293FT cells, which contain CD137 protein on the cell surface.

[0052] Flow cytometry identification of white

[0053] Figure 5 ELISA can be used to identify CD137 protein sensitivity in conjunction with human CD137 monoclonal antibody detection.

[0054] Figure 6 Affinity identification of CD137 protein binding by human CD137 monoclonal antibody.

[0055] The curves in the figure show the reaction at different antibody concentrations. From top to bottom, the antibody concentrations are 250 nM, ...

[0056] 125 nM, 62.5 nM, 31.3 nM, 15.6 nM, 7.8 nM and 0 nM

[0057] Figure 7 A sandwich ELISA method was developed to identify the combination of a self-developed human CD137 monoclonal antibody and the commercially available antibody 4B4-1.

[0058] CD137 epitope characteristics

[0059] Figure 8 The ForteBio molecular interaction analyzer identifies a self-developed monoclonal antibody that binds to human CD137 and recognizes 4B4-1.

[0060] CD137 epitope characteristics Detailed Implementation

[0061] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0062] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0063] the term

[0064] The term "antibody" refers to a protein that contains at least one and preferably two heavy chain (H) variable regions (abbreviated as VH), at least one and preferably two light chain (L) variable regions (abbreviated as VL), and at least one and preferably two heavy chain constant regions and light chain constant regions.

[0065] The "variable region" (V) or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be called "VH," and the variable domain of the light chain can be called "VL." The amino acid sequence within these domains typically varies considerably and contains antigen-binding sites. Each of the three regions in the light or heavy chain variable regions (VL or VH) has a highly variable amino acid composition and sequence, forming a spatial conformation complementary to the antigenic epitope; these are called "complementarity-determining regions" (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The amino acid composition and sequence of the regions outside the CDRs in the V region remain relatively stable and are called backbone regions (FRs). Both VH and VL have four backbone regions, denoted as FR1, FR2, FR3, and FR4, respectively. The main function of the FR regions is to stabilize the spatial conformation of the CDR regions, facilitating precise and specific binding between the antibody's CDR regions and the antigenic determinants. The scope of the skeletal region and CDR has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E. Kabat et al., U.S. Department of Health and Human Services, (1983) and Chothia.

[0066] As used in this article, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0067] As used herein, the term "affinity" refers to the binding force between an antibody and an antigenic epitope or antigenic determinant. Within an antigen, the variable region of an antibody interacts with the antigenic epitope or antigenic determinant through non-covalent forces. Generally, the higher the degree of binding between an antibody and an antigen, the higher the affinity.

[0068] Example

[0069] Example 1: Preparation of mouse anti-human CD137 monoclonal antibody

[0070] 1.1 Animal Immunization

[0071] Balb / c mice aged 6-8 weeks were selected and numbered after one week of normal feeding. Approximately 0.1 ml of blood was collected via the retro-orbital venous plexus. After standing at room temperature for 1 hour, the blood was incubated overnight at 4°C. The supernatant serum was collected after centrifugation at 1500g and stored at -20°C for later analysis. For primary immunization, 100 μg of CD137-His recombinant protein (Sino Biological) was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple sites in the abdomen and back. A booster immunization was performed 3 weeks later, with an equal volume of CD137-His recombinant protein emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites. After three immunizations, a booster immunization was administered. Blood could be collected via the retro-orbital venous plexus 7 days later to determine serum titer. One month later, mice with higher titers were selected for intraperitoneal injection of 100 μg of CD137-His recombinant protein for pulse immunization. Mice were sacrificed on the 3rd day after immunization, and the spleen was collected and rapidly transferred to liquid nitrogen for cryopreservation.

[0072] 1.2 Valence Testing Process

[0073] CD137-His recombinant protein was diluted to 5 μg / ml with coating buffer and added to 100 μl per well of a 96-well microplate. The plate was gently vortexed to mix, sealed tightly with plastic wrap, and incubated overnight at 4°C. The plate was washed, dried, and 200 μl / well of blocking buffer was added to block non-specific binding sites. The plate was incubated for 2 hours. After washing, 100 μl / well of serially diluted serum was added and the plate was incubated for 2 hours. The plate was washed five times, and 100 μl / well of 1:5000 horseradish peroxidase-labeled goat anti-mouse IgG (H+L) was added and the plate was incubated for 1 hour. After washing, 100 μl / well of chromogenic buffer was added and the plate was incubated for 30 minutes. 50 μl / well of stop solution was added. The OD was measured using a microplate reader. 450nm Value. Mouse serum diluted 1:16000, antibody test positive, OD value selected. 450nm Mice with high values ​​were subjected to shock immunization.

[0074] 1.3 Construction of scFv phage antibody library and antibody screening

[0075] Total RNA was extracted from the spleen of immunized mice and reverse transcribed into cDNA. The antibody variable region (V) gene was amplified by RT-PCR using specific primers. Subsequently, the heavy chain and light chain variable region (VH and VL) fragments were spliced ​​to obtain a fused scFv fragment, which was then inserted into a phage vector. Electroporation was performed on competent cells to obtain a phage antibody library with a volume of not less than 10^6 cells / mL. 8 The CD137-His recombinant protein was coated with ELISA to screen the phage library. A "wash-amplify-enrich" cycle was used, and positive clones were generally obtained after two or more rounds. The positive clones detected by ELISA were then amplified by PCR, digested with enzymes, genotyped, and sequenced. Suitable variable region sequences were selected based on the sequencing results.

[0076] 1.4 Construction of antibody expression vector

[0077] Using phage culture or plasmids as templates, the variable region gene was amplified, and the variable region gene fragment was inserted into an expression vector containing the antibody constant region to construct IgG antibody heavy chain (pCMV3-H) and light chain (pCMV3-L) expression vectors. The amplified plasmids were then sent to downstream expression vectors.

[0078] Example 2: CD137 antibody sequence analysis

[0079] 2.1 Amino acid sequence of the CDR region of CD137 monoclonal antibody

[0080] Amino acid sequence analysis of the variable region of the antibodies confirmed five CD137 monoclonal antibodies through phage antibody library screening using scFv, named CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077. Positive clones were sequenced to obtain the light and heavy chain variable region sequences. Analysis using IGBLAST software (https: / / www.ncbi.nlm.nih.gov / igblast / ) revealed that the scFv antibodies belonged to the VH class and Vκ type. Further analysis of the three CDR regions (VH (heavy chain variable region) and VL (light chain variable region) of each antibody using ABodyBuilder software (http: / / opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / abodybuilder / ) in Kabat mode was performed.

[0081] The amino acid sequences of CD137-M001-HCDR1-3 are shown in SEQ ID NO.1-3, respectively; the amino acid sequences of CD137-M001-LCDR1-3 are shown in SEQ ID NO.8-10, respectively.

[0082] The amino acid sequences of CD137-M006-HCDR1-3 are shown in SEQ ID NO.4-6, respectively; the amino acid sequences of CD137-M006-LCDR1-3 are shown in SEQ ID NO.8-10, respectively.

[0083] The amino acid sequences of CD137-M009-HCDR1-3 are shown in SEQ ID NO.4-6, respectively; the amino acid sequences of CD137-M009-LCDR1-3 are shown in SEQ ID NO.8-10, respectively.

[0084] The amino acid sequences of CD137-M051-HCDR1-3 are shown in SEQ ID NO.4, SEQ ID NO.7 and SEQ ID NO.3, respectively; the amino acid sequences of CD137-M051-LCDR1-3 are shown in SEQ ID NO.8-10, respectively.

[0085] The amino acid sequences of CD137-M077-HCDR1-3 are shown in SEQ ID NO.4-6, respectively; the amino acid sequences of CD137-M077-LCDR1-3 are shown in SEQ ID NO.8-9 and SEQ ID NO.11, respectively.

[0086] 2.2 Amino acid sequences of the heavy and light chain variable regions of the CD137 monoclonal antibody

[0087] The amino acid sequences of VH and VL of CD137-M001 are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0088] The amino acid sequences of VH and VL of CD137-M006 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively.

[0089] The amino acid sequences of VH and VL of CD137-M009 are shown in SEQ ID NO.16 and SEQ ID NO.15, respectively.

[0090] The amino acid sequences of VH and VL of CD137-M051 are shown in SEQ ID NO.17 and SEQ ID NO.15, respectively.

[0091] The amino acid sequences of VH and VL of CD137-M077 are shown in SEQ ID NO.16 and SEQ ID NO.18, respectively.

[0092] The five antibody heavy chain constant regions have the same amino acid sequence, as shown in SEQ ID NO.19.

[0093] The five antibody light chain constant regions have the same amino acid sequence, as shown in SEQ ID NO.20.

[0094] 2.3 The nucleotide sequence encoding the antibody is shown below:

[0095] Nucleotide sequence encoding the variable region of the antibody heavy chain (underlined sequences are signal peptide encoding sequences):

[0096] The VH and VL nucleotide sequences of CD137-M001 are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively.

[0097] The VH and VL nucleotide sequences of CD137-M006 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively.

[0098] The VH and VL nucleotide sequences of CD137-M009 are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively.

[0099] The VH and VL nucleotide sequences of CD137-M051 are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively.

[0100] The VH and VL nucleotide sequences of CD137-M077 are shown in SEQ ID NO.29 and SEQ ID NO.30, respectively.

[0101] The five antibodies encode the same nucleotide sequence for the constant region of the heavy chain, as shown in SEQ ID NO.31.

[0102] The five antibodies encode the same nucleotide sequence for the constant region of the light chain, as shown in SEQ ID NO.32.

[0103] Table 1 Sequences VHCDR1-3 and VLCDR1-3

[0104]

[0105]

[0106] Table 2. Sequence listing of CD137-M001, CD137-M006, CD137-M009, CD137-M0051 and CD137-M0077 antibodies.

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Example 3 Antibody Expression, Purification and Identification

[0116] HEK293 cells were transiently transfected simultaneously with the same antibody light and heavy chain expression vectors. One week after transfection, the culture supernatant was collected and purified using a protein A purification column to obtain purified antibodies. The antibody molecular weight was predicted using a protein molecular weight calculator, and the purity and molecular weight of the antibodies were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0117] SDS-PAGE electrophoresis procedure: Take 5 μg of purified antibody and perform electrophoresis using a 10% SDS-PAGE gel under both non-reducing (no β-mercaptoethanol added to protein electrophoresis loading buffer, no boiling of samples) and reducing (β-mercaptoethanol added to protein electrophoresis loading buffer, boiling of samples for 5 min) conditions at 150V for approximately 1 hour. After electrophoresis, stain with Coomassie Brilliant Blue solution and incubate overnight at 4°C. The next day, destain with destaining solution until the bands are clear and the background is clean, then photograph and record the staining results.

[0118] SDS-PAGE detection results ( Figure 1 The predicted molecular weights of the five antibody heavy chains are approximately 48 kDa, the light chains are approximately 24 kDa, and the total molecular weight of the IgG antibody is approximately 145 kDa. SDS-PAGE electrophoresis confirmed that the protein molecular weights were close to the predicted molecular weights, indicating good antibody purity of approximately 95%. In the figure, A: Detection results under non-reducing conditions; B: Detection results under reducing conditions; M: marker; 1: CD137-M001; 2: CD137-M006; 3: CD137-M009; 4: CD137-M051; 5: CD137-M077.

[0119] Example 4: Specificity detection of CD137 antibody

[0120] 4.1 Specificity of ELISA detection of CD137 antibody

[0121] The specificity of CD137 antibodies was identified using recombinant human proteins CD137, PD-1, PD-L1, CTLA4, and TIM-3. The ELISA assay procedure was as follows: Human CD137-His, human PD-1-His, human PD-L1-His, CTLA4-His, and TIM-3-His recombinant proteins were coated with coating buffer at 1 μg / ml and incubated overnight at 4°C. The next day, the plates were washed with Wash Buffer, and 5% skim milk powder was added to block non-specific binding sites (200 μl / well), and the reaction was allowed to proceed for 2 hours. After washing, CD137 antibody (CD137 antibody control 4B4-1 and CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077) diluted to 1 μg / ml with 5% skim milk powder was added (100 μl / well), and the reaction was allowed to proceed for 2 hours. After washing, add horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) diluted 1:5000 and react for 1 h. After washing, add TMB substrate solution, 100 μl / well, and react for 30 min. Add stop solution, 50 μl / well. Measure OD using a microplate reader. 450 Value. Graphpad software analyzes the detection results.

[0122] Main reagent sources and preparation:

[0123] Coating buffer (BD OptEIA Coating Buffer, 51-2713KC) pH 9.5, 0.1M sodium carbonate

[0124] The washing solution (BD OptEIAWash Buffer, 51-9003739) 20× concentrated washing solution should be diluted with deionized water or distilled water to a 1× working solution.

[0125] Developing solution A (BD OptEIA Substrate Reagent A, 51-2606KZ) is a buffer solution containing hydrogen peroxide.

[0126] Developing solution B (BD OptEIA Substrate Reagent B, 51-2607KZ) contains 3,3',5,5'-tetramethylbenzidine (TMB) in an organic solvent.

[0127] Stop solution (BD OptEIA Stop Solution, 51-2608KZ) 1 sulfuric acid

[0128] 5% Milk: Add 5 grams of milk to 100 ml of 1×PBS solution.

[0129] Test results ( Figure 2Antibodies CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 can all effectively recognize recombinant human CD137 protein and do not react with human PD1, PD-L1, CTLA4, and TIM-3 proteins, exhibiting high specificity for human CD137 protein.

[0130] 4.2 Specificity of CD137 antibody detected by Western blotting

[0131] Western blotting was used to identify the binding characteristics of CD137 antibodies to recombinant CD137 protein under both non-reducing and reducing conditions. Commercially available CD137 antibody 4B4-1 was used as a control.

[0132] Western blotting procedure: 1 μg of CD137-His recombinant protein was separated by electrophoresis using a 10% SDS-PAGE gel at 150V for approximately 1 hour under both non-reducing (no β-mercaptoethanol added to the protein electrophoresis loading buffer, no boiling) and reducing (β-mercaptoethanol added to the protein electrophoresis loading buffer, boiling for 5 min) conditions. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane at 300mA for 1 hour. The membrane was then blocked with 5% skim milk at room temperature for 2 hours, followed by incubation at 4°C overnight with 0.5 μg / ml of antibodies (4B4-1, CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077). The membrane was washed three times with PBST solution, 5 min each time. Secondary antibody was added to horseradish peroxidase-labeled goat anti-mouse IgG (H+L) diluted 1:5000, and the reaction was carried out at room temperature for 1 hour. Unbound antibodies were removed by washing with PBST. Chemiluminescent substrate was added, and the results were recorded using a gel imaging system.

[0133] Test results ( Figure 3 The control antibody 4B4-1, as well as the CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 antibodies, primarily recognize recombinant CD137 protein under non-reducing conditions, suggesting that these antibodies recognize CD137 conformational epitopes. In the figure, M: Marker; 1: recombinant CD137 protein under non-reducing conditions; 2: recombinant CD137 protein under reducing conditions.

[0134] 4.3 Flow cytometry detection of CD137 protein on cell membrane surface

[0135] The CD137 protein expressed on the cell membrane surface was identified by flow cytometry after transfection of 293FT cells with the CD137-GFP recombinant plasmid. A commercially available mouse anti-human CD137 antibody (4B4-1) was used as a positive control.

[0136] Flow cytometry assay procedure: 48 h after transfection of 293FT cells with the CD137-GFP recombinant plasmid, single-cell suspensions were prepared by pipetting off the cells. The cells were washed once with PBS, and 1 μg / ml of anti-CD137 antibody (4B4-1, CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 antibodies) were added. The cells were incubated at room temperature for 30 min, washed once with PBS, and then 1:2000 diluted AF647-labeled goat anti-mouse IgG (H+L) antibody was added. The cells were incubated at room temperature in the dark for 30 min. After washing with PBS, the cells were resuspended in 200 μl of PBS solution. The assay was performed using BD LSRFortessa. TM The samples were analyzed using flow cytometry and the results were analyzed using BDFACSDiva software.

[0137] Flow cytometry analysis of CD137 protein on the surface of 293FT cells transfected with CD137-GFP recombinant plasmid ( Figure 4 All CD137 antibodies could bind to the CD137 protein expressed on the cell membrane surface of 293FT cells transfected with the CD137 recombinant plasmid. The proportions of GFP and CD137 double-positive cells (Q2 quadrant) detected by antibodies 4B4-1, CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 were 12.6%, 11.2%, 12.1%, 12.3%, 9.3%, and 10.5%, respectively.

[0138] Example 5: Sensitivity identification of CD137 antibody

[0139] Indirect ELISA detection of CD137 antibody and its sensitivity to CD137 protein.

[0140] Detection Procedure: CD137-His recombinant protein was serially diluted 10-fold (1000-0.001 ng / ml) and coated onto 96-well ELISA plates, 100 μl / well, and incubated overnight at 4°C. Primary antibodies (1 μg / ml) of CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 antibodies and control antibody (4B4-1) were added. The remaining steps were the same as the ELISA detection procedure described above.

[0141] ELISA sensitivity test results ( Figure 5(): By indirect ELISA, the sensitivity of CD137-M001, CD137-M009 and CD137-M051 for detecting CD137-His recombinant protein was 100 ng / ml, and the sensitivity of CD137-M006, CD137-M077 and 4B4-1 for detecting CD137-His recombinant protein was 10 ng / ml.

[0142] Example 6: Affinity Identification of CD137 Antibody

[0143] Antibody affinity was detected using the ForteBio Octet molecular interaction instrument. CD137-hFc recombinant protein was used as the antigen for detection, employing the FORTEBIO anti-human IgG Fc capture (AHC) biosensor, with CD137 antibody 4B4-1 used as the control antibody.

[0144] Affinity assay procedure: The FORTEBIO anti-human IgG Fc capture (AHC) biosensor was pre-wetted in equilibration buffer (0.1% BSA + 0.02% Tween 20 in PBS) for 10 minutes. CD137-hFc recombinant protein was diluted to 5 μg / mL with equilibration buffer and added to the second column of a light-protected 96-well plate (200 μl / well). CD137 antibody was serially diluted from 250 nM to 7.8 nM and added to the fourth column of a light-protected 96-well plate (200 μl / well). A 0 nM antibody blank control was set up, and 200 μl of equilibration buffer was added. The equilibration buffer was then added to the first and third columns (200 μl / well). ForteBio Octet molecular interaction analyzer was used for detection. The sensor was equilibrated in the first column for 60 seconds to obtain the baseline equilibration curve, and then antigen was immobilized in the second column for 100 seconds. The antibody was washed for 120 seconds in the third column, then bound to the antibody in the fourth column for 180 seconds to obtain the binding curve. Finally, the antibody was dissociated in the first column for 300 seconds to obtain the dissociation curve. The ForteBio Octet analysis software was used to fit and analyze the curves to obtain the affinity values.

[0145] Affinity test results ( Figure 6 Antibody affinity was determined using the ForteBio Octet system, with a buffer solution without CD137 protein as a blank control and commercially available CD137 antibody 4B4-1 as a detection control. After data analysis, the antibody affinity K... D The values ​​are 6.45 × 10 -8 M(4B4-1), 2.17×10 -10 M(CD137-M001), 1.32×10 - 10 M(CD137-M006), 1.92×10 -10M(CD137-M009), 1.39×10 -9 M(CD137-M051) and <1.0×10 -12 M(CD137-M077). Curve fit goodness R 2 The values ​​are 0.9824 (4B4-1), 0.9990 (CD137-M001), 0.9988 (CD137-M006), 0.9984 (CD137-M009), 0.9419 (CD137-M051), and 0.9964 (CD137-M077), respectively.

[0146] Example 7: Analysis of the binding epitopes of CD137 protein between a self-developed monoclonal antibody and the commercially available antibody 4B4-1.

[0147] 7.1 Sandwich ELISA Detection: A self-developed monoclonal antibody that binds to human CD137 protein binds to the 4B4-1 antibody epitope.

[0148] Detection Procedure: Capture antibodies (1 μg / ml) of the self-developed antibodies CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077, along with control antibody 4B4-1, were immobilized and incubated overnight at 4°C. The detection reagent, CD137-His recombinant protein, was 1 μg / ml, 100 μl / well, incubated at 37°C for 2 hours. The detection reagent, biotin-labeled antibody 4B4-1-Bio, was 1 μg / ml, incubated at 37°C for 2 hours. For biotin recognition, 1:5000 peroxidase-labeled streptavidin was added, incubated at 37°C for 1 hour. The remaining steps are the same as the ELISA detection procedure described above.

[0149] Sandwich ELISA results for identifying CD137 antibody epitopes ( Figure 7 CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 can all pair with the 4B4-1-Bio antibody, indicating that the independently developed monoclonal antibody that can bind to the human CD137 protein recognizes a different CD137 epitope than the commercially available antibody 4B4-1; however, the paired antibody combination detects the OD of CD137-His. 450 The value is low, which suggests that the binding of CD137-M001, CD137-M006, CD137-M009, CD137-M051 and CD137-M077 to CD137 may interfere with the binding of the 4B4-1 antibody, which is likely due to a conformational change of CD137.

[0150] 7.2 Molecular interaction instrument identification: The monoclonal antibody independently developed to bind to human CD137 protein and the CD137 epitope characteristics of 4B4-1 binding were identified.

[0151] The binding epitopes of the self-developed monoclonal antibody that binds to human CD137 protein and the commercially available antibody 4B4-1 were identified using a ForteBio Octet molecular interaction analyzer. The CD137-hFc recombinant protein was used as the detection antigen, and a ForteBio anti-human IgG Fc capture (AHC) biosensor was employed. In the aforementioned sandwich ELISA assay, the binding of the self-developed monoclonal antibody to CD137-His may have interfered with the binding of 4B4-1. In this experiment, the 4B4-1 antibody first bound to CD137-hFc, and then to the self-developed monoclonal antibody.

[0152] Interaction detection procedure: The ForteBio anti-human IgG Fc capture (AHC) biosensor was pre-wetted in equilibration buffer (0.1% BSA + 0.02% Tween 20 in PBS solution) for 10 minutes. The CD137-hFc recombinant protein was diluted to 2 μg / mL with equilibration buffer and added to the second column of a 96-well plate protected from light, 200 μl / well. The 4B4-1 antibody was diluted to 2 μg / mL with equilibration buffer and added to the fourth well. The self-developed CD137 antibodies CD137-M001, CD137-M006, CD137-M009, CD137-M051, CD137-M077, 4B4-1 and blank control (0 nM; equilibration buffer) were added from top to bottom to the fifth column (4A-4G), diluted to 2 μg / mL with equilibration buffer, 200 μl / well. Equilibration buffer was added to the first and third columns, 200 μl / well. ForteBio Octet molecular interaction analyzer was used for detection. The sensor was equilibrated in the first column for 60 s to obtain a baseline equilibrium curve, followed by antigen immobilization in the second column for 120 s. Washing was performed in the third column for 120 s, followed by reaction with 4B4-1 in the fourth column for 300 s, and finally antibody binding in the fifth column for 300 s. The curves were analyzed using ForteBio Octet analysis software.

[0153] Molecular interaction assay results for identifying CD137 antibody binding to CD137 epitopes ( Figure 8The figure shows the binding curves of CD137-hFC to 4B4-1, forming the 4B4-1-CD137 complex, and then to CD137-M001, CD137-M006, CD137-M009, CD137-M051, CD137-M077, and 4B4-1. Compared to the 4B4-1 binding curve, the binding curves of CD137-M001, CD137-M006, CD137-M009, CD137-M051, and CD137-M077 are significantly higher than the 4B4-1 binding curve, further indicating that these self-developed monoclonal antibodies that can bind to human CD137 protein bind to different CD137 epitopes with 4B4-1.

[0154] Based on the amino acid sequence of the monoclonal antibody or its functional fragment that can bind to human CD137 protein disclosed in this invention, those skilled in the art can easily obtain the monoclonal antibody or its functional fragment that can bind to CD137 using conventional techniques in the art, such as genetic engineering techniques. Regardless of how the monoclonal antibody or its functional fragment that can bind to human CD137 protein is obtained, it falls within the protection scope of this invention.

[0155] Based on the amino acid sequences disclosed in this invention, those skilled in the art can easily obtain the nucleotide sequences encoding these antibodies. Regardless of how the nucleotide molecular sequence changes, as long as it encodes the monoclonal antibody or its functional fragment that can bind to the human CD137 protein, it falls within the protection scope of this invention.

Claims

1. An antibody or a functional fragment thereof capable of binding human CD137, said monoclonal antibody capable of binding human CD137 protein comprising complementarity-determining region (CDR) sequences of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region CDR1, CDR2, and CDR3, respectively: The amino acid sequence of the heavy chain variable region CDR region is shown below: The amino acid sequence of VHCDR1 is SEQ ID NO.1 or SEQ ID NO.4; The amino acid sequence of VHCDR2 is SEQ ID NO.2, SEQ ID NO.5, or SEQ ID NO.7; The amino acid sequence of VHCDR3 is SEQ ID NO.3 or SEQ ID NO.6; The amino acid sequence of the CDR region of the light chain variable region is shown below: The amino acid sequence of VLCDR1 is selected from SEQ ID NO.8; The amino acid sequence of VLCDR2 is SEQ ID NO.9; The amino acid sequence of VLCDR3 is selected from SEQ ID NO.10 or SEQ ID NO.

11.

2. The antibody or functional fragment thereof capable of binding to human CD137 as described in claim 1. in, When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.1, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.2, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.3; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind to human CD137 protein is CD137-M001. The VH and VL amino acid sequences of the monoclonal antibody that can bind to human CD137 protein CD137-M001 are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively; and the VH and VL nucleotide sequences of CD137-M001 are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively. When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M006. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M006 are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively; and the VH and VL nucleotide sequences of CD137-M006 are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively. When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M009. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M009 are shown in SEQ ID NO.16 and SEQ ID NO.15, respectively; and the VH and VL nucleotide sequences of CD137-M009 are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively. When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.7, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.3; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.10, the monoclonal antibody that can bind human CD137 protein is CD137-M051. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M051 are shown in SEQ ID NO.17 and SEQ ID NO.15, respectively; and the VH and VL nucleotide sequences of CD137-M051 are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively. When the amino acid sequence of VHCDR1 is selected from SEQ ID NO.4, the amino acid sequence of VHCDR2 is selected from SEQ ID NO.5, and the amino acid sequence of VHCDR3 is selected from SEQ ID NO.6; and the amino acid sequence of VLCDR1 is selected from SEQ ID NO.8, the amino acid sequence of VLCDR2 is selected from SEQ ID NO.9, and the amino acid sequence of VLCDR3 is selected from SEQ ID NO.11, the monoclonal antibody that can bind human CD137 protein is CD137-M077. The VH and VL amino acid sequences of the monoclonal antibody that can bind human CD137 protein CD137-M077 are shown in SEQ ID NO.16 and SEQ ID NO.18, respectively; and the VH and VL nucleotide sequences of CD137-M077 are shown in SEQ ID NO.29 and SEQ ID NO.30, respectively.

3. The human CD137-binding monoclonal antibody or its functional fragment as described in claim 1 or 2, characterized in that, The antigen-binding fragment includes a functional fragment for antigen binding, and the fragment is selected from one or more of Fab, Fab', F(ab')2, Fv, Fd, scFv, scFv-Fc chimeric fragments and bispecific antibodies.

4. A nucleic acid molecule encoding a human CD137 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2.

5. A vector or host cell comprising a nucleic acid molecule encoding a functional fragment of a human CD137 monoclonal antibody as described in claim 1 or 2.

6. An antibody conjugate comprising a human CD137 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2.

7. A chimeric antigen receptor comprising the human CD137 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2.

8. A composition comprising the human CD137 monoclonal antibody or a functional fragment thereof as described in claim 1 or 2, a nucleic acid molecule, a vector or host cell or antibody conjugate.

9. The use of the human CD137 monoclonal antibody or its functional fragment, nucleic acid molecule, vector or host cell, antibody-drug conjugate, chimeric antigen receptor or its encoded nucleic acid molecule, construct or vector, transformed immune cell and / or composition as described in claims 1-7 in disease diagnosis and in the preparation of medicaments that positively modulate immune cell activity and / or enhance immune responses.

10. A kit for detecting CD137 protein, the kit comprising instructions and detection reagents, the detection reagents comprising the reagents of claims 1-7 capable of binding to human CD137 monoclonal antibodies or functional fragments thereof, nucleic acid molecules, vectors or host cells, antibody conjugates, chimeric antigen receptors or their encoded nucleic acid molecules, constructs or vectors, transformed immune cells and / or compositions.

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