Anti-CD137 antibody and the use thereof

Abstract

Disclosed is an antibody or an antigen binding fragment thereof comprising a heavy chain 5 variable region comprising CDR-H1 of SEQ ID NO:1, CDR-H2 of SEQ ID NO:2, CDR- H3 of SEQ ID NO:3, and a light chain variable region comprising CDR-L1 of SEQ ID NO:4, CDR-L2 of SEQ ID NO:5, CDR-L3 of SEQ ID NO:6, or an amino acid sequence having at least 80% sequence identity with these sequences, wherein the antibody or antigen binding fragment binds to CD137 antagonistically. Also disclosed is a method of 0 treating a cancer or a non-malignant disease in a subject in need thereof, comprising administering to the subject the antibody or antigen binding fragment thereof as disclosed herein.

Description

ANTI-CD137 ANTIBODY AND THE USE THEREOFFIELD OF THE INVENTION

[0001] The present invention generally relates to an anti-CD137 antibody. The present invention also relates to use of the anti-CD137 antibody to treat cancer.BACKGROUND

[0002] The immunosuppressive tumor microenvironment (TME) is one of the main barriers for effective immunotherapies for solid tumors. Foxp3+CD25+CD4+regulatory T cells (Tregs) are among the suppressor cells and play a vital role in hampering anti-tumor responses. Tregs abundantly infiltrate into tumor tissues, and a high abundance of infiltrated Tregs is associated with poor clinical outcomes in various types of cancer2. Reducing the numbers of intratumoral Tregs by monoclonal antibodies enhances antitumor immunity and improves the efficacy of immunotherapy3. Antibody-based Treg depletion is induced by the binding of the fragment crystallizable (Fc) region of the antibody to Fey receptors (FcyR) on immune cells, including NK cells and phagocytes, to trigger antibody dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) against target cells. Antibody-based Treg depletion can also be induced by Complement-dependent cytotoxicity (CDC), an immune mechanism where antibodies bind to antigens on the surface of target cells such as Tregs, which activates the complement system, a group of proteins in the blood that enhances the ability of antibodies and phagocytic cells to clear' the target cells.

[0003] Recently, CD 137 has been identified as a signature marker for intratumoral Tregs. Human and murine Tregs that had infiltrated tumors expressed high amounts of CD1374, while peripheral Treg had low to negligible levels5'6. A high frequency of CD137+Treg correlated with poor prognosis in lung adenocarcinoma patients7. This is in line with CD137+Treg having been more suppressive than CD137" Treg8.

[0004] This association of CD 137 with Treg and immunosuppression is surprising since CD137 is best known as a potent T cell costimulatory molecule. CD137 signalling enhances T cell survival and proliferation, IFN-y secretion and cytotoxicity of Teffs9'10, and is a potent driver for T helper type 1 (Thl) / Type 1 cell (Tcl)-mediated immune responsesu. Agonist anti-CD137 antibodies have been shown to induce tumor regression in multiple preclinical models by stimulating CD 137 signalling to enhance T cellactivities and to reverse the established anergy in cytotoxic T lymphocytes12. Among a range of CD137 agonist in development, Urclumab and Utomilumab arc the most advanced ones and are currently being tested in clinical trials13.

[0005] The ligand for CD137, CD137L, is expressed by antigen presenting cells (APC), and APC use the CD137-CD137L system to costimulate T cells. The CD137 receptor / ligand system has an inbuilt negative feed-back mechanism to prevent overstimulation of the immune response and subsequent autoimmune damage. This consists of the trogocytic transfer of CD 137 from activated T cells to APC, where the transferred CD137 and the endogenous CD137L form a complex that is internalized and degraded by the protea some14. This deprives the T cells from further receiving costimulatory signals through CD 137, and it deprives the APC of the ability to costimulate other T cells via CD137L (Figure 14). Tregs that express higher levels of CD137 than Teffs, and that express it constitutively, also utilize this mechanism to keep immune responses in check15.

[0006] Ectopic CD 137 expression is found on malignant cells of Hodgkin lymphoma (HL)14, rhabdomyosarcoma (RMS)16, nasopharyngeal carcinoma (NPC)17and NKT cell lymphoma18, cancers that usurp this negative regulatory mechanism to evade immune surveillance. Further, Human T cell lymphotropic virus (HTLV)-l induces the expression of CD137 in infected T cells19, and Epstein-Barr virus (EBV) induces CD1.37 in infected NKT cells1S, NPC cells17and HL cells20. CD137+malignant cells are present in 34% (NPC) to 89% (HL) of cancers with varying expression levels14, 17.

[0007] CD137 in the tumor, whether on malignant cells or Tregs, provides the tumor with a selection advantage. However, it may also be a cancer’s Achilles heel, as it can be targeted by immunotherapy. There is a need for effective antibodies that can bind to CD137 on tumor and Tregs to elicit ADCC, ADCP, or CDC to kill them. Therefore, anti- CD137 antibodies of the IgGl isotype were generated and it was demonstrated that they were able to induce cell death specifically in CD137-expressing cancer cells and Tregs. Further, clone P1A1 cross-reacts with murine CD137 and after reconstitution with a murine IgG2a isotype, it was able to kill murine malignant cells and Tregs that express murine CD137 in vitro and in vivo in an HCC and a melanoma lung metastasis model.

[0008] In one aspect, the present disclosure refers to an antibody or an antigen binding fragment thereof comprising:(A) a heavy chain variable region comprising the following CDRs:(a) CDR-H1 : GYSLTSYGIS [SEQ ID NO:1 ] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(b) CDR-H2: WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(c) CDR-H3: ASYYDSSGYAFDI [SEQ ID NO:3] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; and(B) a light chain variable region comprising the following CDRs:(a) CDR-L1: SGDKLEDKYAS [SEQ ID NO:4] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(b) CDR-L2: QDYKRPS [SEQ ID NO:5] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(c) CDR-L3: QTWDRITTSYV [SEQ ID NO:6] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; wherein the antibody or antigen binding fragment binds to CD137 antagonistically.

[0009] In another aspect, the present disclosure refers to a bispecific antibody, comprising (i) a first binding arm comprising a first antigen-binding region binding to CD137 antagonistically, wherein the first antigen-binding region comprises (a) heavy chain variable (VH) region CDR-H1, CDR-H2, and CDR-H3 having the sequences as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable (VL) region CDR- Ll, CDR-L2, and CDR-L3 having the sequences as set forth in SEQ ID NOs:4, 5 and 6, respectively, and (ii) a second binding arm comprising a second antigen-binding region binding to human CD3.

[0010] In another aspect, the present disclosure refers to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein.

[0011] In another aspect, the present disclosure refers to a vector comprising a nucleic acid molecule as disclosed herein.

[0012] In another aspect, the present disclosure refers to a recombinant host cell comprising a nucleic acid molecule as disclosed herein or a vector as disclosed herein.

[0013] hi another aspect, the present disclosure refers to a method for producing an antibody or antigen- binding fragment thereof as disclosed herein, or the bispccific antibody as disclosed herein, the method comprising culturing a recombinant host cell as disclosed herein under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.

[0014] In another aspect, the present disclosure refers to a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, and a pharmaceutically- acccptablc diluent, carrier or excipient.

[0015] hi another aspect, the present disclosure refers to a method of treating a cancer or a non-malignant disease in a subject in need thereof, comprising administering to the subject the antibody or antigen binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, or the pharmaceutical composition as disclosed herein; wherein the antibody or antigen binding fragment thereof or the bispecific antibody induces antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137- expressing cancer cells and / or Treg.

[0016] In another aspect, the present disclosure refers to use of the antibody or antigen binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, or the pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a cancer or a non-malignant disease in a subject in need of, wherein the antibody or antigen binding fragment thereof or the bispecific antibody or the pharmaceutical composition is to be administered to the subject, wherein the antibody or antigen binding fragment thereof induces antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137-expressing cancer cells and / or Treg.

[0017] Advantageously, the antibody of the present invention would be able to treat all types of solid cancers as Trcgs in virtually all solid cancers express CD1375,6. Accordingly, elimination of CD137+intratumoral Tregs would deprive these solid cancers of Treg help.

[0018] Advantageously, the antibody of the present invention is fully human so it can be translated clinically in a straightforward manner.

[0019] Advantageously, the antibody of the present invention has an antagonistic nature to block CD137 signalling, since there is evidence that CD137 signalling enhances Treg activity similarly as it enhances effector T cells (Tcffs) activity.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0021] Figure 1 shows P1A1 induces cytolysis against human CD137-expressing cancer cells. Representative time-course cytolysis curve of NK cells against (A) RD 18, (B) HK1, (C) C666, (D) RD18-CD137, (E) HK1-CD137, (F) C666-CD137. Data are representative for 2 independent experiments for each cell line. All samples were assessed in duplicates in each experiment. Statistical analysis by two-way ANOVA compared to IgGl treatments. Representative cytolysis curve of NK cells at indicated E:T ratios against (G) SNK-1, (H) SNK-6, and (I) HDLM-2 cells. Data are representative for 2 sets of independent experiments for each cell line. All samples were assessed in triplicates in each experiment, ns: not significant, * p value < 0.05, ** p value < 0.01, *** p value < 0.001 using Student’s t-test compared to the IgGl treatments.

[0022] Figure 2 shows that NK cells are activated by P1A1 during ADCC. Representative histogram and normalized mean fluorescence intensities (MFI) of surface marker on the NK cells in the ADCC assays. (A) CD16, (B) CD137, (C) CD25, (D) CD69, (E) 0X40. Percentages of the positive population and MFI are indicated within the histograms. Data was pooled from 9 experiments using the following cell lines: C666 (3 donors), HK1 (2 donors) and RD18 (4 donors). Each dot represents one donor and independent experiment, ns: not significant; * p value < 0.05, ** p value < 0.01 using Student’s t-test compared to the IgGl isotype.

[0023] Figure 3 shows that Pl Al activates NK cells and induces cytolysis against autologous Tregs. An ADCC assays with Trcg as targets and autologous NK cells was conducted. (A) Representative viability scatter plots for PMA and ionomycin stimulated Tregs. Numbers in quadrants indicate percentages of positive cells. (B) Quantification of dead cells among stimulated Tregs. (C) Representative histogram and normalized MFI of surface marker on the NK cells in the ADCC assays. (D) Representative scatter plots and normalized MFI of degranulation markers and intracellular cytokines in autologous NK cells in a Treg degranulation assays. Percentages of the positive population are indicated in the plots. Each dot represents one donor in an independent experiment, ns: not significant; * p value < 0.05, ** p value < 0.01 using Student’s t-test compared to the IgGl isotype.

[0024] Figure 4 shows that murine chimeric P1A1 induces cell death of murine CD137+cells. (A) Representative time-course cytolysis curve of B 16-control and B16- murine CD137 by mouse splenocytes. Data are representative of two independent experiments. All samples were assessed in duplicates in each experiment. Statistical analysis by two-way ANOVA compared to IgGl treatments. (B) Representative scatter plot of phagocytosis of murine A20 cells by mouse macrophage cell line RAW-LKO. Percentages of phagocytosed cells are indicated in the plots. (C) Percentages of phagocytosis of CFSE-labeled A20 and A20-CD137 cells by RAW-LKO cells from 2 independent experiments. All samples were assessed in duplicates in each experiment. (D) Schematic diagram of the B16-murine CD137 lung metastasis model and applied treatments. Expression of murine CD137 on B16-mCD137 was checked. Percentages of murine CD137+cells and MFI arc indicated in the histogram. (E) Representative photographs of tumor nodules in the lungs. Arrows point to melanoma nodules. Quantification of tumor nodules pooled from 3 independent experiments. (F) Comparison of the percentages of CD137-expressing CD4+FOXP3+splenic Tregs. Percentages of positive cells are indicated in the representative scatter plots. Each dot represents one mouse, ns, not significant; * p value < 0.05; ** p value < 0.01, *** p value < 0.001 using Student’s t-test.

[0025] Figure 5 shows that mPlAl inhibits growth of oncogene-induced orthotopic HCC. (A) Schematic diagram of the murine HCC tumor model and applied treatments. (B) Representative tumor morphology. Tumor nodules and liver weights. (C) Comparison of the percentages and absolute counts of FOXP3+CD4+T cells in thetumors. (D) Comparison of percentages of CD137+Tregs in tumors. (E,F) Representative histograms and MFI of PD-1 and CD69 expression on Tregs in tumors. (G-I) Comparison of percentages of CD 137 and levels of IFN-y and TNF-a in CD4+FOXP3 Teff in tumors. (J-K) Comparison of percentages of CD137 and levels of IFN-y and TNF-a in CD8+T cells in tumors. (M-O) Comparison of percentages of CD137 and levels of IFN-y and TNF-a in CD3" NK1.1+NK cells in tumors. Each dot represents one mouse. Data are representative of two independent experiments. * p value < 0.05, ** p value < 0.01, *** p value < 0.001 using Student’s t-test.

[0026] Figure 6 shows that P1A1 is internalized upon binding to CD137. (A) Time-course of Average Red Object Mean Intensity (RCU) of internalized Fabfluor- labelled antibodies. (B) Representative red fluorescence showing internalized antibodies after 24 h incubation. The scale bar is 200 pm Data are representative of two independent experiments. ** p value < 0.01 using 2-way ANOVA.

[0027] Figure 7 shows mapping of the P1A1 binding site. (A) CD137 expression on the nasopharyngeal carcinoma cell line C666-CD137. (B-D) competitive binding of murine chimeric P1A1 (mPlAl) with (B) human P1A1, (C) Urclumab, or (D) Utomulimab. This data is representative of 3 independent experiments. (E) Schematic of Pl Al binding to CD 137.

[0028] Figure 8 shows CD137 expression on cells. All the CD137-transfected cell lines are more than 80% CD137+, whereas controls are devoid of CD 137. (A) C666 and C666-CD137. (B) HK1 and HK1-CD137. (C) RD18 and RD18-CD137. Expression of CD137 on endogenous CD137-expressing cell lines (D) SNK-1; (E) SNK-6 and (F) HDLM-2. (G) CD137 expression on PMA (100 ng / ml) and ionomycin (1 mg / ml) stimulated Tregs and unstimulated primary Tregs. Percentages of CD137+cells and mean fluorescence intensities (MFI) are indicated within the histograms.

[0029] Figure 9 shows that P1A1 has no agonist activities. PBMCs were sub- optimally activated with 0.1 ng / ml anti-CD3 antibody (clone: OKT3) and incubated with 10 pg / ml P1A1 or IgGl isotype for 48 h followed by flow cytometry. Representative histogram and normalized mean fluorescence intensities (MFI) of (A) CD25, (B) CD69 and (C) 0X40. Percentages of positive cells and MFI arc indicated within the histograms. Data was pooled from 3 donors. Each dot represents one donor and independent experiment, ns: not significant using Student’s t-test compared to the IgGl isotype.

[0030] Figure 10 shows effects of P1A1 in ADCC assays with murine cell lines. Human NK cells were coculturcd with 104target cells at E:T ratios of 5:1 and 10 |ag / ml P1A1 or isotype control antibody. (A,B) Cytolysis was monitored using the xCelligence RTCA system. Representative cytolysis curves of (A) B16 and (B) B16-mCD137. Data are representative for 2 independent experiments. All samples were assessed in duplicates in each experiment, ns: not significant, ** p value < 0.01 using two-way ANOVA compared to IgGl treatments. (C) Cytolysis against A20 and A20-mCD137 was analysed by Annexin V and 7-AAD staining after 20 h of coculture. ** p value < 0.01, using Student’s t-test comparing P1A1 to the IgGl isotypc.

[0031] Figure 11 shows that mPlAl binds to murine CD137 and does not block the detection of murine CD137 by flow antibodies. B16-mCD137 cells were incubated with murine chimeric P1 A1 (mPl Al ) followed by PE-conjugated anti-mouse IgG and APC-conjugated anti-mouse CD137 (clone 17B5, Biolegend), and then analysed by flow cytometry. (A) Binding of mPlAl to B16-mCD137. (B) Detection of murine CD137 by flow antibody. Percentages of CD137+cells and mean fluorescence intensities (MFI) arc indicated within the histograms. (C) Scatter plots showing double positive population for mPlAl binding and murine CD 137 detection. Percentages of cells in each quadrant are indicated.

[0032] Figure 12 shows that P1A1 is internalized upon binding to CD137. Representative photographs taken by Incucyte of Figure 6 where Fabfuor-labeled antibodies were incubated with HDLM-2 cells. (A) Brightfield+red showing similar cell counts in the field; (B) Red fluorescence showing internalized antibody. The scale bar is 200 pm.

[0033] Figure 13 shows mapping of the P1A1 binding site. (A-D) Rhabdomyosarcoma cell line RD18-CD137 and (E-H) Nasopharyngeal carcinoma cell line HK1-CD137. (A,E) CD137expression. Competitive binding murine chimeric PlAl(mPlAl) with (B,F) human P1A1, (C,G) Urelumab, or (D,H) Utomulimab.

[0034] Figure 14 shows (A) CD137-mediated T cell costimulation. An APC takes up antigen (e.g. a pathogen) and presents epitopes to a cognate T cell. Engagement of the T cell receptor (TCR) induces expression of CD137 through which the T cell can receive costimulation. (B) CD137-mediated feed-back inhibition. Following engagement of CD137 by CD137L, CD137 is transferred to the APC by trogocytosis, where it forms a complex with CD137L which is internalized and degraded. Faded symbols indicateformer localization. (C). Schematic representation of Anti-CD137 antibodies inducing ADCC.

[0035] Figure 15 shows that P1A1 induces ADCC in cells expressing canine or feline CD137. Human NK cells were cocultured with 104target cells at E:T ratios of 5:1 and 10 pg / ml Pl A1 or isotype control antibody. Antibodies were added at the 20 h time point. Triton was used as a positive control to achieve 100% lysis. (A) Target = HEK293 cells expressing human CD137. (B) Target = HEK293 cells expressing canine CD137. (C) Target = HEK293 cells expressing feline CD137. Cytolysis was monitored using the xCclligcncc RTCA system.

[0036] Figure 16 shows that mPlAl inhibits growth of CD137-expressing B16 melanoma. (A) CD 137 expression on CD137-expressing murine melanoma cell line B 16 (B16-CD137). (B) Tumor nodules in the lungs. Arrows point to melanoma nodules. (C) Percentages of CD137+cells among CD4+FOXP3+splenic Tregs. Each dot represents one mouse. * p value < 0.05; ** p value < 0.01 using Student’ s t-test.

[0037] Figure 17 shows the antagonistic nature of P1A1. - P1A1 blocks CD137 - CD137L interaction. (A.B) P1A1 blocks binding of recombinant CD137L protein to CD137-expressing MCF-7 cells. (A) schematic depiction of experimental set-up. (B) Depicted are the percentages of cells that were not stained by biotinylated recombinant CD137L protein + streptavidin-APC in the presence of Pl Al or the isotype controls human IgG (HuIgG) and HA4. 3H is an antibody that does not recognize CD137 and serves as additional control. (C,D) P1A1 inhibits signaling of CD137 in L-428 cells (C) schematic depiction of experimental set-up. (D) Depicted are the concentrations of TNF in culture supernatants as determined by ELISA in the presence of P1A1 or the isotypc controls human IgG (HuIgG) and HA4. 3H is an antibody that does not recognize CD137 and serves as additional control. (E) Pl Al inhibits T cell activation. T cells isolated from PBMCs were cultured on plates coated with 5 pg / ml of anti-CD3 (clone OKT3) and 5 pg / ml of the isotype control antibody (HA4) or P1A1 or the agonistic anti-CD137 antibody P2B4 (positive control). After 48 h, fFN-y concentrations in supernatants were determined by ELISA. Means ± SD of triplicate measurements are depicted. ** p<0.01 . Data is representative of 3 independent experiments.

[0038] Figure 18 shows that an anti-CD137 P1A1 x anti-CD3 bispecific antibody (BsAb) can function as a T cell engager. (A) An illustration of T cell engager BsAb format; (B) An in vitro cell killing assay was performed using the P1A1 BsAb and anisotype control BsAb, both containing the same anti-CD3 ami. The BsAbs were co- culturcd with naive human CD8 T cells and tumor cells to assess BsAb-induccd tumor cell killing. The assay was conducted with C666, HK1, RD18, and RH41 cell lines, all engineered to express human CD137. Cytotoxicity induced by the BsAb was continuously measured for 72 hours using the xCELLigence RTCA system. The resulting killing curves, showing the percentage of cytotoxicity relative to BsAb concentration at the 72-hour time point, were plotted.DETAILED DESCRIPTION

[0039] Regulatory T cells (Tregs) contribute significantly to the immunosuppressive nature of the tumor microenvironment which is a main barrier for immunotherapies of solid cancers. Reducing Treg numbers enhances anti-tumor immune responses but current depletion strategies also impair effector T cells (Teffs), potentially leading to reduced anti-tumor immunity and / or autoimmune diseases. CD137 has been identified as the most differentially expressed gene between peripheral Tregs and intratumoral Tregs in virtual ly all solid cancers. Further, CD137 is expressed by malignant cells of certain cancers, making it a potential target for tumor immunotherapy. The present invention discloses the development of a fully human anti-human CD137 antibody of the IgGl isotype, clone P1 A1 , that induces antibody-dependent cell-mediated cytotoxicity (ADCC) in CD137+Tregs and cancer cells. P1A1 cross-reacts with murine CD137 which allowed testing murine chimeric P1A1 in syngeneic murine tumor models where P1A1 significantly reduced the number of CD137+Tregs and inhibited tumor growth in a murine hepatocellular carcinoma (HCC) and a melanoma lung metastasis model. P1A1 can also be internalized thus enabling it as a carrier for drugs to target CD137+Tregs and cancer cells. These anti-cancer properties suggest a translation of P1A1 to human immunotherapy.

[0040] In one aspect, the present disclosure refers to an antibody or an antigen binding fragment thereof comprising:(A) a heavy chain variable region comprising the following CDRs:(a) CDR-H1: GYSLTSYGIS [SEQ ID NO:1] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(b) CDR-H2: WMGWISAYNGNTNYAQKLQG [SEQ ID N0:2] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(c) CDR-H3: ASYYDSSGYAFDI [SEQ ID NO:3] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; and(B) a light chain variable region comprising the following CDRs:(a) CDR-L1: SGDKLEDKYAS [SEQ ID NO:4] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(b) CDR-L2: QDYKRPS [SEQ ID NO:5] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(c) CDR-L3: QTWDRITTSYV [SEQ ID NO:6] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; wherein the antibody or antigen binding fragment binds to CD137 antagonistically.

[0041] As used herein, the term " antibody " refers to an immunoglobulin molecule able to bind to a specific epitope on an antigen such as CD137 (also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB). Antibodies can be comprised of a polyclonal mixture, or may be monoclonal in nature. Further, antibodies can be entire immunoglobulins derived from natural sources, or from recombinant sources. The antibodies of the present invention may exist in a variety of forms, including for example as a whole antibody, or as an antibody fragment, or other immunologically active fragment thereof, such as complementarity determining regions. Similarly, the antibody may exist as an antibody fragment having functional antigen-binding domains (i.e. antigen binding fragment), that is, heavy and light chain variable domains. Also, the antibody fragment may exist in a form selected from the group consisting of, but not limited to: Fv, Fab, F(ab)2, scFv (single chain Fv), dAb (single domain antibody), bispecific antibodies, diabodics and triabodics.

[0042] Antibodies are usually heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.

[0043] Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0044] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P- sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC). For the antibody disclosed herein, its constant domains of the fragment crystallizable region (Fc region) bind to Fey receptors (FcyR) on immune cells, including NK cells and phagocytes, to trigger antibody dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) against target cells.

[0045] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" and / or those residues from a "hypervariable loop". "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0046] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual fragment crystallizable ("Fc") fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0047] Typically, greater than 60% sequence identity between two antibodies or antigen binding fragments thereof is considered to be an indication of functional equivalence, provided that either the biological activity of the antibodies or antigen binding fragments thereof (for example, the ability to bind to CD 137 antagonistically and induces ADCC, ADCP or Complement-dependent cytotoxicity (CDC) of CD137- expressing cancer cells and / or Treg) is retained, or the antibody or antigen binding fragment thereof possesses an antigenic determinant in common with the antibody or antigen binding fragment thereof comprising the CDR sequences SEQ ID NO: 1-6 as disclosed herein. Preferably, a functionally equivalent antibody or antigen binding fragment thereof according to this aspect of the invention exhibits a degree of sequence identity with the antibody or antigen binding fragment thereof as disclosed herein, or with a fragment thereof, of greater than 60%. More preferred antibodies or antigen binding fragments thereof have degrees of sequence identity of greater than 70%, 80%, 90%, 95%, 98% or 99%, respectively.

[0048] In one particular example, the antibody or antigen binding fragment binds to CD137 antagonistically and induces ADCC, ADCP or CDC of CD137-expressing cancer cells and / or Treg, and is named "P1A1". P1A1 comprises (A) a heavy chain variable region comprising: (a) CDR-H1: GYSLTSYGIS [SEQ ID NO:1]; (b) CDR-H2: WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]; and (c) CDR-H3: ASYYDSSGYAFDI [SEQ ID NO:3]; and (B) a light chain variable region comprising: (a) CDR-L1: SGDKLEDKYAS [SEQ ID NO:4J; (b) CDR-L2: QDYKRPS [SEQ ID NO:5]; and (c) CDR-L3: QTWDRITTSYV [SEQ ID NO:6]. P1A1 comprises a heavy chain comprising a variable region of SEQ ID NO:7 and a constant region of SEQ ID NO: 9; and a light chain comprising a variable region of SEQ ID NO:8 and a constant region of SEQ ID NO: 10. Pl Al comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:1 1 , and a light chain comprising the amino acid sequence of SEQ ID NO:12. The detailed sequences of this invention is listed in Table 1.

[0049] Table 1 Sequence listing of the P1A1 antibody (lie abet sequence. signature residues)

[0050] The antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H1 having at least 80% sequence identity with the sequence of CDR- H1 of P1A1 (GYSLTSYGIS [SEQ ID NO:1]), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H1 having at least 80% sequence identity with the sequence of CDR-H1 of P1 A1 (GYSLTSYGIS [SEQ ID NO:1 ]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR- H1 having at least 85% sequence identity with the sequence of CDR-H1 of P1A1 (GYSLTSYGIS [SEQ ID NO: 1]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H1 having at least 90% sequence identity with the sequence of CDR-H1 of P1A1 (GYSLTSYGIS [SEQ ID NO:1]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H1 having at least 95% sequence identity with the sequence of CDR- H1 of P1A1 (GYSLTSYGIS [SEQ ID NO:1]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H1 having 100% sequence identity with the sequence of CDR-H1 of P1A1 (GYSLTSYGIS [SEQ ID NO:1]).

[0051] The antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H2 having at least 80% sequence identity with the sequence of CDR- H2 of Pl A1 (WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H2 having at least 80% sequence identity with the sequence of CDR-H2 of P1A1 (WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H2 having at least 85% sequence identity with the sequence of CDR-H2 of P1A1 (WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]). In another example, the antibody or an antigen binding fragment thereof as disclosedherein comprises a CDR-H2 having at least 90% sequence identity with the sequence of CDR-H2 of P1A1 (WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H2 having at least 95% sequence identity with the sequence of CDR- H2 of Pl A1 (WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR- H2 having 100% sequence identity with the sequence of CDR-H2 of P1A1 (WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2]).

[0052] The antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H3 having at least 80% sequence identity with the sequence of CDR- H3 of P1A1 (ASYYDSSGYAFDI [SEQ ID NO:3]), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H3 having at least 80% sequence identity with the sequence of CDR-H3 of P1A1 (ASYYDSSGYAFDI [SEQ ID NO:3J). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H3 having at least 85% sequence identity with the sequence of CDR- H3 of P1A1 (ASYYDSSGYAFDI [SEQ ID NO:3]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H3 having at least 90% sequence identity with the sequence of CDR-H3 of P1 A1 (ASYYDSSGYAFDI [SEQ ID NO:3]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H3 having at least 95% sequence identity with the sequence of CDR-H3 of Pl Al (ASYYDSSGYAFDI [SEQ ID NO:3]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-H3 having 100% sequence identity with the sequence of CDR-H3 of Pl Al (ASYYDSSGYAFDI [SEQ ID NO:3]).

[0053] The antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L1 having at least 80% sequence identity with the sequence of CDR- L1 of P1A1 (SGDKLEDKYAS [SEQ ID NO:4J), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L1 having at least 80% sequence identity with the sequence of CDR-L1 of P1 Al (SGDKLEDKYAS [SEQ ID NO:4]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR- L1 having at least 85% sequence identity with the sequence of CDR-L1 of P1A1(SGDKLEDKYAS [SEQ ID N0:4]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L1 having at least 90% sequence identity with the sequence of CDR-L1 of P1A1 (SGDKLEDKYAS [SEQ ID NO:4]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L1 having at least 95% sequence identity with the sequence of CDR-L1 of Pl Al (SGDKLEDKYAS [SEQ ID NO:4]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR- L1 having 100% sequence identity with the sequence of CDR-L1 of P1A1 (SGDKLEDKYAS [SEQ ID NO:4]).

[0054] The antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L2 having at least 80% sequence identity with the sequence of CDR- L2 of Pl Al (QDYKRPS [SEQ ID NO:5]), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L2 having at least 80% sequence identity with the sequence of CDR-L2 of P1A1 (QDYKRPS [SEQ ID NO:5]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L2 having at least 85% sequence identity with the sequence of CDR-L2 of P1A1 (QDYKRPS [SEQ ID NO:5]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L2 having at least 90% sequence identity with the sequence of CDR-L2 of P1A1 (QDYKRPS [SEQ ID NO:5]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises CDR-L2 having at least 95% sequence identity with the sequence of CDR-L2 of Pl Al (QDYKRPS [SEQ ID NO:5]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L2 having 100% sequence identity with the sequence of CDR-L2 of Pl Al (QDYKRPS [SEQ ID NO:5]).

[0055] The antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L3 having at least 80% sequence identity with the sequence of CDR- L3 of P1A1 (QTWDRITTSYV [SEQ ID NO:6J), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L3 having at least 80% sequence identity with the sequence of CDR-L3 of Pl Al (QTWDRITTSYV [SEQ ID NO:6]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises CDR- L3 having at least 85% sequence identity with the sequence of CDR-L3 of P1A1(QTWDRITTSYV [SEQ ID NO:6]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR-L3 having at least 90% sequence identity with the sequence of CDR-L3 of P1A1 (QTWDRITTSYV [SEQ ID NO: 6]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises CDR-L3 having at least 95% sequence identity with the sequence of CDR-L3 of P1A1 (QTWDRITTSYV [SEQ ID NO:6]). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a CDR- L3 having 100% sequence identity with the sequence of CDR-L3 of P1A1 (QTWDRITTSYV [SEQ ID NO:6]).

[0056] The antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain variable region having at least 80% sequence identity with the sequence of heavy chain variable region of Pl Al (SEQ ID NO:7), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain variable region having at least 80% sequence identity with the sequence of heavy chain variable region of P1A1 (SEQ ID NO:7). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain variable region having at least 85% sequence identity with the sequence of heavy chain variable region of P1A1 (SEQ ID NO:7). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain variable region having at least 90% sequence identity with the sequence of heavy chain variable region of P1A1 (SEQ ID NO:7). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain variable region having at least 95% sequence identity with the sequence of heavy chain variable region of P1A1 (SEQ ID NO:7). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain variable region having 100% sequence identity with the sequence of heavy chain variable region of Pl Al (SEQ ID NO:7).

[0057] The antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain variable region having at least 80% sequence identity with the sequence of light chain variable region of P1A1 (SEQ ID NO:8), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain variable region having at least 80% sequence identity with the sequence of light chain variable region of P1A1(SEQ ID N0:8). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain variable region having at least 85% sequence identity with the sequence of light chain variable region of P1A1 (SEQ ID NO:8). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain variable region having at least 90% sequence identity with the sequence of light chain variable region of P1A1 (SEQ ID NO:8). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain variable region having at least 95% sequence identity with the sequence of light chain variable region of P1A1 (SEQ ID NO:8). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain variable region having 100% sequence identity with the sequence of light chain variable region of P1 A1 (SEQ ID NO:8).

[0058] The antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain constant region having at least 80% sequence identity with the sequence of heavy chain constant region of Pl Al (SEQ ID NO:9), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain constant region having at least 80% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain constant region having at least 85% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain constant region having at least 90% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain constant region having at least 95% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain constant region having 100% sequence identity with the sequence of heavy chain constant region of Pl Al (SEQ ID NO:9).

[0059] Depending on the amino acid sequence of the constant domain of their heavy chains, full length antibodies can be assigned to different "classes". There are five major classes of full length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into "subclasses" (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called a, 8, e, y, and p, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0060] The isotype of the antibody Pl A1 as disclosed herein, as well as the antibody or an antigen binding fragment thereof as disclosed herein comprising a heavy chain constant region having at least 80% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9), for example at least 80%, 90%, or 95% sequence identity, is IgGl. The antibody with a human IgGl isotypc is able to induce ADCC, ADCP, and / or CDC and kill human malignant cells and Tregs that express human CD 137. In another example, the isotype of the antibody as disclosed herein, as well as the antibody or an antigen binding fragment thereof as disclosed herein comprising a heavy chain constant region having at least 80% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9), for example at least 80%, 90%, or 95% sequence identity, is IgG2a, which is a murine isotype. The antibody with a murine IgG2a isotypc is able to induce ADCC, ADCP, and / or CDC and kill murine malignant cells and Tregs that express murine CD137. In another example, the isotype of the antibody as disclosed herein, as well as the antibody or an antigen binding fragment thereof as disclosed herein comprising a heavy chain constant region having at least 80% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9), for example at least 80%, 90%, or 95% sequence identity, is IgGl or IgG2, which are canine isotypes. The antibody with a canine IgGl or IgG2 isotype is able to induce ADCC, ADCP, and / or CDC and kill canine malignant cells and Tregs that express canine CD 137. In another example, the isotype of the antibody as disclosed herein, as well as the antibody or an antigen binding fragment thereof as disclosed herein comprising a heavy chain constant region having at least 80% sequence identity with the sequence of heavy chain constant region of P1A1 (SEQ ID NO:9), for example at least 80%, 90%, or 95% sequence identity, is IgGl, which is a feline isotype. The antibody with a feline IgGl isotype is able to induce ADCC, ADCP, and / or CDC and kill feline malignant cells and Tregs that express feline CD137.

[0061] The antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain constant region having at least 80% sequence identity with the sequence of light chain constant region of Pl Al (SEQ ID NO: 10), for example at least80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain constant region having at least 80% sequence identity with the sequence of light chain constant region of P1A1 (SEQ ID NO: 10). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain constant region having at least 85% sequence identity with the sequence of light chain constant region of P1A1 (SEQ ID NO:10). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain constant region having at least 90% sequence identity with the sequence of light chain constant region of Pl Al (SEQ ID NO: 10). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain constant region having at least 95% sequence identity with the sequence of light chain constant region of Pl A 1 (SEQ ID NO: 10). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain constant region having 100% sequence identity with the sequence of light chain constant region of Pl Al (SEQ ID NO: 10).

[0062] The "light chains" of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ), based on the amino acid sequences of their constant domains. In one example, the light chain constant region of the antibody or an antigen binding fragment thereof as disclosed herein is a kappa (K) light chain. In another example, the light chain constant region of the antibody or an antigen binding fragment thereof as disclosed herein is a lambda (X) light chain. In a particular example, the P1A1 antibody disclosed herein has a lambda (X) light chain.

[0063] In one example, the antibody or antigen- binding fragment thereof as disclosed herein further comprises an Fc region. In another example, the antibody or antigenbinding fragment thereof as disclosed herein does not have an Fc region.

[0064] The antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain having at least 80% sequence identity with the sequence of heavy chain of P1A1 (SEQ ID NO: 11), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain having at least 80% sequence identity with the sequence of heavy chain of Pl Al (SEQ ID NO:1 1 ). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain having at least 85% sequence identity with the sequence of heavy chain of Pl Al (SEQ ID NO: 11). In anotherexample, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain having at least 90% sequence identity with the sequence of heavy chain of P1A1 (SEQ ID NO: 11). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain having at least 95% sequence identity with the sequence of heavy chain of Pl Al (SEQ ID NO:1 1 ). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a heavy chain having 100% sequence identity with the sequence of heavy chain of PlAl (SEQ ID NO:11).

[0065] The antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain having at least 80% sequence identity with the sequence of light chain of P1A1 (SEQ ID NO: 12), for example at least 80%, 90%, or 95% sequence identity. In one example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain having at least 80% sequence identity with the sequence of light chain of P1A1 (SEQ ID NO: 12). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain having at least 85% sequence identity with the sequence of light chain of Pl Al (SEQ ID NO: 12). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain having at least 90% sequence identity with the sequence of light chain of Pl A 1 (SEQ ID NO: 12). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain having at least 95% sequence identity with the sequence of light chain of P1A1 (SEQ ID NO: 12). In another example, the antibody or an antigen binding fragment thereof as disclosed herein comprises a light chain having 100% sequence identity with the sequence of light chain of P1A1 (SEQ ID NO: 12).

[0066] The antibody or an antigen binding fragment thereof with at least 80% sequence identity with the sequence of with one or more of the CDR sequences, or heavy chain variable region, or light chain variable region, or heavy chain constant region, or light chain constant region, or heavy chain, or light chain of P1A1 has an amino acid sequence which differs from the sequence of one or more of the CDR sequences, or heavy chain variable region, or light chain variable region, or heavy chain constant region, or light chain constant region, or heavy chain, or light chain ofP1 Al . Ordinarily, amino acid sequence variants will possess at least about 70% homology with the antibody P1A1, and preferably, they will be at least about 80%, more preferably at least about 90%, at leastabout 95%, about 100% homologous with the antibody P1A1. The amino acid sequence variants possess substitutions, deletions, and / or additions at certain positions within or adjacent to the amino acid sequence of the antibody Pl Al. Examples of amino acid sequence variants herein include acidic variant (e.g. deamidated antibody variant), basic variant, the antibody with an amino-terminal leader extension (e.g. VHS-) on one or two light chains thereof, antibody with a C-terminal lysine residue on one or two heavy chains thereof, etc, and includes combinations of variations to the amino acid sequences of heavy and / or light chains.

[0067] As used herein, "antigen binding fragment" comprise a portion of a full length antibody such as Pl Al, preferably comprising the antigen- binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s). In one example, the antigen binding fragment as disclosed herein is a Fv fragment selected from the group consisting of a single chain Fv (scFv) or disulphide-bonded Fv. In another example, the antigen binding fragment as disclosed herein is a Fab-like fragment selected from the group consisting of a Fab fragment, Fab' fragment, and a F(ab')2 fragment.

[0068] "Fv" is the minimum antibody fragment which contains a complete antigenrecognition and antigen- binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VE dimer. Collectively, the six hypcrvariablc regions confer antigen- binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0069] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VE domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0070] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domainincluding one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab1in which the cysteine rcsiduc(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments originally are produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0071] In one example, the antibody or an antigen binding fragment thereof as disclosed herein is soluble in an aqueous medium such as water or a cell culture medium. Being soluble makes the antibody or an antigen binding fragment thereof as disclosed herein able to be developed and translated to clinical applications easily.

[0072] hi one example, the antibody or an antigen binding fragment thereof as disclosed herein is a human antibody or antigen binding fragment thereof. Advantageously, a fully human antibody can be translated clinically in a straightforward manner.

[0073] In another example, the antibody or an antigen binding fragment thereof as disclosed herein is a humanized antibody or antigen binding fragment thereof. "Humanized" forms of non-human (c.g., rodent) antibodies arc chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0074] The antibody or an antigen binding fragment thereof with at least 80% sequence identity with the sequence of with one or more of the CDR sequences, one ormore of heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, heavy chain, and light chain of Pl Al, bind to the same or an overlapping epitope on CD137 with Utomilumab, which is at border regions of the CRD3 and CRD4 domains.

[0075] The antibody or an antigen binding fragment thereof with at least 80% sequence identity with one or more of the CDR sequences, one or more of heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, heavy chain, and light chain of P1A1, have the same function as P1A1, that is, binding to CD137 on target cells such as cancer cells and / or Trcg, and inducing antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137- expressing cancer cells and / or Treg.

[0076] The antibody or an antigen binding fragment thereof as disclosed herein binds to CD137 antagonistically, thus can be termed an "antagonistic antibody". As used herein, an "antagonistic antibody" refers to an antibody (such as P1A1) that binds to a specific target (such as CD 137), and inhibits its biological activity. The antagonistic antibody acts as antagonists by blocking or interfering with the target’s normal function, often preventing it from interacting with its natural ligand and / or initiating downstream signaling pathways. This is in contrast to an agonist antibody, which binds to a target and mimics the action of its natural ligand, activating the target and inducing a biological response. In the present invention, the antibody or an antigen binding fragment thereof as disclosed herein, such as P1A1, specifically binds to CD137, which is expressed on a cancer cell, and / or an intratumoral Trcg, thus inhibits CD137 signalling in Trcg and reduces the activity of Treg, resulting in a less immunosuppressive tumor environment. Further, in the present invention, the antibody or an antigen binding fragment thereof as disclosed herein, such as P1A1, specifically binds to CD137, which is expressed on a cancer cell, and / or an intratumoral Treg, thus inhibits CD137 signalling in cancer cells and reduces the tumor- supporting signaling, resulting in reduced cancer growth and metastatic spread. In the present invention, the antibody or an antigen binding fragment thereof as disclosed herein, such as P1A1, interferes with CD137 - CD137L interaction by blocking the binding of CD137L to CD 137, and preventing CD 137 signalling. In addition, the antagonistic antibody as disclosed herein blocks the interaction of CD137 on the cancer cell and / or intratumoral Treg with CD137L expressed on antigen presentingcells (APC). Cancer cells and / or intratumoral Treg, upon binding to CD137L on APC, would utilize a negative feedback mechanism to allow trogocytic transfer of CD 137 from cancer cells and / or intratumoral Treg to APC, where the transferred CD137 and the endogenous CD137L form a complex that is internalized and degraded by the proteasome (Fig. 14B). By blocking CD137 on cancer cells and / or intratumoral Treg from interacting with CD137L on APC, APC are free to interact with CD137 expressed on effector T cells (Teffs) for anti-tumor immunity. Its antagonistic nature provides distinct advantages to P1A1 since there is evidence that CD137 signalling enhances Treg activity similarly as it enhances Toff activity.

[0077] Pl Al bound to CD 137 on target cells such as cancer cell and intratumoral Treg and then engage and activate NK cells and phagocytes via Fey receptor CD 16, triggering cytotoxic responses of NK cells and phagocytes, such as antibody dependent cell- mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) against target cells. As a result, the antibody or an antigen binding fragment thereof as disclosed herein, such as Pl Al selectively enhanced cytotoxicity of NK cells against CD137-cxprcssing cells such as cancer cell and intratumoral Treg. By inducing ADCC, ADCP and CDC against CD137-expressing cancer cells, the antibody or an antigen binding fragment thereof as disclosed herein, such as Pl A1 , reduces the population of tumor cells, reduces tumor size and inhibits tumor growth directly. By inducing ADCC, ADCP and CDC against CD137-expressing intratumoral Treg, the antibody or an antigen binding fragment thereof as disclosed herein, such as P1A1, reduces the population of intratumoral, CD137-expressing Tregs, thereby resulting in an overall less immunosuppressive tumor microenvironment and indirectly inhibiting tumor growth.

[0078] In another aspect, the present disclosure refers to a bispecific antibody, comprising (i) a first binding arm comprising a first antigen-binding region binding to CD137 antagonistically, wherein the first antigen-binding region comprises (a) heavy chain variable (VH) region CDR-H1, CDR-H2, and CDR-H3 having the sequences as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable (VL) region CDR- Ll, CDR-L2, and CDR-L3 having the sequences as set forth in SEQ ID NOs:4, 5 and 6, respectively, and (ii) a second binding arm comprising a second antigen-binding region binding to human CD3.

[0079] The term "bispecific" means that the antibody with two different antigenbinding regions for binding to two distinct antigenic determinants, for example two binding regions each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) binding to different antigens or to different epitopes on the same antigen. Typically, a bispecific antibody comprises two antigen binding regions, each of which is specific for a different antigenic determinant. In one example, the first antigen-binding region of the bispecific antibody as disclosed herein binds to CD137 antagonistically, and it comprises (a) heavy chain variable (VH) region CDR-H1, CDR-H2, and CDR-H3 having the sequences as set forth in SEQ ID NOs:l, 2, and 3, respectively, and light chain variable (VL) region CDR-L1, CDR-L2, and CDR-L3 having the sequences as set forth in SEQ ID NOs:4, 5 and 6, respectively, or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity, as shown below:CDR-H1: GYSLTSYG1S [SEQ ID NO:1J or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;CDR-H2: WMGWISAYNGNTNYAQKLQG [SEQ ID NO:2] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;CDR-H3: ASYYDSSGYAFDI [SEQ ID NOG] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; andCDR-L1: SGDKLEDKYAS [SEQ ID NO:4] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;CDR-L2: QDYKRPS [SEQ ID NOG] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;CDR-L3: QTWDRITTSYV [SEQ ID NO:6] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity.

[0080] In another example, the second antigen-binding region of the bispccific antibody as disclosed herein binding to human CD3 comprises the VH sequence of SEQ ID NO: 17, and the VL sequence of SEQ ID NO: 18. In another example, the second antigen-binding region of the bispecific antibody as disclosed herein binding to humanCD3 is an scFv. and the VH sequence and VL sequence are connected by a linker comprising the amino acid sequence of SEQ ID NO: 19. In another example, the second antigen-binding region of the bispecific antibody as disclosed herein binding to human CD3 comprises an amino acid sequence of SEQ ID NO:20. The above sequences, as well as nucleic acid sequences encoding the same, i.e. SEQ ID NO:21 -24, are displayed in Table 2.

[0081] Table 2. Sequences of the second antigen-binding region of the bispecific antibody as disclosed herein binding to human CD3

[0082] The present invention also provides bi specific antibodies comprising functional variants of the VL regions, VH regions, or one or more CDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a bispecifc antibody still allows each arm of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or the specificity / selectivity of the parent bispccific antibody and in some cases such a bispccific antibody may be associated with comparable or greater affinity and / or the specificity / selectivity than the parent bispecific antibody. Exemplary variants include those which differ from VHand / or VL and / or CDR regions of the parent bispecific antibody sequences mainly by conservative substitutions; for instance 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

[0083] In the present invention, the first antigen-binding region binds to CD 137 expressed on cancer cells and / or Treg, while the second antigen-binding region binds to human CD3 on a T cell, and the bispecific antibody acts as a T cell engager for directing the T cell to target and kill the cancer cells and / or Treg. As used herein, the term "T cell engager" refers to the bispecific antibody as disclosed herein (also referred to as bispecific T-cell engager), consisting of two antigen-binding regions from different antibodies. One binds to a tumor cell or Treg via a tumor- specific antigen such as CD137, and the other binds to T cells via the CD3 receptor. Thus, the T cell engager is designed to direct the body's immune system, specifically T cells, to target and destroy cancer cells and / or Treg that express CD137. In some examples, the two antigen-binding regions can be singlechain variable fragments (scFvs). In some examples, the two antigen-binding regions can be Fab. In some examples, the two antigen-binding regions can be Fab'. A "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-lerminus of the VH with the C- terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. The term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteins from the antibody hinge region. In some examples, the first antigen-binding region comprising the antigen binding fragment, for binding to CD137, is a Fab fragment comprising the CDR sequences as disclosed herein, and the second antigen-binding region binding to human CD3 is a scFv.

[0084] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage).

[0085] In one example, the bispecific antibody as disclosed herein comprises a Fc domain that is an IgGl, particularly hlgGl-KiH-LALA. Thus, the bispccific antibody is a human antibody, which allows easy clinical translation. In another example, the IgGl is hlgGl-KiH-LALA. The "knob-into-hole" (KiH) technology is a method used in the production of bispecific antibodies. This technique involves engineering the CH3 domains of antibodies to create a "knob" on one heavy chain and a "hole" on the other. This design promotes the formation of heterodimers, which are antibodies with two different binding sites, enhancing their therapeutic potential. In another example, the bispccific antibody as disclosed herein comprises a Fc domain that is an IgG2. In another example, the bispecific antibody as disclosed herein comprises a Fc domain that is an IgG4.

[0086] In another aspect, the present disclosure refers to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as disclosed herein or the bispecific antibody as disclosed herein.

[0087] As used herein, the term "nucleic acid molecule" also includes analogues of DNA and RNA, such as those containing modified backbones. The term "nucleic acid" includes a deoxyribonucleotide or ribonucleotide polymer in either single- or doublestranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. In one example, the nucleic acid molecule is a cDNA molecule. The terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence” and polynucleotide etc. are used interchangeably herein unless the context indicates otherwise.

[0088] Nucleic acid molecules encoding an antibody or antigen-binding fragment thereof as disclosed herein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide - mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a nonvariant version of the antibody.

[0089] The nucleic acid molecules encoding an antibody or antigen-binding fragment thereof as disclosed herein may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid molecule is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g. other cellular nucleic acids (e.g. the other parts of the chromosome) orproteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art.

[0090] The nucleic acid encoding Pl Al heavy chain is SEQ ID NO: 13. The nucleic acid encoding Pl Al heavy chain variable region is SEQ ID NO: 14. The nucleic acid sequence encoding Pl Al light chain is SEQ ID N:15. The nucleic acid encoding Pl Al light chain variable region is SEQ ID NO: 16.

[0091] In one example, the nucleic acid molecule encodes a VH sequence of the second antigen-binding region of the bispecific antibody binding to human CD3 and comprises a nucleotide sequence of SEQ ID NO:21. In another example, the nucleic acid molecule encodes a VL sequence of the second antigen-binding region of the bispecific antibody binding to human CD3 and comprises a nucleotide sequence of SEQ ID NO:22. In one example, the nucleic acid molecule encodes a linker connecting the VH sequence and VL sequence of the second antigen-binding region of the bispecific antibody binding to human CD3 and comprises a nucleotide sequence of SEQ ID NO:23. In another example, the nucleic acid molecule encodes the sequence of the second antigen-binding region of the bispccific antibody binding to human CD3 and comprises a nucleotide sequence of SEQ ID NO:24. Details of these sequences are listed in Table 2.

[0092] In another aspect, the present disclosure refers to a vector comprising a nucleic acid molecule as disclosed herein.

[0093] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they arc operatively linked. Such vectors arc referred to herein as "recombinant expression vectors" (or simply, "expression vectors") in general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is themost commonly used form of vector. However, also included are other forms of expression vectors, such as viral vectors (c.g. replication defective retroviruses, baculoviruses, Simian Viruses 40 (SV40), polyomavirus, herpesvirus, papovirus, adenoviruses and adeno-associated viruses), which serve equivalent functions. In one example, the vector is an expression vector.

[0094] In another aspect, the present disclosure refers to a recombinant host cell comprising a nucleic acid molecule as disclosed herein or a vector as disclosed herein.

[0095] The term "recombinant host cell" (or simply "host cell"), as used herein, refers to a cell that comprises a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0096] For expression of the antibody or antigen binding fragment thereof, the expression vector(s) encoding the heavy and light chains of the antibody or antigen binding fragment thereof of as disclosed herein is transfected into a recombinant host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic recombinant host cell, e.g. electroporation, calcium-phosphate precipitation, DEAE-Dextran transfection and the like. Although it is theoretically possible to express the antibody or antigen binding fragment thereof of the invention in either prokaryotic or eukaryotic host cells, expression of the antibody or antigen binding fragment thereof in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody or antigen binding fragment thereof.

[0097] In one example, the recombinant host cell is a bacterial cell selected from the group consisting of E. coli, B. subtilis. Pseudomonas, Lactobacillus, Spirilla, Vibrio, Salmonella, Acetobacter, Cyanobacteria, Spirochetes, Chlamydias, Caulobacter, Mycoplasma, Aliivibrio, Bacteroides, Synechocystis, Azotobacter, Streptomyces,Thermococcus, Sulfolobus, Streptococcus, Methanococcus, Halobacterium, other protcobactcria and a gram-positive bacterium. In another example, the recombinant host cell is a mammalian cell. Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells), NSO (Mouse myeloma) cells, COS and Vero (both green African monkey kidney) cells, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO- Rb50, Hep G2, DUKX-X11, J558L, Baby hamster kidney (BHK) cells and SP2 cells. In another example, the recombinant host cell is a human cell selected from the group consisting of human embryonic kidney HEK293 cells, human Jurkat cells, and HcLa (Human cervical cancer) cells.

[0098] In another aspect, the present disclosure refers to a method for producing an antibody or antigen-binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, the method comprising culturing a recombinant host cell as disclosed herein under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.

[0099] When recombinant expression vectors encoding antibody genes arc introduced into host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0100] hi another aspect, the present disclosure refers to a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof as disclosed herein or the bispecific antibody as disclosed herein, and a pharmaceutically- acceptable diluent, carrier or excipient.

[0101] The diluent, carrier or excipient must be pharmaceutically "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. These are typically molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to the subject.

[0102] Examples of pharmaceutically acceptable carriers or diluents are demineralized or distilled water; saline solution; vegetable based oils such as peanut oil,safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3- butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0103] As used herein, the term "effective amount" refers to a therapeutically effective amount including a sufficient but non-toxic amount of the antibody or antigen binding fragment thereof as disclosed herein to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity' of the condition being treated, the particular agent or composition being contacted or administered, the mode of contact or administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. For example, an effective amount to result in therapeutic amount may be an amount sufficient to result in the improvement of the pathological symptoms of a target disease or an amount sufficient to result in protection against a target infectious disease. An effective dosage of the antibody or antigen-binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, may be in the range of about 100 ng / kg to about 100 mg / kg, about 100 ng / kg to about 90 mg / kg, about 100 ng / kg to about 80 mg / kg, about 100 ng / kg to about 70 mg / kg, about 100 ng / kg to about 60 mg / kg, about 100 ng / kg to about 50 mg / kg, about 100 ng / kg to about 40 mg / kg, about 100 ng / kg to about 30 mg / kg, about 100 ng / kg to about 20 mg / kg, about 100 ng / kg to about 10 mg / kg, about 90 ng / kg to about 100 mg / kg, about 80 ng / kg to about 100 mg / kg, about 70 ng / kg to about 100 mg / kg, about 60 ng / kg to about 100 mg / kg, about 50 ng / kg to about 100 mg / kg, about 40ng / kg to about 100 mg / kg, about 30 ng / kg to about 100 mg / kg, about 20 ng / kg to about 100 mg / kg, or about 0.1 mg / kg to about 100 mg / kg, and includes any subranges therein, as well as individual numbers within the ranges and subranges.

[0104] In another aspect, the present disclosure refers to a method of treating method of treating a cancer or a non-malignant disease in a subject in need thereof, comprising administering to the subject the antibody or antigen binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, or the pharmaceutical composition as disclosed herein; wherein the antibody or antigen binding fragment thereof or the bispccific antibody induces antibody-dependent cellular cytotoxicity (ADCC), antibodydependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137-expressing cancer cells and / or Treg.

[0105] In another aspect, the present disclosure refers to use of the antibody or antigen binding fragment thereof as disclosed herein, or the bispecific antibody as disclosed herein, or the pharmaceutical composition as disclosed herein, in the manufacture of a medicament for treating a cancer or a non-malignant disease in a subject in need of, wherein the antibody or antigen binding fragment thereof or the bispccific antibody or the pharmaceutical composition is to be administered to the subject, wherein the antibody or antigen binding fragment thereof induces antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137-expressing cancer cells and / or Treg.

[0106] As used herein, the term "treating" or "treatment", or grammatically equivalent terms, include any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Hence, “treatment” includes prophylactic and therapeutic treatment.

[0107] In some examples, the cancer is a solid cancer or solid tumor. In some examples, the cancer is a solid cancer selected from the group consisting of nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL), rhabdomyosarcoma (RMS), hepatocellular carcinoma (HCC), melanoma, stomach cancer, brain cancer, eye cancer (such as retinoblastoma), breast cancer, lung cancer, colorectal cancer, kidney cancer (such as Wilms tumor), pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, neuroblastoma, bone cancer (such as osteosarcoma and Ewing sarcoma), skin cancer, bladder cancer, adrenocortical carcinoma, glioblastoma, biliary cancer, coloncancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, osteosarcoma, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, renal cancer, renal cell carcinoma, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer.100108] In some examples, the cancer is a hematological malignancy selected from the group consisting of diffuse large B cell lymphoma (DLBCL), NK / T cell lymphoma and primary effusion lymphoma.

[0109] hr some examples, the cancer is selected from the group consisting of nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL), rhabdomyosarcoma (RMS), hepatocellular carcinoma (HCC), melanoma, diffuse large B cell lymphoma (DLBCL), NK / T cell lymphoma and primary effusion lymphoma, which express CD 137 and can be targeted by the antibody or antigen binding fragment thereof as disclosed herein. In some examples, the CD137-expressing cancer cells are cells of a cancer selected from the group consisting of nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL), rhabdomyosarcoma (RMS), hepatocellular carcinoma (HCC), melanoma, diffuse large B cell lymphoma (DLBCL), NK / T cell lymphoma and primary effusion lymphoma. The CD137-expressing cancer cells can be in vivo cancer cells, primary cancer cells derived directly from a cancer sample, or a cell line. In one example, the RMS cells can be selected from the group consisting of in vivo cancer cells, primary cancer cells derived directly from a cancer sample, or a cell line such as RD 18 that expresses CD 137. In another example, the NPC cells can be selected from the group consisting of in vivo cancer cells, primary cancer cells derived directly from a cancer sample, or a cell line such as C666, HK1, HONE-1, that express CD137. In another example, the Natural killer / T cell lymphoma (NKTCL) cells can be selected from the group consisting of in vivo cancer cells, primary cancer cells derived directly from a cancer sample, or a cell line such as SNK-1, SNK-6 that express CD137. In another example, the Hodgkin's lymphoma (HL) cells can be selected from the group consisting of in vivo cancer cells, primary cancer cells derived directly from a cancer sample, or a cell line such as HDLM- 2, KM-H2, L-428, L-540, SUP-HD1 , L-1236 that express CD137. In another example, the melanoma cells can be selected from the group consisting of in vivo cancer cells,primary cancer cells derived directly from a cancer sample, or a cell line such as B 16 that expresses CD137.

[0110] hi some examples, the non-malignant disease is characterised by having CD137-expressing cells, such as mononucleosis and post-transplantation lymphoproliferative disease. Mononucleosis is a viral infection also known as glandular fever and most commonly caused by the Epstein-Barr virus (EBV) or cytomegalovirus (CMV) and spreads through saliva, transmittable by sharing drinks, food, or utensils with someone who is infected. Post-transplantation lymphoproliferative disease is a complication that can occur after an organ or stem cell transplant. It involves the uncontrolled proliferation of lymphocytes, often due to the Epstein-Barr virus (EBV) taking advantage of the weakened immune system caused by immunosuppressive medications. In one example, for treating mononucleosis, the antibody or antigen binding fragment thereof as disclosed herein can target CD137 on the EBV-infected B cells and kill said infected B cells, thus controlling the spread of EBV.

[0111] The antibody or antigen binding fragment thereof as disclosed herein bind to CD137 on cancer cells as disclosed herein, and / or CD137 on intratumoral Trcgs in virtually all solid cancers, thus triggering ADCC, ADCP, or CDC on the cancer cells expressing CD137 and / or intratumoral Tregs expressing CD137, thereby induce cell death and eliminate the cancer cells and / or intratumoral Tregs expressing CD137.

[0112] As used herein, "administering" refers to the physical introduction of the antibody or antigen binding fragment thereof as disclosed herein or the pharmaceutical composition as disclosed herein to a subject as disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for antibodies described herein include intratumoral, intravenous, intranodal, intradermal, subcutaneous, intraperitoneal, intramuscular, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intratumoral, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralcsional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. Alternatively, the antibody or antigen binding fragment thereof asdisclosed herein or the pharmaceutical composition as disclosed herein can be administered via a non-parcntcral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one example, the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein is administered via intratumoral administration. In another example, the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein is administered via intravenous administration. In another example, the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein is administered via intradermal administration. In another example, the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein is administered via intranodal administration. In another example, the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein is administered via subcutaneous administration.

[0113] The term “subject”, “host”, and “patient” are used interchangeably. As used herein, “subject” refers to patients of human or other mammal and includes any individual it is desired to examine or treat using the methods of the invention. However, it will be understood that “patient” does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (eg. sheep, cows, horses, donkeys, pigs), laboratory test animals (eg. rabbits, mice, rats, guinea pigs, hamsters), companion animals (eg. cats, dogs) and captive wild animals (eg. foxes, deer, dingoes). As used herein, a subject is preferably a mammal, such as a non-primatc or a primate, most preferably a human.

[0114] The antibody or antigen binding fragment thereof as disclosed herein may be administered with one or more further therapeutic agents. In one example, the one or more further therapeutic agents is a cancer therapeutic agent selected from the group consisting of tacrolimus, a dendritic cell therapy, an immune checkpoint inhibitor, a chimeric antigen receptor T or NK cell, and an immunostimulatory antibody. In another example, the further therapeutic agent is tacrolimus, which limits T cell activation, and thereby reduces CD137 expression on T cells, but allows activation of monocytcs / macrophagcs that mediate ADCC, via intracellular signaling pathways in monocytes and macrophages, particularly the p38 MAPK, ERK, and Akt pathways. In another example, the further therapeutic agent is a dendritic cell therapy, comprising the process of (1) extraction ofdendritic cells (DCs) from the patient's blood; (2) loading the DCs with cancer-specific antigens; and (3) reinfusion of the antigen-loaded dendritic cells into the patient, so that they can present the antigens to T cells once back in the body, stimulating a targeted immune response against the cancer cells. In another example, the further therapeutic agent is an immune checkpoint inhibitor such as PD-1 Inhibitors (e.g. Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo)), PD-L1 Inhibitors (e.g. Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (hnfinzi)), CTLA-4 Inhibitors (e.g. Ipilimumab (Yervoy), Tremelimumab (Imjuno)), and LAG-3 Inhibitors (e.g. Rclatlimab (used in combination with nivolumab as Opdualag)). The immune checkpoint inhibitors block the checkpoint proteins such as PD-1 from binding with their partners such as PD-L1 expressed by some cancer cells (which turns off the immune response). This prevents the "off" signal from being sent, allowing T cells to attack cancer cells. In another example, the further therapeutic agent is a chimeric antigen receptor T or NK cell, which are T cells or NK cells extracted from a patient's blood and genetically modified to express chimeric antigen receptors (CARs) that target specific cancer antigens. In another example, the further therapeutic agent is an immunostimulatory antibody designed to enhance the body's immune response, particularly against cancer, by targeting specific proteins on immune cells such as T cells to boost their activity to attack cancer cells. In some examples, an immunostimulatory antibody can be selected from the group comprising Ipilimumab (Yervoy-Targets CTLA-4, enhancing T cell activation), Pembrolizumab (Keytruda) and Nivolumab (Opdivo) (Target PD-1, preventing cancer cells from evading the immune system), and 0X40 Agonists. In some examples, the antibody or antigen binding fragment thereof or the pharmaceutical composition as disclosed herein when administered with the one or more further therapeutic agents as disclosed herein, produces a synergistic anticanccr effect.[001 15] In one example, the antibody or antigen binding fragment thereof or the pharmaceutical composition is administered before the administration of the one or more further therapeutic agents as disclosed herein. In another example, the antibody or antigen binding fragment thereof or the pharmaceutical composition is administered at the same time as the administration of the one or more further therapeutic agents as disclosed herein. In another example, the antibody or antigen binding fragment thereof or the pharmaceutical composition is administered after the administration of the one or more further therapeutic agents as disclosed herein.

[0116] In one example, the one or more further therapeutic agents is to be administered using the antibody or antigen binding fragment thereof as disclosed herein as a carrier, wherein the one or more further therapeutic agents are conjugated with the antibody or antigen binding fragment thereof to form a conjugate, and wherein the conjugate is internalized by the CD137-expressing cancer cells and / or Treg upon binding. The antibody and the one or more further therapeutic agents are chemically linked through a linker to form an antibody-drug conjugate (ADC), wherein the linker is selected from the group consisting of an Acid-cleavable Linker which is sensitive to the acidic environment of endosomes and lysosomes, an Enzymc-Clcavablc Linker which is clcavablc by specific enzymes present in the target cells such as cathepsin B, a Reduction-Cleavable Linker which can be broken down by the reducing environment inside cells, often involving disulfide bonds, and a Non-Cleavable Linker, which remain intact until the ADC is internalized and degraded by the lysosomes of the target cell to release the drug. In one example, the enzyme-cleavable linker is a Valine-Citrulline (Val-Cit) Linker that is stable in the bloodstream but cleavable by cathepsin B inside the cell. In another example, the non-clcavablc linker is a Malcimidocaproyl (MC) Linker, which relics on lysosomal degradation to release the drug.

[0117] The ADC circulates in the bloodstream until the antibody component binds to its specific antigen on the surface of a cancer cell. Once bound, the ADC is internalized by the cancer cell through endocytosis. Inside the cell, the linker is cleaved or degraded, releasing the cytotoxic or radioactive therapeutic agent or drug. The released drug then exerts its toxic effects, leading to the death of the cancer cell or intratumoral Treg.

[0118] In another example, the one or more further therapeutic agents is selected from the group consisting of a cytotoxic compound selected from the group consisting of Maytansinoids, Auristatins, Duocarmycins, Calicheamicins, Pyrrolobenzodiazepines, SN-38, Doxorubicin, and Camptothecin derivatives, and a radioactive compound selected from the group consisting of Iodine-131 (1-131), Yttrium-90 (Y-90), Lutetium-177 (Lu- 177), lndium-111 (In-111), Technetium- 99m (Tc-99m), Rhenium-186 (Re-186), Rhenium- 188 (Re- 188), Copper-64 (Cu-64), Zirconium-89 (Zr-89) and Actinium-225 (Ac-225). Cytotoxic compounds arc substances that arc toxic to cells, meaning they can damage or kill cells. They are commonly used in chemotherapy to target and destroy rapidly dividing cancer cells. Radioactive compounds, or radiopharmaceuticals, emit radiation that damages the DNA of cancer cells, leading to cell death. The cytotoxiccompound and / or the radioactive compound upon delivered to the cancer cells and / or Trcg after internalization of the conjugated antibody or antigen binding fragment thereof, can boost the cell damage or death of the target cancer and / or Treg cells particularly intratumoral Treg.[001 19] The antibody or antigen binding fragment thereof, or the bispecific antibody, or the pharmaceutical composition is administered at a frequency from once every three days to once every three months. The administration frequency is selected from the group consisting of once every day, once every two days, once every' three days, once every four days, once every five days, once every six days, once every' seven days, once every eight days, once every nine days, once every ten days, once every 15 days, once every 20 days, once every 30 days, once every 40 days, once every 50 days, once every 60 days, once every 70 days, once every 80 days, once every 90 days, and other frequencies depending on the need and reaction of an individual subject.

[0120] The antibody or antigen binding fragment thereof, or the bispecific antibody, or the pharmaceutical composition is administered for a period of 1 day to 1 year. The administration duration is selected from the group consisting of 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 15 days, 20 days, 25 days, 30 days, 40 days, 50 days, 60 days, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer depending on the need and reaction of an individual subject.

[0121] The antibody or antigen binding fragment thereof, or the bispecific antibody, or the pharmaceutical composition is administered at a dose of 0.1-100 mg / kg body weight. The administration dosage is selected from the group consisting of 0.1-100 mg / kg bodyweight, 0.1-90 mg / kg bodyweight, 0.1-80 mg / kg bodyweight, 0.1-70 mg / kg bodyweight, 0.1-60 mg / kg bodyweight, 0.1-50 mg / kg bodyweight, 0.1-40 mg / kg bodyweight, 0.1-30 mg / kg bodyweight, 0.1-20 mg / kg bodyweight, 0.1-10 mg / kg bodyweight, 0.1-5 mg / kg bodyweight, 0.1-3 mg / kg bodyweight, 0.1-1 mg / kg body weight, 1-100 mg / kg body weight, 2-100 mg / kg body weight, 3-100 mg / kg body weight, 5-100 mg / kg body weight, 8-100 mg / kg body weight, 10-100 mg / kg body weight, 20-100 mg / kg bodyweight, 30-100 mg / kg body weight, 40-100 mg / kg body weight, 50-100 mg / kg bodyweight, 60-100 mg / kg body weight, 70-100 mg / kg body weight, 80-100 mg / kg body weight, 90-100 mg / kg body weight, 1 -90 mg / kg bodyweight, 10-80 mg / kg bodyweight, 20-70 mg / kg bodyweight, 30-60 mg / kg body weight, 40-50 mg / kg bodyweight, about 0.1 mg / kg bodyweight, about 1 mg / kgbody weight, about 2 mg / kg body weight, about 3 mg / kg body weight, about 5 mg / kg bodyweight, about 8 mg / kg bodyweight, about 10 mg / kg bodyweight, about 20 mg / kg bodyweight, about 30 mg / kg bodyweight, about 40 mg / kg bodyweight, about 50 mg / kg bodyweight, about 60 mg / kg bodyweight, about 70 mg / kg bodyweight, about 80 mg / kg bodyweight, about 90 mg / kg bodyweight, about 100 mg / kg bodyweight, or other dosage depending on the need and reaction of an individual subject.

[0122] As used in this application, the singular form “a,” “an," and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a primer" includes a plurality of primers, including mixtures and combinations thereof.

[0123] As used herein, the term “comprising" means “including " Variations of the word "comprising", such as “comprise” and “comprises," have correspondingly varied meanings. Thus, for example, a composition “comprising" X may consist exclusively of X or may include one or more additional unrecited components.

[0124] As used herein, the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, or + / - 1% of the stated value, or + / - 0.5% of the stated value.

[0125] Throughout this disclosure, certain embodiments may be disclosed in a range format, ft should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0126] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it is recognized that various modifications arc possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0127] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0128] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0129] Other embodiments are within the following claims and non-limiting examples.EXAMPLES

[0130] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the disclosure.

[0131] Example 1 - Materials and Methods

[0132] Cell lines

[0133] The human Hodgkin lymphoma (HL) cell line HDLM-2 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), and was authenticated by DSMZ using DNA-typing, PCR analysis and cytogenetic testing. The murine B cell lymphoma line A20 and the melanoma cell line B 16 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The human NK / T cell lines SNK-1 and SNK-6 were a kind gift from Dr. Norio Shimizu of Tokyo Medical and Dental University, Japan. The human rhabdomyosarcoma (RMS) cell line RD18, human nasopharyngeal carcinoma (NPC) cell lines HK1 and C666, human HL cell line HDLM- 2 and murine B lymphoma cell line A20 were cultured in RPMI 1640 with 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin (R10-PS). SNK-1 and SNK-6cells were cultured in RIO supplemented with 500 lU / mL IL-2. The murine melanoma cell line B16 was cultured in DMEM with 10% FBS and 1% streptomycin and penicillin (D10-PS). All cell lines were maintained in a humidified 37°C and 5% CO2 incubator. CD137-overexpressing (OE) stable cell lines were generated by lentiviral transduction.

[0134] Isolation of peripheral blood mononuclear cells (PBMCs), NK cells and Tregs

[0135] All human blood samples were obtained from healthy donors at Health Sciences Authority Singapore with approval from the Institutional Review Board, NUS, Singapore (IRB number: 13-079E) in accordance with the guidelines of the Health Sciences Authority of Singapore. PBMCs were isolated using density gradient centrifugation by Lymphoprep™, followed by removal of red blood cells by ACK lysis buffer. PBMCs were resuspended in PBS with 2% FBS.

[0136] NK cells were isolated from PBMCs using EasySep™ Human NK Cell Isolation Kit (Stemcell Technologies) according to the manufacturer's protocol. The isolated NK cells were cultured and expanded in NK MACS® medium supplemented with 5% human AB scrum and 500 lU / mL IL-2 in a humidified 37 °C and 5% CO2 incubator.

[0137] Tregs were isolated using EasySep™ Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stemcell Technologies). The isolated Tregs were cultured in R10-PS with 500 lU / ml IL-2 in a humidified 37°C and 5% CO2 incubator.

[0138] Antibody generation

[0139] Anti-CD137 antibodies were generated by panning a phage display library with the recombinant extracellular domain of human CD 137 protein. Positive binders were confirmed by flow cytometry with HEK293 cells that were transfected with full length human CD 137 protein and were then reconstitutes as human IgGl.

[0140] ADCC assay

[0141] Isolated NK cells were rested in R10-PS overnight in a humidified 37°C and 5% CO2 incubator before being used in the ADCC assay. For human RMS cell lines and murine melanoma cell lines, 5xl04NK cells were harvested and cocultured with 104target cells (RD18 I RD18-CD137 / B 16 / B16-murinc CD137) at an effector to target (E:T) ratio of 5:1 in the presence of 10 pg / ml antibodies. For human NPC cell lines, 10sNK cells were harvested and cocultured with 104target cells (HK1 1 HK1-CD137 I C666 / C666-CD137) at an E:T ratio of 10:1 in the presence of 10 pg / ml antibodies. Cytolysisof target cells were monitored for 24 h in a real-time manner using xCELLigence real- time cell analyser (RTCA).

[0142] For human HL and NK / T cell lines, the cytolysis was determined using the DELFIA EuTDA cytotoxicity assay (Perkin Elmer) according to manufacturer's protocol. Briefly, target cells were loaded with BATDA and co-cultured at 104cells / well in triplicate with effector cells (NK cells) at the different E:T ratios in the presence of 10 pg / ml antibodies. Supernatants were collected after 2 h, incubated with Europium solution and measured using SpectraMax iD5 plate reader. Specific lysis was calculated according to the standard formula in the manufacturer’s instructions.

[0143] For autologous Tregs, 10 NK cells were harvested and cocultured with 104Tregs at an E:T ratio of 10:1 in the presence of 10 pg / ml antibodies for 20 h. Percentage of cytolysis of target cells were checked by Annexin-V and 7-AAD staining in the CD4+population using flow cytometry.

[0144] For murine B lymphoma cell line A20, target cells were first labelled by CFSE, and then 5xl04NK cells were harvested and cocultured with 104CFSE-labelled target cells at an E:T ratio of 5:1 in the presence of 10 pg / ml antibodies for 20 h. Cytolysis of target cells were checked by Annexin-V and 7-AAD staining in the CFSE+population using flow cytometry.

[0145] Antibodies used in the ADCC assays included anti-CD137 TgG1 antibody, clone P1A1 and its isotype control (IgGl). Cetuximab is an anti-EGFR monoclonal antibody of the IgGl isotype, and was included as a positive control for assays with human RMS and NPC cell lines.

[0146] Degranulation assay

[0147] 105NK cells were cocultured with 104autologous Tregs at an E:T ratio of 10: 1 in the presence of 10 pg / ml antibodies for 1 h. Afterwards, Brefeldin A and anti-CD107a antibody were added to the coculture and incubated for another 3 h. Cells were then harvested, stained for intracellular IFN-y and TNF-a and analysed by flow cytometry.

[0148] Murine ADCC assay and ADCP assay

[0149] 104B 16-control and B16-murine CD137 were seeded as target cells. Splcnocytcs were pre-activated with 100 lU / ml rm-IL2 for 48 h, washed and added as effector cells at an E:T ratio of 10:1 in the presence of 10 pg / ml mTgG2a or mPl Al antibodies. Cytolysis of target cells were monitored for 24 h in a real-time manner using xCELLigence real-time cell analyser (RTCA).

[0150] 4xl04RAW264.7-mCD137L-KO (RAW-LKO) cells were cocultured with 104CFSE labelled A20 or A20-murinc CD137 cell lines in the presence of 10 pg / ml m!gG2a or mPlAl antibodies for 2 h. Percentages of ADCP were analysed by the CFSE signal in CD1 lb+population using flow cytometry.

[0151] Jncucyte antibody internalization assay

[0152] HDLM-2 cells were seeded into a 96-well flat plate. P1A1 and IgGl were labelled with Incucyte Human FabFluor-pH Red Antibody Labelling Dye (Sartorius, Gottingen, Germany) at a molar ratio of 1:3 (test antibody: labelling Fab reagent), following the manufacturer’s instructions. Labelled antibodies were added to cells at a final concentration of 4 pg / ml and plate was placed in the Incucyte S3 Live-Cell Analysis System for 24 h. Images were captured every 30 min at 20X magnification. Triplicates were performed for each treatment. Analysis of antibody internalization was performed using the Incucyte Cell-by-Cell Analyzer.

[0153] Tumor models

[0154] The HCC mouse model was established by hydrodynamic tail vein injection (HDTV). 2 ml solution of sterile saline and plasmids, totalling 30 pg CRISPR-Cas9 vector system carrying a single guide RNA (sgRNA) targeting Trp53 and a transposon system carrying a c-Myc vector (Kindly provided by Dr. Wong from The University of Hong Kong21) were injected into the lateral tail vein of 7 - 8-week old male C57BL / 6 mice within 6 - 8 seconds. 4 weeks post HDTV, mice were administrated 250 pg anti-CD137 antibody P1A1 (of which the human IgGl Fc domain was replaced by the the Fc domain of murine IgG2a (mPlAl)) or mouse IgG2a isotype control (mIgG2a, Biolegend) i.p. 3 times weekly for 2 weeks. 2 days after the last dose mice were sacrificed. Tumors and spleens were harvested for analysis.

[0155] For the B16 lung metastasis model, 105B16-CD137 cells were injected into the lateral tail vein of 7-week old male C57BL / 6 mice on day 0. One week after injection, mice were i.p. administrated 200 pg mPlAl or mIgG2a (isotype) twice weekly for 2 weeks. On day 21 mice were sacrificed and lungs were harvested and the tumor nodules were counted.

[0156] Flow cytometry analysis ofNK cells

[0157] Expression of markers on NK cells in the ADCC assays were assessed by flow cytometry. In the cases where intracellular staining was applied, Brefeldin A were added into the coculture 4 h prior to harvest. The antibodies used are: CD56 (clone CMSSB),

[0158] Statistical Analysis

[0159] Statistical evaluation was performed using GraphPad Prism 9.0 (GraphPad Software). Statistical significance was determined by using two-tailed unpaired Student’s t-test or Two-way ANOVA.

[0160] Example 2 - Results

[0161] Anti-CD137 antibodies enhance NK cell-mediated cytolysis against human CD137+cancer cells

[0162] Ectopic expression of CD137 is found on human cancer cells, including RMS and NPC. To assess the ability of the anti-CD137 antibodies in killing CD137+cancer cells, a kinetic ADCC assay was done on the CD137-expressing RMS cell line RD18 and the NPC cell lines C666 and HK1 (Fig 8A-C). CD137-ncgativc cell lines were included as negative controls. Cetuximab, an anti-EGFR monoclonal antibody of the IgGl isotype, was used as positive control, since RD 18, HK1 and C666 display a high expression of EGFR22 23.

[0163] The positive control Cetuximab efficiently induced cytolysis of the target cell lines regardless of CD 137 expression (Fig. 1A-F), indicating the effectiveness of the NK cells as effectors. The anti-CD137 antibody P1A1 significantly enhanced the cytolytic activities of NK cells against CD137+cancer cells compared to the isotypc control (Fig. 1D-F). This enhancement in cytolysis was not observed in CD137 cancer cells (Fig. 1A- C), demonstrating that cytotoxicity of Pl Al is CD137-specific.

[0164] P1A1 also induced NK cell cytotoxicity against cells that endogenously express CD137, such as the NKTCL cell lines SNK-1 and SNK-6 and HL cell line HDLM-2 (Fig. 1G-I), even in the SNK-1 and SNK-6 cells in which the endogenous expression level of CD 137 is lower (Fig 8D-F).

[0165] NK cells were activated in ADCC assays against human CD137+cancer cells

[0166] NK cells in the ADCC assays were harvested for analysis by flow cytometry. There was a significant drop in CD16 (FcyRIIIa) levels on NK cells in the ADCC assays with CD137+cell lines and P1A1 (Fig. 2A), indicating the ligation of the Fc region ofP1A1 to CD16. P1A1 induced significantly higher expression of the activation markers CD137 and CD25 on NK cells in the assays with CD137+cells, while the change was minimal in the assays with CD137 control cells (Fig. 2B,C). Expression of the activation markers, CD69 and 0X40, was also slightly increased in the assays with CD137+cells but this change did not reach statistical significance (Fig. 2D,E). This enhancement of NK cell activity by P1A1 was most likely due to Fey receptor engagement since P1A1 has no agonist activity towards CD137 (Fig. 9). Importantly, the binding of P1A1 to CD137 on target cells is required for the subsequent activation of NK cells. Pl Al did not activate NK cells (Fig. 2B-E) or enhance NK cell-mediated killing of targets in the assays where target cells did not express CD137 (Fig. 1A-C).

[0167] P1A1 enhanced NK cell-mediated cytolysis against autologous human CD137+ Tregs

[0168] Tregs were isolated from PBMCs and more than 90% were FOXP3+(not shown). Since peripheral human Tregs express low levels of CD137 during the resting state, isolated Tregs were preactivated with Phorbol 12-myristate 13-acetate (PMA) and ionomycin for 24 h which induced CD137 expression from 15.9 to 94.4% of the cells (Fig 8G). These Tregs were then cocultured with autologous NK cells in the presence of Pl Al. Consistent with assays using malignant cells as targets, P1A1 increased the cytolysis against CD137+Tregs (Fig. 3A,B) by engaging and activating NK cells. This was reflected by significantly reduced CD 16 and significantly higher CD 137, CD25 and 0X40 expression (Fig. 3C). Moreover, P1A1 also enhanced the degranulation of NK cells as well as their IFN-y and TNF-a secretion (Fig. 3D).

[0169] Murine chimeric P1A1 enhances cytolysis against murine CD137+cancer cells

[0170] P1A1 cross-reacts with murine CD137 (Fig. 11). Therefore, whether Pl Al can induce ADCC in murine cells that express murine CD137 was tested. Indeed, P1A1 significantly enhanced the cytolytic activity of human NK cells against murine CD137+B 16 melanoma cells (Fig. 10A,B) and against the murine B lymphoma cell line A20 (Fig. 10C).

[0171] In addition, P1A1 also recognizes canine and feline CD137 and is able to mediate NK cell killing in HEK293 cells expressing dog or cat CD1 7 (Fig. 15).

[0172] In order to test P1A1 in vivo in a murine tumor model, its human IgGl Fc domain was replaced with the Fc domain of murine lgG2a. Mouse IgG2a is an analogueof human IgGl, which is able to engage murine Fey receptors and to induce ADCC and ADCP. This murine chimeric P1A1 (mPlAl) bound specifically to murine CD137 on CD137-transduced B16 cells (Fig. 11A). The specificity is comparable to commercially available flow cytometry antibody against murine CD 137 (Fig. 1 IB), and mPlAl did not block the detection of murine CD137 by another anti-CD137 antibody through flow cytometry. mPlAl induced ADCC selectively in murine CD137-expressing B16 cells when activated murine splenocytes were used as effector cells (Fig. 4A). In addition to ADCC, mPlAl was able to induce ADCP in CD137-expressing cells. RAW 264.7 mCD137L knockout cells (RAW-LKO) were used as effector cells to eliminate the interaction of effector and target cells via CD137L and CD137. Again, the rnPlAl- mediated phagocytosis occurred selectively in murine CD137-expressing A20 cells (Fig. 4B,C).

[0173] Next, a CD137-expressing lung metastasis model was established using the B16 melanoma cell line. A CD137-expressing clone of B16 (Fig. 4D) was i.v. injected into syngeneic C57BL / 6 mice which gave rise to lung metastases. Treatment with mPlAl significantly reduced the number of tumor nodules in the lungs (Fig. 4E). This anti-tumor activity of mPlAl may have been a direct killing of murine CD137-expressing B 16 cells. In addition, mPlAl also eliminated Tregs in the melanoma models (Fig. 4F) which may have contributed to the overall reduction in lung metastases.

[0174] P1A1 reduces the number of CD137+Tregs and inhibits tumor growth in mice

[0175] Apart from direct CD 137 -expressing cancer cell elimination, whether mPlAl could modify the tumor microenvironment by reducing the population of intratumoral, CD137-expressing Tregs, thereby indirectly inhibiting tumor growth, was evaluated. A transgenic HCC mouse model was used (Fig. 5A), which has been characterised to have high numbers of CD137+intratumoral Tregs. Treatment with mPlAl significantly reduced tumor nodules and tumor size (Fig. 5B). mPlAl administration induced a noticeable reduction in intratumoral Treg numbers (Fig. 5C). Although both CD137+Tregs and Teffs were reduced in the tumor (Fig. 5D,G), mPlAl resulted in an overall less immunosuppressive tumor microenvironment. With mPlAl treatment, intratumoral Tregs displayed a lower activation state as reflected by reduced CD69 and PD-1 expression (Fig. 5E,F). Concomitantly, CD4+Teffs, CD8+T cells and NK cells exhibited higher activity as indicated by higher IFNv and TNF-a secretion (Fig. 5H,I,K,L,N,O).

[0176] In addition, the lung metastasis model for the B 16 melanoma cell line was used as a model for a CD137-cxprcssing cancer. A CD137-cxprcssing clone of B 16 was generated (Fig. 16A) that was intravenously injected into the tail vein of syngeneic C57BL / 6 mice which gives rise to lung metastases. Treatment with mPlAl significantly reduced the number of tumor nodules in the lungs (Fig. 16B). This anti-tumor activity of mPl Al may have been a direct killing of CD137-expressing B 16 cells as shown in Figure 4. However, similarly as in the HCC model (Fig. 5), mPlAl also eliminated Treg in the melanoma model which may have contributed to the overall reduction in lung metastases (Fig. 16C).

[0177] Pl Al is internalized

[0178] To assess the potential of P1A1 as an antibody drug conjugate (ADC) carrier to target cytotoxic or radioactive compounds to CD137+Tregs and cancer cells, we evaluated its ability to undergo internalization using the endogenously CD137-expressing human HL cell line HDLM-2. P1A1 and IgGl isotype antibody were labelled with a Fab fragment-conjugated pH sensitive fluorophore that binds to the Fc domain of human IgG heavy chain. The increased red fluorescence detected in cells with P1A1 treatment demonstrated that binding of P1A1 to CD137 leads to its internalization (Fig. 6 and Fig. 12).

[0179] Mapping of the Pl A 1 binding site

[0180] In order to identify the CD137 domain to which P1A1 binds, competitive binding assays were conducted with anti-CD137 antibodies with well-characterised binding sites. Human nasopharyngeal carcinoma C666 cells that express human CD137 (Fig. 7A) were first incubated with murine chimeric P1A1 (to block the P1A1 binding site), and then with Urelumab or Utomilumab13. Their extent of binding was tested by staining with a secondary antibody that recognizes the human Fc domain and therefore only measures binding by Urelumab or Utomilumab but not by mPlAl. Pretreatment of the cells with mPl Al blocked binding of human Pl Al which served as a positive control (Fig. 7B). However, binding of P1A1 was not blocked by Urelumab, indicating that they have different binding sites (Fig. 7C). Binding was significantly reduced by preincubation of the cells with Utomilumab, demonstrating that the binding site of P1A1 and Utomilumab overlap (Fig. 7D). These data were verified with two other CD137- expressing cells lines; the rhabdomyosarcoma cell line RD 18 and the nasopharyngeal carcinoma line HK-1 (Fig. 13). Since the binding site of Utomilumab has been mappedto the border regions of the CRD3 and CRD4 domains, it can be concluded that P1A1 binds to the same or an overlapping epitope on CD13713.

[0181] P1A1 blocks CD137 - CD137L interaction

[0182] Since the border region of the CRD3 and CRD4 domains of CD 137 is essential for ligand binding, it was suspected that Pl Al may interfere with CD137 - CD137L interaction. Indeed, P1A1 blocked binding of recombinant CD137L protein to CD137- transfected MCF7 cells (Fig. 17A). In addition, P1A1 prevented CD137 signalling. Treatment of the CD137-expressing Hodgkin Lymphoma cell line L-428 with recombinant CD137L protein induces secretion of TNF. This TNF release was blocked by P1A1, while P1A1 had no effect on the CD 137-negative L-428 control cells (Fig. 17B). Similarly, when P1A1 was added to preactivated PBMC, cell activation was blocked as evidenced by a significant reduction in IFN-y secretion (Fig. 17C). This is in line with Pl Al showing no agonist activity in NK cells, and characterizes Pl Al as an antagonistic anti-CD137 antibody.

[0183] anti-CD137 Pl Al x anti-CD3 bispecific antibody (BsAb) as T cell engager

[0184] To assess if an anti-CD137 P1A1 x anti-CD3 bispecific antibody (BsAb) (Fig. 18A) can function as a T cell engager, the P1A1 BsAb was constructed and produced. Its functionality was tested in vitro using primary human T cells against the tumor cell lines (C666, HK1, RD 18, and RH41) that were engineered to express human CD 137. The tumor cells were cultured overnight in xCELLigence 96-well E-plates for cell adhesion, followed by the addition of freshly isolated naive human CD8 T cells at an effector-to- target (E:T) ratio of 10:1. The P1A1 BsAb, along with an isotype control BsAb, was then introduced. The cell index values were continuously measured over 72 hours using the xCELLigence RTCA system to monitor cytotoxicity.

[0185] The results revealed that the P1A1 BsAb induced dose-dependent cytotoxicity across all four CD137+cell lines, with varying levels of efficacy (Fig. 18B). Compared to the isotype control, the Pl Al BsAb triggered a dose-dependent tumor cell killing effect, achieving >80% cytotoxicity in HK1-CD137 and RH41-CD137 cells at 100 pM Pl A1 BsAb, and >50% cytotoxicity in C666-CD137 and RD18-CD137 cells at the same concentration. These findings confirm that the P1A1 T cell-engaging BsAb effectively targets and kills CD137-expressing tumor cells, proving its potential as an effective anti-CD137 T cell engager.

[0186] Example 3- Discussion

[0187] CD137 is well-established as a co-stimulatory molecule on Teff cells. Its signalling induced by its ligand, CD137L, induces potent anti-tumor immune responses9'10. However, the expression of CD137 on intratumoral Tregs and the ectopic expression on several types of cancer cells facilitate evasion from immune surveillance by triggering a negative feed-back mechanism, where CD137L on APCs is downregulated via CD137 trogocytosis24. This mechanism is conserved between human and mouse2\ and resembles the downregulation of CD80 and CD86 by Treg-expressed CTLA-426. Therefore, it is hypothesized that targeting CD 137 will (1) directly deplete malignant cells in CD137-cxprcssing cancers, and (2) deplete CD137+intratumoral Tregs in solid cancers. While CD137 expression has so far only been documented for HL, NKTCL, NPC and RMS, CD137+Treg have been identified in all solid cancers tested5> 6. Accordingly, an ADCC-inducing anti-CD137 antibody like P1 A1 would be widely applicable for cancer immunotherapy.

[0188] Several studies have shown that monoclonal antibodies targeting surface markers such as CCR4, CCR8, CD25 and CTLA-4 are effective in reducing intratumoral Treg numbers in tumor-bearing mouse models27-30. However, Tcffs were also depleted due to the off-target effects of anti-CD25 antibodies27. Of note, the anti-CTLA-4 antibodies Ipilimumab and Tremelimumab, which are approved for cancer therapy, do not deplete Treg in tumors31. Therefore, targets that are more selective for intratumoral Tregs are needed. CD137 is such a target as it is highly expressed by Tregs inside the tumor but only marginally by peripheral Tregs. CD137 can also be expressed by Teff and NK cells but that is only at comparatively low levels and only transiently following activation. Furthermore, targeting CD137-cxprcssing Tregs for tumor immunotherapy has already been validated in vivo in murine cancer models. However, these studies were conducted with rat anti-murine CD 137 antibodies of IgG2a isotype4, 5, and are therefore not as easily translatable as P1A1 which is a fully human antibody of human IgGl isotype that targets human CD 137.

[0189] Existing humanized anti-CD137 antibodies Utomilumab of the lgG2 isotype and Urelumab of the IgG4 isotype are being evaluated in clinical trials and show encouraging signs of efficacy. Their mechanism of action is different from that of P1A1 as Utomilumab and Urelumab are agonists that induce T cell costimulation by crosslinking CD137 on T cells13. Since IgG2 and IgG4 have a low affinity to Feyreceptors, Utomilumab and Urelumab are not able to induce depletion of CD137+Treg and malignant cells.

[0190] Adagene has developed an anti-CD137 antibody with an IgGl Fc domain (ADG-206). This antibody can induce ADCC just as Pl Al. However, ADG-206 is an agonistic antibody, while Pl Al is antagonistic. Its antagonistic nature provides distinct advantages to P1A1 since there is evidence that CD137 signalling enhances Treg activity similarly as it enhances Teff activity. Zhang et al., 2007, reported that an agonistic antiCD 137 antibody promoted the proliferation of Treg, which maintained Foxp3 expression and the ability to suppress conventional CD4+T cells36. Similarly, Elpck ct al., 2007, found that Treg upregulate CD137 expression in response to IL-2, and that CD137 stimulation expands Treg. These Treg were characterized by increased expression of CD25 and TGF-[>:and they not only suppressed T cell proliferation but also prevented the rejection of allogeneic islets37. Recently, Lubrano et al., 2020, reported that CD137 stimulation activates Treg via the NF-KB pathway38. In addition, a recent study showed that using anti-CD137 agonist antibody promoted the expansion and accumulation of exhausted T cells in the tumor, which may shift the microenvironment toward a more immunosuppressive state and dampen antitumor immunity Pichler32.

[0191] Further, an agonistic anti-CD137 antibody is more likely to promote the growth of cancers that express CD137 on malignant cells. Crosslinking of CD137 on Hodgkin and Reed- Sternberg cells induces IL- 13 release, which is the most potent growth factor for HL14. In nasopharyngeal carcinoma and rhabdomyosarcoma, CD137 crosslinking induces the release of IL-6 and IL-8, two cytokines that are intimately involved in metastasis of these two cancers16, 17.

[0192] hi the ADCC assays with human Tregs and human cancer cell lines, P1A1 bound to CD137 on target cells and then engaged and activated NK cells via Fey receptor CD16, triggering cytotoxic responses of NK cells (Fig. 14C). As a result, P1A1 selectively enhanced cytotoxicity of NK cells against CD137-expressing cells.

[0193] To assess the effects of P1A1 in immunocompetent murine cancer models, the human IgGl Fc domain of Pl Al was replaced with that of murine IgG2a which is the functional equivalent to human IgGl in terms of binding to human FcyR and its efficacy in inducing ADCC and ADCP. In immunocompetent mice with melanoma lung metastasis expressing CD137, treatment with this murine chimeric P1A1 reduced lung tumors by directly targeting CD137+cancer cells. In an immunocompetent mouse modelwith oncogene-induced orthotopic liver cancer, Pl Al treatment also reduced tumor size, this time by modifying the tumor microenvironment rather than direct cytotoxic effects on the cancer cells. With P1A1 treatment, the number of CD137+Tregs in the tumor microenvironment was significantly reduced. Concurrently, there was an increase in the Teff activation status as evidenced by increased IFN-y and TNF-a expression, implying the tumor microenvironment became more favorable for antitumor responses. Although depletion of Tregs in the two tumor models was not complete, this was likely due to the lower affinity of P1A1 to murine CD137. The affinity of P1A1 to human CD137 is two orders of magnitude higher than to murine CD 137, which promises a higher efficacy in human cancer therapy trials. Actually, total elimination of Tregs may be unnecessary, as selectively removing CD137+Tregs can already elicit significant anti-tumor effects. This data confirms earlier observations that CD137+Tregs are more suppressive than CD137" Tregs8.

[0194] There are other cell surface molecules on Tregs that are being targeted for Treg elimination such as CD2533, 34or CCR833for cancer immunotherapy. It is currently impossible to determine which target will be the most therapeutically effective. Even though none of these targets is selective for Tregs, what is in favour of targeting CD 137 are the facts that (1) CD137 is the most differentially expressed gene between intra- and extratumoral Tregs, (2) CD137 is expressed at higher levels in Tregs than in Teff, and (3) is expressed constitutively in Tregs but only transiently in Teff. Furthermore, CD137 is even expressed by malignant cells in some cancers.

[0195] The anti-cancer activity of P1A1 is not solely dependent on inducing ADCC or ADCP. It can be internalized upon binding to CD137 and could potentially be used as a carrier for cytotoxic drugs or radiochemicals.

[0196] In summary, P1A1 is a fully human antibody that targets human CD137 and is able to exert cytotoxicity against CD137-expressing malignant cells and Tregs in the tumor microenvironment of solid cancers.

[0197] Industrial Applicability

[0198] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.References1. Wolf D, Wolf AM, Rumpold H, Ficgl H, Zcimct AG, Mullcr-Holzncr E, Dcibl M, Gastl G, Gunsilius E, Marth C. The expression of the regulatory T cell-specific forkhead box transcription factor FoxP3 is associated with poor prognosis in ovarian cancer. Clin Cancer Res 2005; 11:8326-31.2. O'Callaghan DS, Rexhepaj E, Gately K, Coate L, Delaney D, O’Donnell DM, Kay E, O'Connell F, Gallagher WM, O'Byrne KJ. Tumour islet Foxp3+ T-cell infiltration predicts poor outcome in nonsmall cell lung cancer. Eur Respir J 2015; 46:1762-72.3. Iglesias-Escudero M, Arias -Gonzalez N, Martinez-Caceres E. 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Claims

Claims1. An antibody or an antigen binding fragment thereof comprising:(A) a heavy chain variable region comprising the following CDRs:(a) CDR-H1: GYSLTSYGIS [SEQ ID NO:1] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(b) CDR-H2: WMGWISAYNGNTNYAQKLQG [SEQ ID N0:2J or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(c) CDR-H3: ASYYDSSGYAFDI [SEQ ID NO:3] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; and(B) a light chain variable region comprising the following CDRs:(a) CDR-L1: SGDKLEDKYAS [SEQ ID N0:4J or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(b) CDR-L2: QDYKRPS [SEQ ID NO:5] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;(c) CDR-L3: QTWDRITTSYV [SEQ ID NO:6] or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; wherein the antibody or antigen binding fragment binds to CD137 antagonistically.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising the CDRs of SEQ ID NOs 1, 2 and 3; and a light chain variable region comprising the CDRs of SEQ ID NOs: 4, 5 and 6.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising:(A) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; and(B) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8; or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity.

4. The antibody or antigen-binding fragment thereof of claim 3, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising tire amino acid sequence of SEQ ID NO:8.

5. The antibody or antigen-binding fragment thereof of any one of the preceding claims comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:9; or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain constant region is of an immunoglobulin subtype IgGl.

7. The antibody or antigen-binding fragment thereof of any one of the preceding claims comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 10; or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the light chain constant region is of a kappa (K) or lambda (X) light chain.

9. The antibody or antigen-binding fragment thereof of any one of the preceding claims, further comprising an Fc region.

10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising:(A) a heavy chain comprising a variable region of SEQ ID NO:7 and a constant region of SEQ ID NO: 9; and(B) a light chain comprising a variable region of SEQ ID NO:8 and a constant region of SEQ ID NO: 10.

11. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising:(A) a heavy chain comprising the amino acid sequence of SEQ ID NO:11; or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity; and(B) a light chain comprising the amino acid sequence of SEQ ID NO: 12; or an amino acid sequence having at least 80% sequence identity therewith, for example at least 80%, 90%, or 95% sequence identity;12. The antibody or antigen-binding fragment thereof of claim 11, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

13. The antibody or antigen binding fragment of claim 1, wherein the antigen binding fragment is a Fv fragment selected from the group consisting of a single chain Fv (scFv) or disulphide-bonded Fv; or a Fab-like fragment selected from the group consisting of a Fab fragment, Fab' fragment, and a F(ab')2 fragment.

14. A bispecific antibody, comprising(i) a first binding arm comprising a first antigen-binding region binding to CD 137 antagonistically, wherein the first antigen-binding region comprises (a) heavy chain variable (VH) region CDR-H1, CDR-H2, and CDR-H3 having the sequences as set forth in SEQ ID NOs:l, 2, and 3, respectively, and light chain variable (VL) region CDR-L1, CDR-L2, and CDR-L3 having the sequences as set forth in SEQ ID NOs:4, 5 and 6, respectively , and(ii) a second binding ami comprising a second antigen-binding region binding to human CD3.

15. The bispecific antibody of claim 14, wherein the first antigen-binding region binding to CD 137 antagonistically is a Fab fragment , and the second antigen-binding region binding to human CD3 is a scFv.

16. The bispecific antibody of claim 15, wherein the second antigen-binding region binding to human CD3 comprises the VH sequence of SEQ ID NO: 17, and the VL sequence of SEQ ID NO: 18.

17. The bispccific antibody of claim 14, wherein the first antigen-binding region binds to CD 137 expressed on cancer cells and / or Treg, wherein the second antigen-binding region binds to human CD3 on a T cell, and wherein the bispecific antibody acts as a T cell engager for directing the T cell to target and kill the cancer cells and / or Treg.

18. The bispecific antibody of claim 14, wherein the bispecific antibody comprises a Fc domain that is an IgGl, particularly hlgGl-KiH-LALA.

19. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of any one of claims 1-13, or the bispccific antibody of any one of claims 14- 18.

20. The nucleic acid molecule according to claim 19, wherein the molecule is a cDNA molecule.

21. The nucleic acid molecule of claim 19 or 20, encoding an antibody heavy chain comprising a nucleotide sequence of SEQ ID NO: 13, or encoding an antibody heavy chain variable region comprising a nucleotide sequence of SEQ ID NO: 14.

22. The nucleic acid molecule of any of claims 19-21, encoding an antibody light chain comprising a nucleotide sequence of SEQ ID NO: 15, or encoding an antibody light chain variable region comprising a nucleotide sequence of SEQ ID NO: 16.

23. The nucleic acid molecule according to claim 19 or 20, encoding a VH sequence of the second antigen-binding region of the bispecific antibody binding to human CD3 comprising a nucleotide sequence of SEQ ID NO: 21, or encoding a VL sequence of the second antigen-binding region of the bispecific antibody binding to human CD3 comprising a nucleotide sequence of SEQ ID NO: 22.

24. A vector comprising a nucleic acid molecule of any one of claims 19-23.

25. The vector of claim 24, wherein the vector is an expression vector.

26. A recombinant host cell comprising a nucleic acid molecule of any one of claims 19- 23 or a vector of claim 24 or 25.

27. The recombinant host cell of claim 26, wherein the recombinant host cell is selected from the group consisting of a bacterial cell, a mammalian cell, and a human cell.

28. The recombinant host cell of claim 27, wherein the recombinant host cell is human embryonic kidney (HEK-293) or Chinese hamster ovary (CHO) cell.

29. A method for producing an antibody or antigen-binding fragment thereof of any one of claims 1-13, or the bispecific antibody of any one of claims 14-18, the method comprising culturing a recombinant host cell of any one of claims 26-28 under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.

30. A pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1-13, or the bispecific antibody of any one of claims 14-18, and a pharmaceutically-acceptable diluent, carrier or excipient.

31. The pharmaceutical composition of claim 30, adapted for a delivery route selected from the group consisting of parenteral deliver}', intravenous delivery, topical delivery, intradermal delivery, intranodal delivery, intratumoral delivery, and subcutaneous delivery.

32. The antibody or antigen-binding fragment thereof of any one of claims 1-13, or the bispccific antibody of any one of claims 14-18, or the pharmaceutical composition of claim 30 or 31, for use in medicine.

33. A method of treating a cancer or a non-malignant disease in a subject in need thereof, comprising administering to the subject the antibody or antigen binding fragment thereof of any one of claims 1-13, or the bispecific antibody of any one of claims 14- 18, or the pharmaceutical composition of claim 30 or 31; wherein the antibody or antigen binding fragment thereof or the bispecific antibody induces antibodydependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137-expressing cancer cells and / or Treg.

34. Use of the antibody or antigen binding fragment thereof of any one of claims 1 -13, or the bispecific antibody of any one of claims 14-18, or the pharmaceutical composition of claim 30 or 31, in the manufacture of a medicament for treating a cancer or a non-malignant disease in a subject in need of, wherein the antibody or antigen binding fragment thereof or the bispccific antibody or the pharmaceutical composition is to be administered to the subject, wherein the antibody or antigen binding fragment thereof induces antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or Complement-dependent cytotoxicity (CDC) of CD137-expressing cancer cells and / or Treg.

35. The method of claim 33 or the use of claim 34, wherein the cancer is selected from the group consisting of nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL), rhabdomyosarcoma (RMS), hepatocellular carcinoma (HCC), melanoma, stomach cancer, brain cancer, eye cancer (such as retinoblastoma), breast cancer, lung cancer, colorectal cancer, kidney cancer (such as Wilms tumor), pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, neuroblastoma, bone cancer (such as osteosarcoma and Ewing sarcoma), skin cancer, bladder cancer, adrenocortical carcinoma, glioblastoma, diffuse large B cell lymphoma (DLBCL), NK / T cell lymphoma and primary effusion lymphoma.

36. The method of claim 33 or the use of claim 34, wherein the CD137-cxprcssing cancer cells are cells of a cancer selected from the group consisting of nasopharyngeal cancer (NPC), Hodgkin's lymphoma (HL), rhabdomyosarcoma (RMS), hepatocellular carcinoma (HCC) and melanoma.

37. The method of claim 33 or the use of claim 34, wherein the Treg is an intra-tumoral Trcg.

38. The method of claim 33 or the use of claim 34, wherein the non-malignant disease is characterised by having CD137-expressing cells, such as mononucleosis and posttransplantation lymphoproliferative disease.

39. The method of any one of claims 33 and 35-38 or the use of any one of claims 34-38, wherein the antibody or antigen binding fragment thereof or the bispecific antibody or the pharmaceutical composition is administered via intratumoral, intravenous, intranodal, intradermal or subcutaneous administration.

40. The method of any one of claims 33 and 35-39 or the use of any one of claims 34-39, wherein the subject is human.41 . The method of any one of claims 33 and 35-40 or the use of any one of claims 34-40, wherein one or more further therapeutic agents is to be administered.

42. The method or use of claim 41, wherein the one or more further therapeutic agents is a cancer therapeutic agent selected from the group consisting of tacrolimus, a dendritic cell therapy, an immune checkpoint inhibitor, a chimeric antigen receptor T or NK cell, and an immunostimulatory antibody.

43. The method or use of claim 42, wherein the antibody or antigen binding fragment thereof or the pharmaceutical composition is administered before, at the same time, or after the administration of the one or more further therapeutic agents.

44. The method or use of claim 41, wherein the one or more further therapeutic agents is to be administered using the antibody or antigen binding fragment thereof of any one of statements 1-13 as a carrier, wherein the one or more further therapeutic agents are conjugated with the antibody or antigen binding fragment thereof to form a conjugate, and wherein the conjugate is internalized by the CD137-expressing cancer cells and / or Treg upon binding.

45. The method or use of claim 44, wherein the one or more further therapeutic agents is selected from the group consisting of a cytotoxic compound selected from the group consisting of Maytansinoids, Auristatins, Duocarmycins, Calicheamicins, Pyrrolobcnzodiazcpincs, SN-38, Doxorubicin, and Camptothccin derivatives, and a radioactive compound selected from the group consisting of Todine-131 (1-131 ), Yttrium-90 (Y-90), Lutetium-177 (Lu-177), Indium-I l l (In-111), Technetium-99m(Tc-99m), Rhenium-186 (Re-186), Rhenium-188 (Re-188), Copper-64 (Cu-64), Zirconium-89 (Zr-89) and Actinium-225 (Ac-225).

46. The method of any one of claims 33 and 35-45 or the use of any one of claims 34-45, wherein the antibody or antigen binding fragment thereof or the bispecific antibody or the pharmaceutical composition is administered at a frequency from once every three days to once a month.

47. The method of any one of claims 33 and 35-46 or the use of any one of claims 34-46, wherein the antibody or antigen binding fragment thereof or the bispecific antibody or the pharmaceutical composition is administered for a period of 1 day to 1 year.

48. The method of any one of claims 33 and 35-47 or the use of any one of claims 34-47, wherein the antibody or antigen binding fragment thereof or the bi specific antibody or the pharmaceutical composition is administered at a dose of 0.1 - 100 mg / kg body weight.

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