Antibodies targeting CD137 and methods of use thereof

Abstract

The present invention relates to isolated antibodies that specifically bind to human CD137, as well as pharmaceutical compositions and methods of using the same. The invention further relates to nucleic acids comprising nucleotide sequences encoding the antibodies, vectors comprising the nucleic acids, host cells comprising the nucleic acids or the vectors, and methods of producing the antibodies.

Description

[Technical field]

[0001] The present invention relates to isolated antibodies that specifically bind to human CD137, and pharmaceutical compositions and methods of use thereof. The present invention further relates to nucleic acids encoding said antibodies, vectors comprising said nucleic acids, host cells comprising said nucleic acids or said vectors, and methods of producing said antibodies. [Background technology]

[0002] The tumor necrosis factor receptor superfamily (TNFRSF) is a protein superfamily of receptors characterized by their ability to bind tumor necrosis factor (TNF) through cysteine-rich pseudorepeats in the extracellular domain (Locksley et al., 2001, Cell. 104: 487-501). Currently, 27 TNF family members have been identified. TNFRSF members and their ligands are primarily expressed in immune cells and play an immunomodulatory role in T cell-mediated immune responses. TNFRSF members play a role in enhancing dendritic cell survival and T cell priming capacity, optimal generation of effector T cells, optimal antibody responses, and amplification of inflammatory responses.

[0003] CD137 (4-1BB, TNF receptor superfamily 9, TNFRSF9) is a surface glycoprotein of the TNFR superfamily. It is an inducible costimulatory T cell receptor. CD137 expression is activation-dependent and encompasses a broad subset of immune cells, including activated NK and NKT cells, regulatory T cells, dendritic cells (DCs) including follicular DCs, stimulated mast cells, differentiating myeloid cells, monocytes, neutrophils, eosinophils (Wang et al, Immunol Rev. 229(1): 192-215(2009)), and activated B cells (Zhang et al, J Immunol. 184(2):787-795(2010)). Furthermore, expression of CD137 has been demonstrated in tumor vasculature (Broil K et al., Am J Clin Pathol. 115(4):543-549 (2001); Seaman et al, Cancer Cell 11(6):539-554 (2007)) and atherosclerotic endothelium (Olofsson et al, Circulation 117(10): 1292 1301 (2008)).

[0004] CD137 ligand (CD137L, 4-1BBL, or tnfsf9), a molecule of the TNF family, is the known intercellular natural ligand for CD137 (Alderson, MR, et al., Eur. J. Immunol. 24:2219-2227 (1994); Pollok K., et al., Eur. J. Immunol. 24:367-374 (1994); Goodwin, RG, et al., Eur. J. Immunol. 23: 2631-2641 (1993)). The ligand for CD137 forms a homotrimer, and signaling through CD137 proceeds from a tethering molecule on the cell surface that is crosslinked by the trimerized ligand (Won, EY, et al., J. Biol. Chem. 285: 9202-9210 (2010)). Higher order clustering of CD137 has been suggested to be necessary to mediate signal transduction. CD137 binds to the adaptors TRAF-2 and TRAF-1 at its cytoplasmic tail, resulting in coimmunoprecipitation. This is enhanced by CD137 activation in T cells (Saoulli, K., et al., J. Exp. Med. 187: 1849-1862 (1998); Sabbagh, L., et al., J. Immunol. 180: 8093-8101 (2008)). Recruitment of TRAF-1 and TRAF-2 by CD137 results in downstream activation of NF-kB and the mitogen-activated protein (MAP) kinase cascade, including ERK, JNK, and p38 MAP kinase. NF-kB activation leads to upregulation of Bfl-1 and Bcl-XL, which are pro-survival members of the Bcl-2 family. The pro-apoptotic protein Bim is downregulated in a TRAF-1 and ERK-dependent manner (Sabbagh et al., J Immunol. 180(12):8093-8101 (2008)).It has been suggested that the primary action of CD137 is to position two or more TRAF-2 molecules in molecular proximity to each other (Sanchez-Paulete, AR, et al., Eur. J. Immunology 46(3): 513-522 (2016)). Based on this, it was hypothesized that the main factor driving CD137 signaling is the relative density of TRAF-2-assembled CD137 moieties within micropatches of the plasma membrane (Sanchez-Paulete, AR, et al., Eur. J. Immunology 46(3): 513-522 (2016)). Overall, it was proposed that CD137 signaling is driven by multimerization and that cross-linking of CD137 molecules is a key factor for CD137 costimulatory activity.

[0005] CD137 co-stimulates T cells to eradicate established tumors, primary CD8 + T cells (primary CD8 + Expanded antigen-specific CD8 T cell response + It performs effector functions such as enhancing the memory pool of T cells and inducing interferon gamma (IFN-γ) synthesis. CD8 +The critical role of CD137 stimulation in T cell function and survival may be exploited for the treatment of tumors through manipulation of CD137 / CD137L interactions. Indeed, in vivo efficacy studies in mice showed that treatment with anti-CD137 antibodies led to tumor regression in multiple tumor models. For example, agonistic anti-mouse CD137 antibodies were demonstrated to induce immune responses against P815 mast cell tumors, as well as the poorly immunogenic tumor model Ag104 (I. Melero et al., Nat. Med., 3(6):682-5 (1997)). Several studies have reported the efficacy of CD137 agonistic mAbs in preventive and therapeutic settings, both as monotherapy and in combination therapy, as well as anti-tumor protective T cell memory responses (Lynch et al., Immunol Rev. 222:277-286 (2008)). CD137 agonists also inhibit autoimmune responses in various autoimmune models (Vinay et al, J Mol Med 84(9):726-736 (2006)).

[0006] Two anti-CD137 antibodies currently in clinical trials are urelumab (Bristol-Myers Squibb), a fully human IgG4 mAb, and utomilumab (PF-05082566, Pfizer), a fully human IgG2 mAb (Chester C., et al., Cancer Immunol Immunother Oct;65(10):1243-8 (2016)). The use of therapeutic antibodies acting on CD137 is a very promising treatment strategy, but is associated with difficulties such as low efficacy, high toxicity, and adverse events of anti-CD137 agonist antibodies. CD137 agonist antibodies have been shown to cause changes in the immune system and organ function, increasing the risk of toxicity. High doses of CD137 agonist antibodies in naive and tumor-bearing mice have been reported to induce T cell infiltration into the liver and elevations in aspartate aminotransferase and alanine aminotransferase consistent with hepatic inflammation (Niu L, et al. J Immunol 178(7):4194-4213 (2007); Dubrot J, et al., Int J Cancer 128(1):105-118 (2011)). The first clinical studies of human therapeutic use of CD137 agonist antibodies also showed elevated liver enzymes and increased incidence of hepatitis (Sznol M., et al., J Clin Oncol 26(115S):3007 (2008); Ascierto PA, et al., Semin Oncol 37(5):508-516 (2010); Chester C., et al., Cancer Immunol Immunother Oct;65(10):1243-8 (2016)). Potentially fatal hepatitis was observed in a Bristol-Myers Squibb (BMS) phase II anti-CD137 trial, National Clinical Trial (NCT) 00612664, in previously treated stage III / IV melanoma.This study and several others (NCT00803374, NCT00309023, NCT00461110, NCT00351325) were terminated due to adverse events (Chester C., et al., Cancer Immunol Immunother Oct;65(10):1243-8 (2016)). Such adverse events were likely due to systemic overstimulation of T cells.

[0007] Therefore, there is a need in the art to generate improved therapeutic anti-human CD137 antibodies with higher potency, without the inherent side effects of common anti-proliferative drugs, and in particular with lower toxicity comparable to currently available CD137 antibodies. Summary of the Invention

[0008] It is an object of the present invention to provide antibodies that specifically bind to human CD137 protein and have beneficial properties for therapeutic use, such as improved affinity, efficacy, safety, and improved biophysical properties such as improved solubility, developability, and stability, etc. In particular, CD137 antibodies that are not directly and independently of other cell surface molecules trigger CD137 signaling upon binding.

[0009] In one embodiment, the invention relates to novel CD137 antibodies.

[0010] In one embodiment, the invention relates to a pharmaceutical composition comprising an isolated antibody of the invention and a pharma- ceutically acceptable carrier.

[0011] In another aspect, the invention relates to the antibody of the invention, or the composition of the invention, for use as a medicament.

[0012] In one aspect, the invention relates to an antibody of the invention, or a composition of the invention, for use in treating cancer in a subject in need thereof.

[0013] In one aspect, the invention relates to the use of an antibody of the invention, or a composition of the invention, in the manufacture of a medicament for use in the treatment of cancer in a subject in need thereof.

[0014] In another aspect, the invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody of the invention, or a composition of the invention.

[0015] In yet another aspect, the invention relates to a nucleic acid encoding an antibody of the invention. In a further aspect, the invention relates to a vector comprising said nucleic acid. In a further aspect, the invention relates to a host cell comprising said nucleic acid or said vector.

[0016] In another aspect, the invention relates to a method for producing an antibody of the invention comprising the step of culturing a host cell containing a nucleic acid or vector of the invention.

[0017] The aspects, advantageous features, and preferred embodiments of the present invention summarized in the following items, each alone or in combination, further contribute to solving the object of the present invention: 1. A set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the set of CDRs is HCDR1' is an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 7, and 10; HCDR2' is an amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 8, and 11; HCDR3' is an amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 9, and 12; LCDR1' is an amino acid sequence selected from any one of SEQ ID NOs: 16, 19, and 22; LCDR2' is an amino acid sequence selected from any one of SEQ ID NOs: 17, 20, and 23; and LCDR3' is an amino acid sequence selected from any one of SEQ ID NOs: 18, 21, and 24; An isolated antibody, having binding specificity for human CD137, which antibody has no more than 10 amino acid substitutions compared to a set of CDRs. 2. A set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the set of CDRs comprises: HDR'1 is as set forth in SEQ ID NO:1; HDR'2 is as set forth in SEQ ID NO:2; HDR'3 is as set forth in SEQ ID NO:3; LDR'1 is as set forth in SEQ ID NO:16; LDR'2 is as set forth in SEQ ID NO:17; LDR'3 is as set forth in SEQ ID NO:18; 2. The antibody of item 1, having no more than 10 amino acid substitutions compared to one set of CDRs. 3. A heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises, in order, three complementarity determining regions HCDR1, HCDR2, and HCDR3; and (b) the VL comprises, in order, three complementarity determining regions LCDR1, LCDR2, and LCDR3; The antibody according to item 1 or item 2. 4. (a) said HCDR1 comprises, preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs:1, 4, 7 and 10, preferably SEQ ID NO:1; (b) said HCDR2 comprises, preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs:2, 5, 8 and 11, preferably SEQ ID NO:2; (c) said HCDR3 comprises, preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs:3, 6, 9 and 12, preferably SEQ ID NO:3; (d) said LCDR1 comprises, preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs:16, 19 and 22, preferably SEQ ID NO:16; (e) said LCDR2 comprises, preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs:17, 20 and 23, preferably SEQ ID NO:17; (f) said LCDR3 comprises, preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs:18, 21 and 24, preferably SEQ ID NO:18. The antibody according to item 3. 5. The antibody comprises: (a) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively; (b) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 4, 5, and 6, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 19, 20, and 21, respectively; (c) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 7, 8, and 9, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively; (a) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 10, 11, and 12, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 22, 23, and 24, respectively. The antibody according to item 3, comprising: 6. (a) HCDR1 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:1; (b) HCDR2 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:2; (c) HCDR3 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:3; (d) LCDR1 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:16; (e) LCDR2 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:17; and (f) LCDR3 comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:18. The antibody of item 3, 7. The antibody according to any one of items 3 to 6, wherein the VH comprises a VH3 framework FR1, FR2, FR3, and FR4. 8. The antibody according to any one of items 3 to 7, wherein the VL comprises a framework FR4 selected from Vκ framework FR1, FR2 and FR3, specifically Vκ1 or VK3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and Vκ FR4, specifically Vκ1 FR4 or Vκ3 FR4, and Vλ FR4; specifically, the Vλ FR4 comprises an amino acid sequence having at least 60, 70, 80 or 90% identity to an amino acid sequence selected from any one of SEQ ID NOs: 33 to 39, preferably a Vλ FR4 set forth in any one of SEQ ID NOs: 33 to 39, preferably a Vλ FR4 set forth in SEQ ID NO: 33 or SEQ ID NO: 34, more preferably a Vλ FR4 set forth in SEQ ID NO: 33. 9. The antibody according to any one of items 3 to 8, wherein the VH comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 15, preferably SEQ ID NO: 13 or 15, more preferably SEQ ID NO: 13; and / or the VL comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, and 27, preferably SEQ ID NO: 25 or 27, more preferably SEQ ID NO: 25. 10. The antibody according to any one of items 3 to 9, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 15, preferably SEQ ID NO: 13 or 15, more preferably SEQ ID NO: 13; and / or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, and 27, preferably SEQ ID NO: 25 or 27, more preferably SEQ ID NO: 25. 11. The antibody according to any one of items 1 to 10, comprising: (a) a VH sequence of SEQ ID NO: 13 and a VL sequence of SEQ ID NO: 25; (b) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26; or (c) a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 27. 12. The antibody is a) binds to human CD137 with a dissociation constant (KD) of less than 50 nM, specifically less than 10 nM, more specifically less than 5 nM, as measured by surface plasmon resonance; and b) optionally binds to cynomolgus CD137 with a KD of less than 50 nM, particularly less than 10 nM, more particularly less than 5 nM, as measured by surface plasmon resonance; and c) optionally cross-compete with urelumab; 12. The antibody according to any one of items 1 to 11. 13. The antibody according to any one of items 1 to 12, wherein the antibody does not inhibit the interaction between CD137 and its ligand CD137L, in particular as measured by competitive ELISA. 14. The antibody according to any one of items 1 to 13, wherein the antibody does not bind to human CD40 and / or does not bind to human OX40, in particular as measured by SPR. 15. The antibody described above: a) in the case of scFv format, has a melting temperature (Tm) of at least 50°C, e.g. at least 55°C, preferably at least 60°C, more preferably at least 64°C, as determined by differential scanning fluorimetry, particularly where said antibody is formulated in 50 mM phosphate-citrate buffer, 150 mM NaCl, pH 6.4; and / or b) in the case of an scFv format, the antibody of the invention, when at a starting concentration of 10 mg / ml, in particular where the antibody of the invention is formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4, has a loss of less than 7%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, preferably less than 2%, of monomer content after storage at 4° C. for at least 2 weeks, in particular at least 4 weeks; and / or d) in the case of scFv format, the antibodies of the invention, when at a starting concentration of 10 mg / ml, in particular where the antibodies of the invention are formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4, have a loss of monomer content of less than 5%, preferably less than 3%, more preferably less than 1%, after 5 successive freeze-thaw cycles. 15. The antibody according to any one of items 1 to 14. 16. An isolated antibody that binds to the human CD137 extracellular domain at an epitope located in the distal part of the extracellular domain of CD137, in particular within the cysteine-rich domains CRD1 and / or CRD2, more particularly within amino acid residues 24 to 86 of SEQ ID NO:32, provided that amino acid residue Asn42 of CD137 is not a critical residue for binding. 17. The antibody according to any one of items 1 to 16, wherein the isolated antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a Fab, an Fv, an scFv, a dsFv, an scAb, an STAB, and an alternative scaffold-based binding domain, preferably an Fv, or an scFv, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites engineered into the constant region of the antibody (e.g., F-star's Modular Antibody Technology™). 18. The antibody according to any one of items 1 to 17, which is a single-chain antibody (single-chain variable fragment; scFv). 19. The antibody described in item 18, wherein the scFv has an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, preferably SEQ ID NO:29 or SEQ ID NO:31, more preferably SEQ ID NO:29. 20. The isolated antibody of item 17, wherein the antibody is an IgG selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, preferably IgG4. 21. The isolated antibody of any one of paragraphs 1 to 20, wherein the antibody is humanized. 22. The antibody according to any one of items 1 to 21, wherein the antibody is a multispecific molecule, in particular a multispecific molecule having at least a second functional molecule. 23. The antibody may be a single chain diabody (scDb), tandem scDb (Tandab), linear dimeric scDb (LD-scDb), circular dimeric scDb (CD-scDb), bispecific T cell inducer (BiTE; tandem di-scFv), tandem tri-scFv, tribody (Fab-(scFv)2) or bibody (Fab-(scFv)1), Fab, Fab-Fv2, Morrison (IgG CH3-scFv fusion (Morrison L), or IgG CL-scFv fusion (Morrison H), triabody, scDb-scFv, bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc-scFv fusion, scFv-HSA-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusion, e.g. bsAb (scFv bound to the C-terminus of the light chain), Bs1Ab (scFv bound to the N-terminus of the light chain), Bs2Ab (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the light chain), BsAb (scFv bound to the N-terminus of the light chain), BsAb (scFv bound to the C ... 23. The antibody of item 22, in a format selected from the group consisting of: s3Ab (scFv attached to the C-terminus of the heavy chain), Ts1Ab (scFv attached to the N-terminus of both the heavy and light chains), Ts2Ab (dsscFv attached to the C-terminus of the heavy chain), bispecific antibodies based on heterodimeric Fc domains, such as Knob-into-Hole antibodies (KiHs); Fv, scFv, scDb, tandem-di-scFv, tandem tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD, MATCH, and DuoBodies fused to the N-terminus and / or C-terminus of either chain of a heterodimeric Fc domain or other heterodimerization domain. 24. A pharmaceutical composition comprising the antibody of any one of items 1 to 23 and a pharma- ceutically acceptable carrier. 25. The antibody of any one of items 1 to 23 or the composition of item 24 for use as a medicament. 26. The antibody of any one of items 1 to 23 or the composition of item 24 for use in the manufacture of a medicament for use in the treatment of cancer. 27. The antibody of any one of items 1 to 23 or the composition of item 24 for use in treating cancer. 28. Use of the antibody of any one of items 1 to 23 or the composition of item 24 for the treatment of cancer in a subject in need thereof. 29. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody according to any one of items 1 to 23 or the composition according to item 24. 30. A nucleic acid encoding the antibody according to any one of items 1 to 23. 31. A vector comprising the nucleic acid according to item 30. 32. A host cell comprising the nucleic acid according to item 30 or the vector according to item 31. 33. A method for producing the antibody according to any one of items 1 to 23, comprising the step of culturing a host cell containing the nucleic acid according to item 30 or the vector according to item 31. 34. A kit comprising the antibody according to any one of items 1 to 23 or the composition of item 24. [Brief description of the drawings]

[0018] [Figure 1] Heatmap of epitope binding results of rabbit IgG clone 38-27-A11 with urelumab and utomilumab. Binding levels normalized to theoretical Rmax expressed as a percentage (%) of test article molecules (columns) relative to immobilized molecules (rows). No binding (dark grey) means the same epitope, light grey means that a second molecule (test article) is able to bind and has a different epitope than the immobilized molecule. [Diagram 2] There is no inhibition of CD137 binding to CD137L in competitive ELISA. The absorbance measured in the competitive ELISA assessing the binding of CD137L to CD137 is expressed as a function of increasing concentrations of PRO1359 or PRO1360, respectively. The inhibitory antibody goat anti-human CD137 served as a reference. [Diagram 3] Binding of PRO1359 and PRO1360 to human CD137-expressing Jurkat cells assessed by flow cytometry (FC). The geometric mean of the resulting fluorescent signal is expressed as a function of the molecule concentration in ng / ml. Urelumab was used as a reference. [Figure 4] (A) Structure of CD137. CRD1-4 indicate cysteine-rich domains 1 to 4. (B) Designed constructs of CD137 ECD. [Diagram 5] The trispecific scDb-scFv molecules PRO1480 and PRO1481 were tested in a CD137 activation assay in the presence of HCC827 cells stimulated with IFNγ (10 ng / ml) for 6 and 24 h. In this experiment, PRO885 served as a reference molecule to assess the relative activation of CD137 signaling. The trispecific scDb-scFv molecule PRO1186 was taken on each plate and its activity was compared to the other scDb-scFv molecules. Luminescence was read 6 or 24 h after addition of Jurkat reporter cells and sigmoidal 4PL fit (GraphPad Prism) was used to fit only the concentrations of test molecules that increased the RLU values. [Figure 6] Ex vivo T cell activation assay. PBMCs were stimulated with 10 ng / ml SEA and treated with serial dilutions of the scDb-scFv construct PRO1480 for 96 h. T cell activation was assessed by quantification of IL-2 in harvested supernatants by ELISA. Treatment with PRO1480 resulted in significant IL-2 secretion when compared to a mixture of reference molecules. Data were fitted using sigmoidal 4PL fit (GraphPad Prism). [Figure 7]IL-2 secretion of human PBMC stimulated with or without Staphylococcal Enterotoxin A (SEA) (96 h, 1 mg / ml HSA). PBMC were stimulated with 10 ng / ml SEA and treated with serial dilutions of the reference molecules avelumab and urelumab and a mixture of scDb-scFvs PRO1480 and PRO1186 for 96 h. T cell activation was assessed by quantification of IL-2 in harvested supernatants by ELISA. Treatment with PRO1480 resulted in significant IL-2 secretion when compared to treatment with PRO1486. ​​Data were fitted using sigmoidal 4PL fit (GraphPad Prism). [Figure 8] Titers and maximal values ​​of T cell activation by different anti-CD137 antibody fragments. The titers and maximal values ​​of T cell activation by the antibody constructs scDb-scFv PRO1480 and PRO1186 are expressed by the area under the curve (AUC) of IL-2 secretion and the maximal stimulation of IL-2 secretion, respectively, as shown in FIG. 7. [Figure 9] Schematic diagram of CD137 binding of various anti-CD137 antibody fragments. The extracellular domain (ECD) of CD137 is composed of four cysteine-rich domains (CRD1-4). (A) The anti-CD137 antibody PRO1480 binds to an epitope on CD137 contained in the distal part of the ECD, CRD1 and / or CRD2. (B) In contrast, the anti-CD137 antibody PRO1186 binds to an epitope on CD137 contained in the part of the ECD proximal to the cell, CRD4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention provides antibodies that specifically bind to human CD137 protein, as well as pharmaceutical compositions, methods of making, and methods of using such antibodies and compositions.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] The terms "comprising" and "including" are used herein in their open, non-limiting sense unless otherwise indicated. Thus, with respect to such latter embodiments, the term "comprising" includes the narrower term "consisting of."

[0022] The terms "a" and "an" and "the" and similar references in the context of describing the present invention (particularly in the context of the claims below) should be construed to encompass both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. When the plural is used for compounds, salts, etc., this is construed to mean a single compound, salt, etc.

[0023] In a first aspect, the present invention relates to an antibody that specifically binds to human CD137.

[0024] In one embodiment, the disclosure provides an isolated antibody having binding specificity for human CD137, comprising a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the set of CDRs are: HCDR1' is an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 7, and 10, preferably the amino acid sequence of SEQ ID NO: 1; HCDR2' is an amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 8, and 11, preferably the amino acid sequence of SEQ ID NO: 2; HCDR3' is an amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 9, and 12, preferably the amino acid sequence of SEQ ID NO: 3; and LCDR1' is an amino acid sequence selected from any one of SEQ ID NOs: 16, 19, and 22. LCDR1' is an amino acid sequence selected from any one of SEQ ID NOs: 17, 20, and 23, preferably the amino acid sequence of SEQ ID NO: 17; and LCDR3' is an amino acid sequence selected from any one of SEQ ID NOs: 18, 21, and 24, preferably the amino acid sequence of SEQ ID NO: 18, having 10 or less amino acid substitutions, for example 9 or less amino acid substitutions, 8 or less amino acid substitutions, 7 or less amino acid substitutions, 6 or less amino acid substitutions, 5 or less amino acid substitutions, 4 or less amino acid substitutions, 3 or less amino acid substitutions, 2 or less amino acid substitutions, 1 or 0 amino acid substitutions, preferably 0 amino acid substitutions, compared to a set of CDRs.

[0025] In a specific embodiment, the present invention relates to an isolated antibody having binding specificity for human CD137, comprising HCDR1, HCDR2, and HCDR3 sequences having at least 90% identity to the sequences of SEQ ID NOs:1, 2, and 3, respectively, LDCR1 and LDCR3 sequences having at least 90% identity to the sequences of SEQ ID NOs:16 and 18, and an LDCR2 sequence having at least 85% identity to the sequence of SEQ ID NO:17.

[0026] The term "antibody" as used herein includes: whole antibodies or single chains thereof; and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof; and molecules comprising antibody CDRs, VH regions, or VL regions (including, but not limited to, multispecific antibodies). A naturally occurring "whole antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0027] As used herein, the terms "antigen-binding fragment", "antigen-binding fragment thereof", "antigen-binding portion" and the like refer to one or more fragments of an intact whole antibody that retain the ability to specifically bind to a given antigen (e.g., CD137). The antigen-binding function of an antibody can be performed by a fragment of an intact antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody are Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; F(ab)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; and alternative scaffold-based binding domains, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites integrated into the constant region of an antibody (e.g., F-star's Modular Antibody Technology™). The term "complementarity determining region ("CDR")" refers to those sequences described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme), and Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670 ("AHo" numbering). For example, in the classical format, Kabat numbers the CDR amino acid residues of the heavy chain variable domain (VH) as 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDRs of the light chain variable domain (VL) as 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3). The R amino acid residues are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues of the VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).Combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of the human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of the human VL. In TMGT, the CDR amino acid residues of the VH are numbered approximately 26-35 (HCDR1), 51-57 (HCDR2), and 93-102 (HCDR3), and the CDR amino acid residues of the VL are numbered approximately 27-32 (LCDR1), 50-52 (LCDR2), and 89-97 (LCDR3) ("Kabat" numbering). In IMGT, the CDRs of an antibody can be determined using the program IMGT / DomainGapAlign.

[0028] In the context of the present invention, unless otherwise stated, the numbering system proposed by Honegger & Pluckthun ("AHo") is used (Honegger & Pluckthun, J. Mol. Biol. 309 (2001) 657-670).Furthermore, the following residues are defined as CDRs according to the AHo numbering scheme: LCDR1 (also called CDR-L1): L24-L42; LCDR2 (also called CDR-L2): L58-L72; LCDR3 (also called CDR-L3): L107-L138; HCDR1 (also called CDR-H1): H27-H42; HCDR2 (also called CDR-H2): H57-H76; HCDR3 (also called CDR-H3): H108-H138. For clarity, the numbering system by Honegger & Pluckthun takes into account the length diversity found in naturally occurring antibodies in both different VH and VL subfamilies, especially in the CDRs, and provides for sequence gaps, and therefore in a given antibody variable domain, not all positions 1 to 149 are typically occupied by an amino acid residue.

[0029] Preferably, the "antigen-binding region" comprises at least amino acid residues 4-138 of the variable light (VL) chain and 5-138 of the variable heavy (VH) chain (in each case numbering according to Honegger & Pluckthun), more preferably amino acid residues 3-144 of VL and 4-144 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1-149 of VL and 1-149 of VH). Antigen-binding moieties can also be incorporated into maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, scDb-scFv, v-NAR and bis-scFv (see, e.g., Holliger and Hudson, 2005, Nature Biotechnology, 23, 1126, 36). Antigen-binding portions of antibodies can be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antigen-binding portions can be incorporated into single-chain molecules that contain a pair of tandem Fv segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., 1995 Protein Eng. 8(10):1057-1062; and U.S. Pat. No. 5,641,870).

[0030] The term "binding specificity" as used herein refers to the ability of an individual antibody binding site to react with one antigenic determinant, but not with a different antigenic determinant. As used herein, the term "specifically binds" or "is specific" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. In its most common form (and when no defined reference is mentioned), "specific binding" refers to the ability of an antibody to distinguish between a desired target and unrelated molecules, for example, as determined according to a specificity assay method known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, SPR (surface plasmon resonance) testing, and peptide scanning. For example, a standard ELISA assay can be performed. Scoring can be done by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). Reaction in a particular well is recorded by absorbance at, for example, 450 nm. A typical background (=negative reaction) may be about 0.1 OD; a typical positive reaction may be about 1 OD. This means that the ratio of positive to negative scores can be 10-fold or more. In a further example, an SPR assay can be performed, where a difference between background and signal of at least 10-fold, preferably at least 100-fold, indicates specific binding. Usually, the determination of binding specificity is not performed using a single reference molecule, but a set of about 3-5 unrelated molecules such as milk powder, transferrin, etc. The antibody of the present invention has binding specificity for human CD137. In a particular embodiment, the antibody of the present invention has binding specificity for human CD137 and does not bind to human CD40 and / or does not bind to human OX40, particularly as determined by SPR.

[0031] Suitably, the antibody of the present invention is an isolated antibody. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CD137 is substantially free of antibodies that specifically bind to antigens other than CD137). However, an isolated antibody that specifically binds to CD137 may cross-react with other antigens, such as CD137 molecules of other species. Thus, in one embodiment, an antibody of the present invention has binding specificity for human CD137 and Macaca fascicularis (also known as Cynomolgus monkey or "Cynomolgus") CD137. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0032] Preferably, the antibody of the present invention is a monoclonal antibody. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to antibodies whose amino acid sequences are substantially identical or derived from the same genetic source. A monoclonal antibody composition exhibits binding specificity and affinity for a particular epitope or binding specificity and affinity for particular epitopes.

[0033] The antibodies of the present invention include, but are not limited to, chimeric antibodies and humanized antibodies.

[0034] The term "chimeric antibody" refers to an antibody in which (a) the constant region or a portion thereof has been altered, replaced or exchanged such that the antigen binding site (variable region) is linked to the constant region of a different or altered class, effector function and / or species, or a completely different molecule, thereby giving the chimeric antibody new properties, e.g., enzymes, toxins, hormones, growth factors, drugs, etc.; or (b) the variable region or a portion thereof has been altered, replaced or exchanged with a variable region having a different or altered antigen specificity. For example, a murine antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. The replacement with a human constant region allows the chimeric antibody to retain its specificity in recognizing the antigen while reducing its antigenicity in humans compared to the original murine antibody.

[0035] As used herein, a "humanized" antibody is an antibody that retains the reactivity of a non-human antibody but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (i.e., the constant and variable region framework portions). Additional framework region modifications can be made within the human framework sequences, as well as within the CDR sequences derived from the germline of another mammalian species. The humanized antibodies of the present invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo, or conservative substitutions to promote stability or production). See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239: 1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec. Immun., 31: 169-217, 1994. Other examples of human engineering techniques include, but are not limited to, the Xoma technology disclosed in U.S. Patent No. 5,766,886.

[0036] The term "recombinant humanized antibody" as used herein includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, e.g., antibodies isolated from a host cell that has been transformed to express the humanized antibody, such as, for example, transfectomas, and antibodies prepared, expressed, created or isolated by any other means including splicing all or part of the human immunoglobulin gene, sequence, into other DNA sequences.

[0037] Preferably, the antibody of the present invention is humanized. Preferably, the antibody of the present invention is humanized and comprises CDRs of rabbit origin.

[0038] The term "CD137" refers in particular to human CD137 of UniProt ID number Q07011, reproduced herein as SEQ ID NO:32. Advantageously, the antibodies of the invention target CD137, in particular human CD137 as shown in UniProt ID number Q07011, reproduced herein as SEQ ID NO:32. Advantageously, the antibodies of the invention target human and cynomolgus (Macaca fascicularis) CD137. The antibodies of the invention specifically bind to CD137. In a particular embodiment, the antibodies of the invention have binding specificity for human CD137 and do not bind to human CD40 and / or do not bind to human OC40, in particular as determined by SPR. Preferably, the antibodies of the invention do not block the CD137 / CD137L interaction.

[0039] Suitably, the antibody of the present invention is a CD137 agonist. An "activator" or "activating antibody" or "agonist" or "agonist antibody" is one that enhances or initiates signal transduction by the antigen to which it binds. In the context of the present invention, the term "CD137 agonist" encompasses an antibody of the present invention that can activate CD137 signal transduction upon clustering of its CD137 antigen-binding fragments, e.g., the binding of at least two of said CD137 antigen-binding fragments allows multimerization of the bound CD137 molecules and their activation. In some embodiments, an agonist antibody activates signal transduction without the presence of a natural ligand.

[0040] Antibodies of the present invention include, but are not limited to, humanized monoclonal antibodies isolated as described herein, including in the Examples. Examples of such anti-human CD137 antibodies are the antibodies whose sequences are set forth in Table 1. Additional details regarding the generation and characterization of the antibodies described herein are provided in the Examples.

[0041] The isolated antibody of the present disclosure having binding specificity for human CD137 has a heavy chain variable region (VH) and a light chain variable region (VL), where: (a) the VH comprises three complementarity determining regions HCDR1, HCDR2, and HCDR3, in that order, and (b) the VL comprises three complementarity determining regions LCDR1, LCDR2, and LCDR3, in that order. For clarity, the CDR regions are not linked to each other but are adjacent to the framework regions FR1 to FR4.

[0042] The present invention provides antibodies that specifically bind to CD137 protein, said antibodies comprising a VH CDR having the amino acid sequence of any one of the VH CDRs listed in Table 1. Specifically, the present invention provides antibodies that specifically bind to CD137, said antibodies comprising one, two, or three VH CDRs having the amino acid sequence of any of the corresponding VH CDRs listed in Table 1.

[0043] The present invention provides an isolated antibody having binding specificity for human CD137 comprising a heavy chain variable region (VH), wherein said VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 in order, said HCDR1 having the amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 7 and 10, preferably SEQ ID NO: 1, said HCDR2 having the amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 8 and 11, preferably SEQ ID NO: 2, and said HCDR3 having the amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 9 and 12, preferably SEQ ID NO: 3. Specifically, the present invention provides an antibody having binding specificity for human CD137 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively.

[0044] The present invention also provides antibodies that specifically bind to CD137 protein, said antibodies comprising a VL CDR having the amino acid sequence of any one of the VL CDRs listed in Table 1. Specifically, the present invention provides antibodies that specifically bind to CD137, said antibodies comprising one, two, or three VL CDRs having the amino acid sequence of any of the corresponding VL CDRs listed in Table 1.

[0045] The present invention provides an isolated antibody having binding specificity for human CD137 comprising a light chain variable region (VL), wherein said VL comprises three complementarity determining regions LCDR1, LCDR2 and LCDR3 in order, said LCDR1 having an amino acid sequence selected from any one of SEQ ID NOs: 16, 19 and 22, preferably SEQ ID NO: 16, said LCDR2 having an amino acid sequence selected from any one of SEQ ID NOs: 17, 20 and 23, preferably SEQ ID NO: 17, and said LCDR3 having an amino acid sequence selected from any one of SEQ ID NOs: 18, 21 and 24, preferably SEQ ID NO: 18. Specifically, the present invention provides an antibody having binding specificity for human CD137 comprising the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 16, 17 and 18, respectively.

[0046] Suitably, the present invention provides an isolated antibody having binding specificity for human CD137 comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) said VH comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 in order, said HCDR1 having an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 7 and 10, said HCDR2 having an amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 8 and 11, and said HCDR3 having an amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 9 and 12; and (b) The VL comprises three complementarity determining regions LCDR1, LCDR2, and LCDR3 in order, wherein the LCDR1 has an amino acid sequence selected from any one of SEQ ID NOs: 16, 19, and 22, the LCDR2 has an amino acid sequence selected from any one of SEQ ID NOs: 17, 20, and 23, and the LCDR3 has an amino acid sequence selected from any one of SEQ ID NOs: 18, 21, and 24.

[0047] Specifically, the present invention provides antibodies having binding specificity for human CD137 and comprising (a) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and (b) LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively.

[0048] Other antibodies of the invention comprise amino acids that are mutated but have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity in the CDR regions to the CDR regions shown in the sequences set forth in Table 1. In one embodiment, other antibodies of the invention comprise mutated amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids are mutated in the CDR regions when compared to the CDR regions shown in the sequences set forth in Table 1.

[0049] The term "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. "Percent (%) identity" and "homology" with respect to nucleic acid, peptide, polypeptide or antibody sequences are defined as the percentage of nucleic acid / amino acid residues of a candidate sequence that are identical to the nucleic acid / amino acid residues of a particular nucleic acid, peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, or ALIGN software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0050] For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0051] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, respectively, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0052] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) as incorporated into the ALIGN program (version 2.0) using a weight residue table of PAM120, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) as incorporated into the GAP program of the GCG software package (available at www.gcg.com) using either a Blossom 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0053] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses its conservatively modified variants.

[0054] Suitably, the isolated antibody of the invention having binding specificity for human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) said VH comprises, in order, three complementarity determining regions HCDR1, HCDR2, and HCDR3; said HCDR1 having at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOs: 1, 4, 7, and 10, preferably SEQ ID NO: 1; the HCDR2 has at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOs: 2, 5, 8, and 11, preferably SEQ ID NO: 2; said HCDR3 has at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOs: 3, 6, 9, and 11, preferably SEQ ID NO: 3; and / or (b) said VL comprises, in order, three complementarity determining regions LCDR1, LCDR2 and LCDR3; the HCDR1 has at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOs: 16, 19, and 22, preferably SEQ ID NO: 16; the HCDR2 has at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOs: 17, 20, and 23, preferably SEQ ID NO: 17; The HCDR3 has at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to any one of SEQ ID NOs: 18, 21, and 24, preferably SEQ ID NO: 18.

[0055] In one embodiment, an antibody of the invention with binding specificity for human CD137 comprises: (a) HCDR1, HCDR2, and HCDR3 with at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NOs: 1, 2, and 3, respectively, and / or LCDR1, LCDR2, and LCDR3 with at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with SEQ ID NOs: 16, 17, and 18, respectively. In one embodiment, an antibody of the invention with binding specificity for human CD137 comprises: (a) HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and / or LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively.

[0056] In a further embodiment, the present invention provides an isolated antibody that specifically binds to CD137 (e.g., human CD137 protein), wherein the antibody comprises a VH domain and a VL domain. In the context of the present invention, the terms "VH" (variable heavy chain), "VL" (variable light chain), "Vκ" and "Vλ" refer to a family of antibody heavy and light chain sequences grouped according to sequence identity and homology. Methods for determining sequence homology, for example by using homology search matrices such as BLOSUM (Henikoff, S. & Henikoff, JG, Proc. Natl. Acad. Sci. USA 89 (1992) 10915-10919), and grouping of sequences by homology, are well known to those skilled in the art. For VH, Vκ and Vλ, for example, Knappik et al., J. Mol. Biol. 296 (2000) 57-86, group VH into VH1A, VH1B, VH2 to VH6, Vκ into Vκ1 to Vκ4, and Vλ into Vλ1 to Vλ3. In vivo, antibody Vκ, Vλ and VH chains are the result of random rearrangement of germline κ chain V and J segments, germline λ chain V and J segments, and heavy chain V, D and J segments, respectively. Which subfamily a particular antibody variable chain belongs to is determined by the corresponding V segment, particularly the framework regions FR1 to FR3. Thus, in the present application, any VH sequence characterized by a particular set of only framework regions HFR1 to HFR3 can be combined with any HFR4 sequence, for example an HFR4 sequence taken from one of the heavy chain germline J segments, or an HFR4 sequence taken from a rearranged VH sequence.

[0057] Suitably, the present invention provides an isolated antibody (e.g., human CD137 protein) that specifically binds to CD137, wherein said antibody comprises a VH3 domain. Particular examples of VHs belonging to the VH3 family are shown under SEQ ID NO: 13 or SEQ ID NO: 14. In particular, the framework regions FR1 to FR3 taken from SEQ ID NO: 13 or SEQ ID NO: 14 belong to the VH3 family (Table 1, first three regions not in bold). Suitably, a VH belonging to the VH3 family as used herein is a VH comprising FR1 to FR3 having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity to FR1 to FR3 of SEQ ID NO: 13 or SEQ ID NO: 14. More particularly, the framework regions FR1 to FR4 taken from SEQ ID NO: 13 or SEQ ID NO: 14 belong to the VH3 family (Table 1, regions not in bold). Preferably, a VH belonging to the VH3 family used in this specification is a VH having FR1 to FR4 that has at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity to FR1 to FR4 of SEQ ID NO: 13 or SEQ ID NO: 14.

[0058] Suitably, the present invention provides an isolated antibody that specifically binds to CD137 (e.g., human CD137 protein), wherein said antibody comprises Vκ framework FR1, FR2, and FR3, in particular framework FR4 selected from Vκ1 or Vκ3 frameworks, preferably Vκ1 framework FR1-3, and Vκ FR4, preferably Vκ1 FR4 or Vκ3 FR4 framework, and Vλ FR4. Suitable Vκ1 framework FR1-FR3 are as set forth in SEQ ID NO:25 or SEQ ID NO:26 (Table 1, FR regions not bolded). Suitable Vκ1 framework FR1-FR3 comprise an amino acid sequence corresponding to FR1-FR3 and having at least 60, 70, 80, 90 percent identity with the amino acid sequence obtained from SEQ ID NO:25 or SEQ ID NO:26 (Table 1, FR regions not bolded).

[0059] A suitable Vλ FR4 is as set forth in SEQ ID NO: 33 to SEQ ID NO: 39. In one embodiment, the present invention provides an isolated antibody that specifically binds to CD137 (e.g., human CD137 protein), wherein the antibody comprises a Vλ FR4 comprising an amino acid sequence having at least 60, 70, 80, 90 percent identity to an amino acid sequence selected from any of SEQ ID NO: 33 to SEQ ID NO: 39, preferably SEQ ID NO: 33 or SEQ ID NO: 34, more preferably SEQ ID NO: 33.

[0060] In one embodiment, the antibody of the invention that has binding specificity for human CD137 comprises: (i) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively; (ii) VH3 domain framework sequences FR1 to FR4; and (iii) A VL framework comprising Vκ frameworks FR1, FR2, and FR3, specifically Vκ1 or Vκ3 FR1 to FR3, preferably Vκ1 FR1 to FR3, and a Vκ FR4, specifically Vκ1 FR4 or Vκ3 FR4, and a Vλ FR4, specifically an amino acid sequence having at least 60, 70, 80, 90% identity to an amino acid sequence selected from any of SEQ ID NOs: 33 to 39, preferably a Vλ FR4 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, more preferably SEQ ID NO: 33, more specifically a Vλ FR4 comprising an amino acid sequence selected from any of SEQ ID NOs: 33 to 39, preferably a Vλ FR4 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, more preferably SEQ ID NO: 33.

[0061] In one embodiment, the invention therefore provides an antibody that has binding specificity for human CD137 and comprises a VL comprising: (i) the CDR domains CDR1, CDR2, and CDR3; (ii) human Vκ framework regions FR1-FR3, specifically, human Vκ1 framework regions FR1-FR3; (iii) (a) a human Vλ germline sequence of FR4, in particular a Vλ germline sequence selected from the list of SEQ ID NO: 33 to 39, preferably SEQ ID NO: 33 or SEQ ID NO: 34, more preferably SEQ ID NO: 33; and (b) a Vλ-based sequence having one or two mutations, specifically one mutation, compared to the closest human Vλ germline sequence to the FR4 comprising an amino acid sequence selected from any of SEQ ID NO: 33 to SEQ ID NO: 39, preferably SEQ ID NO: 33 or SEQ ID NO: 34, more preferably SEQ ID NO: 33.

[0062] In a more preferred embodiment, the antibody of the invention that has binding specificity for human CD137 comprises: (i) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively; (ii) VH3 domain framework sequences FR1 to FR4; and (iii) A VL domain comprising a VL framework comprising Vκ1 framework FR1, FR2, and FR3, and a Vλ FR4 comprising an amino acid sequence having at least 60, 70, 80, or 90% identity to an amino acid sequence selected from any one of SEQ ID NOs: 33 to 39, specifically any one of SEQ ID NOs: 33 to 39, preferably SEQ ID NO: 33 or SEQ ID NO: 34, and more preferably SEQ ID NO: 33.

[0063] The present invention provides isolated antibodies that specifically bind to CD137 (eg, human CD137 protein), wherein the antibodies comprise a VH domain listed in Table 1.

[0064] The present invention also provides an isolated antibody that specifically binds to CD137, wherein the antibody comprises (or, alternatively, consists of) a VH amino acid sequence listed in Table 1, and wherein about 10 or fewer amino acids in the framework sequences (e.g., sequences that are not CDRs) are mutated (wherein the mutations are additions, substitutions, or deletions, as various non-limiting examples).

[0065] The present invention also provides an isolated antibody that specifically binds to CD137, wherein the antibody comprises a VH amino acid sequence listed in Table 1, wherein about 20 or less amino acids in a framework sequence (e.g., a sequence that is not a CDR) are mutated (wherein the mutations are additions, substitutions, or deletions, as various non-limiting examples).

[0066] Other antibodies of the invention include amino acids that are mutated but have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity in the VH region to the VH regions shown in the sequences set forth in Table 1.

[0067] The present invention provides isolated antibodies that specifically bind to a CD137 protein, the antibodies comprising a VL domain listed in Table 1.

[0068] The present invention also provides an isolated antibody that specifically binds to CD137, wherein the antibody comprises a VL amino acid sequence listed in Table 1, wherein about 10 or less amino acids in a framework sequence (e.g., a sequence that is not a CDR) are mutated (wherein the mutations are additions, substitutions, or deletions, as various non-limiting examples).

[0069] The present invention also provides an isolated antibody that specifically binds to CD137, wherein the antibody comprises a VL amino acid sequence listed in Table 1, wherein about 20 or less amino acids in a framework sequence (e.g., a sequence that is not a CDR) are mutated (wherein the mutations are additions, substitutions, or deletions, as various non-limiting examples).

[0070] Other antibodies of the invention include amino acids that are mutated but have at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity in the VL region to the VL regions shown in the sequences set forth in Table 1.

[0071] Suitably, the invention provides an isolated antibody that specifically binds to CD137, wherein said antibody comprises a heavy chain variable region comprising amino acids that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, preferably at least 90%, identical to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14 or SEQ ID NO:15, preferably SEQ ID NO:13, and specifically wherein said antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively. In one embodiment, the invention provides an isolated antibody or one that specifically binds to human CD137, wherein said antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:13, wherein said heavy chain variable region comprises G51C (AHo numbering). In a further embodiment, the present invention provides an isolated antibody that specifically binds to human CD137, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:14, wherein the heavy chain variable region comprises V2S, Y105F, and Q141P (AHo numbering).

[0072] In another embodiment, the invention provides an isolated antibody that specifically binds to CD137, wherein the antibody comprises a light chain variable region comprising amino acids that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, preferably at least 90%, identical to the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:26 or SEQ ID NO:27, preferably SEQ ID NO:25, and specifically wherein the antibody comprises LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively. In one embodiment, the invention provides an isolated antibody that specifically binds to human CD137, wherein the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:25, wherein the light chain variable region comprises T141C (AHo numbering). In a further embodiment, the present invention provides an isolated antibody that specifically binds to human CD137, wherein the antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:26, wherein the light chain variable region comprises I2F, M4L, and A51P (AHo numbering).

[0073] The present invention also provides an isolated antibody that specifically binds to CD137, wherein the antibody comprises a heavy chain variable region comprising amino acids that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, preferably at least 90%, identical to an amino acid sequence selected from the group consisting of SEQ ID NO:13, 14, and SEQ ID NO:15, preferably SEQ ID NO:13; and a light chain variable region comprising amino acids that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, preferably at least 90%, identical to an amino acid sequence selected from the group consisting of SEQ ID NO:25, 26, and 27, preferably SEQ ID NO:25.

[0074] In one embodiment, an antibody of the invention having binding specificity for human CD137 comprises: a heavy chain variable region comprising an amino acid sequence selected from any of SEQ ID NOs: 13, 14, and 15, preferably SEQ ID NO: 13; and a light chain variable region comprising an amino acid sequence selected from any of SEQ ID NOs: 25, 26, and 27, preferably SEQ ID NO: 25.

[0075] Thus, the present invention provides an isolated antibody that specifically binds to human CD137, the antibody comprising: a heavy chain variable region comprising amino acids that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, preferably at least 90%, identical to the amino acid sequence of SEQ ID NO:13; and a heavy chain variable region comprising amino acids that are at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, preferably at least 90%, identical to the amino acid sequence of SEQ ID NO:25. and wherein the antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and / or the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, preferably wherein the antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18.

[0076] In a further embodiment, the isolated antibody of the invention, which has binding specificity for human CD137, comprises: (a) the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 25; (b) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 26; or (c) the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 27. In a preferred embodiment, the isolated antibody of the invention, which has binding specificity for human CD137, comprises the VH sequence of SEQ ID NO: 13 and the VL sequence of SEQ ID NO: 25.

[0077] In one embodiment, the antibody of the invention that has binding specificity for human CD137 comprises: (a) a VH sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively, the amino acid sequence of SEQ ID NO: 13, and a VL sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 25; (b) a VH sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively, the amino acid sequence of SEQ ID NO: 14, and a VL sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 26, preferably wherein the VH comprises V2S, Y105F, and Q141P (AHo numbering), and the VL comprises I2F, M4L, and A51P (AHo numbering); or (c) the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, a VH sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:15, and a VL sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:27, preferably wherein the VH comprises G51C (AHo numbering) and the VL comprises T141C (AHo numbering).

[0078] In a preferred embodiment, an antibody of the present invention having binding specificity for human CD137 comprises HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively, a VH sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 15, and a VL sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 27, preferably wherein the VH comprises a G51C mutation (AHo numbering) and the VL comprises a T141C mutation (AHo numbering). Advantageously, the antibodies of the invention are mutated to form an artificial interdomain disulfide bridge in the framework regions, in particular where a pair of cysteines replaces Gly51 (AHo numbering) on ​​the VH and Thr141 (AHo numbering) on ​​the VL. Surprisingly, it was found that such antibodies of the invention comprising an interdomain disulfide bridge have a significantly increased thermal stability.

[0079] The term "artificial" with respect to disulfide bridges ("SS bridges" or "diS") means that the SS bridges are not naturally formed by wild-type antibodies, but by engineered variants of the parent molecule, in which at least one foreign amino acid contributes to the disulfide bond. The site-specific engineering of artificial disulfide bridges is clearly distinct from those naturally available in natural immunoglobulins or modular antibodies such as those described in WO 2009 / 000006. This is because at least one of the bridgehead sites of the artificial disulfide bridges is usually located apart from the position of the Cys residues of wild-type antibodies, thus providing alternative or additional disulfide bridges within the framework regions. The artificial disulfide bridges of the invention can be engineered within antibody domains ("intra-domain bridges"), which stabilize the beta-sheet structure, or cross-link domains ("inter-domain bridges") or chains of domains ("inter-chain bridges"), to constrain the structure of the multispecific antibody according to the invention and support its interaction with potential binding partners.

[0080] In one embodiment, the antibody of the invention that has binding specificity for human CD137 comprises: (a) a VH sequence of SEQ ID NO: 13 and a VL sequence of SEQ ID NO: 25; (b) the VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO: 26; or (c) the VH sequence of SEQ ID NO:15 and the VL sequence of SEQ ID NO:27.

[0081] In a preferred embodiment, an antibody of the invention having binding specificity for human CD137 comprises the VH sequence of SEQ ID NO:13 and the VL sequence of SEQ ID NO:25.

[0082] In one embodiment, the antibody that specifically binds to CD137 is an antibody set forth in Table 1. In one embodiment, the antibody that specifically binds to CD137 is as set forth in SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In one embodiment, the antibody that specifically binds to CD137 is as set forth in SEQ ID NO:30. In one embodiment, the antibody that specifically binds to CD137 is as set forth in SEQ ID NO:29: or SEQ ID NO:31. In a preferred embodiment, the antibody that specifically binds to CD137 is as set forth in SEQ ID NO:29.

[0083] Other antibodies of the invention include those in which the amino acids or nucleic acids encoding the amino acids have been mutated but have at least 60, 70, 80, 90, or 95 percent identity to the sequences set forth in Table 1. In one embodiment, the antibodies include mutated amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the variable regions while retaining substantially the same therapeutic activity when compared to the variable regions set forth in the sequences set forth in Table 1. The term "substantially the same activity" as used herein refers to activity exhibited by substantially the same activity, which is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100%, at least 110%, or at least 120%, or at least 130%, or at least 140%, or at least 150%, or at least 160%, or at least 170%, or at least 180%, or at least 190%, for example up to 200%, of the activity determined for a parent antibody, e.g., an antibody of the present disclosure, particularly an antibody of the present disclosure set forth in Table 1.

[0084] Given that each of these antibodies can bind to CD137 and that antigen-binding specificity is provided primarily by the CDR1, 2, and 3 regions, VH CDR1, 2, and 3 sequences and VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched), provided that each antibody contains a VH CDR1, 2, and 3 and a VL CDR1, 2, and 3 to generate other CD137 binding binding molecules of the invention. Such "mixed and matched" CD137 binding antibodies can be tested using binding assays known in the art and those described in the Examples (e.g., ELISA). When VH CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequences of a particular VH sequence should be replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the CDR1, CDR2, and / or CDR3 sequences of a particular VL sequence should be replaced with structurally similar CDR sequences. It will be readily apparent to one of ordinary skill in the art that novel VH and VL sequences can be generated by mutating one or more VH and / or VL CDR region sequences with structurally similar sequences from the CDR sequences presented herein for the monoclonal antibodies of the invention.

[0085] In yet another embodiment, the invention provides antibodies comprising an amino acid sequence that is homologous to a sequence set forth in Table 1, said antibodies binding to CD137 and retaining the desired functional properties of those antibodies set forth in Table 1.

[0086] For example, the present invention provides an isolated monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:13, 14, and 15, preferably at least 80 percent, at least 90 percent, or at least 95 percent identical to the amino acid sequence of SEQ ID NO:13; the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:25, 26, and 27, preferably at least 80 percent, at least 90 percent, or at least 95 percent identical to the amino acid sequence of SEQ ID NO:25; and wherein the antibody specifically binds to human CD137.

[0087] In one embodiment, the VH and / or VL amino acid sequences may be 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to a sequence set forth in Table 1. In one embodiment, the VH and / or VL amino acid sequences may be identical except for amino acid substitutions at no more than 1, 2, 3, 4 or 5 amino acid positions.

[0088] In one embodiment, an antibody of the invention has a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, where one or more of these CDR sequences have a particular amino acid sequence based on an antibody described herein or conservative modifications thereof, where the antibody retains the desired functional properties of a CD137 binding antibody of the invention.

[0089] The term "conservatively modified variant" or "conservative variant" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to sequences that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variants are "silent variants," which are one type of conservatively modified variant. All nucleic acid sequences herein that encode a polypeptide also describe all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon of a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0090] In the case of polypeptide sequences, "conservatively modified variants" or "conservative variants" include individual substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservatively substituted for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine, threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In one embodiment, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0091] Accordingly, the present invention provides an isolated monoclonal antibody consisting of a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: The heavy chain variable region CDR1 preferably consists of an amino acid sequence selected from any one of SEQ ID NOs: 1, 4, 7, 10, preferably SEQ ID NO: 1, or a conservative variant thereof; the heavy chain variable region CDR2 preferably consists of an amino acid sequence selected from any one of SEQ ID NOs: 2, 5, 8, 11, preferably SEQ ID NO: 2, or a conservative variant thereof; the heavy chain variable region CDR3 preferably consists of an amino acid sequence selected from any one of SEQ ID NOs: 3, 6, 9, 12, preferably SEQ ID NO: 3, or a conservative variant thereof; the light chain variable region CDR1 preferably consists of an amino acid sequence selected from any one of SEQ ID NOs: 16, 19, 22, preferably SEQ ID NO: 16, or a conservative variant thereof; the light chain variable region CDR2 preferably consists of an amino acid sequence selected from any one of SEQ ID NOs: 17, 20, 23, preferably SEQ ID NO: 17, or a conservative variant thereof; the light chain variable region CDR3 preferably consists of an amino acid sequence selected from any one of SEQ ID NOs: 18, 21, 24, preferably SEQ ID NO: 18, or a conservative variant thereof; Here, the antibodies specifically bind to CD137 and can activate CD137 signaling with or without additional cross-linking.

[0092] In one embodiment, the antibody of the present invention is optimized for expression in mammalian cells and has a heavy chain variable region and a light chain variable region, wherein one or more of these sequences are an antibody or a conservative modification thereof described herein, wherein the antibody retains the desired functional properties of the CD137 binding antibody of the present invention. Thus, the present invention provides an isolated monoclonal antibody optimized for expression in mammalian cells, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 13, 14, and 15, preferably SEQ ID NO: 13, or a conservative modification thereof; the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 25, 26, and 27, preferably SEQ ID NO: 25, or a conservative modification thereof; wherein the antibody is capable of specifically binding to CD137 and activating CD137 signaling with or without additional cross-linking.

[0093] In one embodiment, an antibody of the invention is optimized for expression in mammalian cells having a full-length heavy chain sequence and a full-length light chain sequence, where one or more of these sequences have a specific amino acid sequence based on an antibody described herein or conservative modifications thereof, and where the antibody retains the desired functional properties of the CD137 binding antibodies of the invention.

[0094] As used herein, the term "optimized" refers to a nucleotide sequence that has been modified to encode an amino acid sequence using codons that are preferred in a production cell or organism, typically a eukaryotic cell, such as a Pichia cell, a Chinese hamster ovary cell (CHO), or a human cell. An optimized nucleotide sequence is designed to retain, completely or as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence. The optimized sequences herein are designed to have codons that are preferred in mammalian cells. However, optimized expression of these sequences in other eukaryotic or prokaryotic cells is also contemplated herein. The amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0095] Another type of variable region modification is to mutate amino acid residues in the VH and / or VL CDR1, CDR2 and / or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation". Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and the effect on antibody binding, or other functional properties of interest, can be evaluated in in vitro or in vivo assays, as described herein and provided in the Examples. Conservative modifications can be introduced (as explained above). Mutations may be amino acid substitutions, additions, or deletions. Furthermore, typically no more than one, two, three, four, or five residues in the CDR regions are altered.

[0096] An "affinity matured" antibody is one that has one or more changes in one or more variable domains thereof that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have those changes. In one embodiment, an affinity matured antibody has nanomolar or even picomolar affinity for a target antigen. Affinity matured antibodies are produced by methods known in the art. For example, Marks et al, Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of hypervariable region (HVR) and / or framework residues has been described, for example, by Barbas et al. Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Jackson et al, J. Immunol. 154(7):3310-9(1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0097] Antibodies of the invention can also be prepared using antibodies having one or more of the VH and / or VL sequences set forth herein as starting materials to engineer engineered antibodies, which may have altered properties from the starting antibody. Antibodies can be engineered by modifying one or more residues in one or both variable regions (i.e., VH and / or VL), e.g., within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues in the constant regions, e.g., to alter the effector functions of the antibody.

[0098] One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than sequences outside the CDRs. Because the CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that contain CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad. Sci. USA 86: 10029-10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762, and 6,180,370 to Queen et al.).

[0099] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences or rearranged antibody sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as Kabat, EA, et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al., 1992 J. Mol. Biol. 227:776-798; and Cox, JPL et al., 1994 Eur. J Immunol. 24:827-836, the contents of each of which are expressly incorporated herein by reference. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "IMGT" database (available on the Internet at www.imgt.org; see Lefranc, MP et al., 1999 Nucleic Acids Res. 27:209-212; the contents of each of which are expressly incorporated herein by reference).

[0100] Exemplary framework sequences for use in the antibodies of the present invention are those structurally similar to the framework sequences used by the selected antibodies of the present invention, e.g., the consensus sequences and / or framework sequences used by the monoclonal antibodies of the present invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3 sequences, can be grafted onto framework regions having sequences identical to those found in the germline immunoglobulin genes from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations compared to the germline sequences. For example, in certain instances, it has been found to be beneficial to mutate residues within framework regions to maintain or enhance the antigen-binding ability of the antibody (see U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0101] A wide variety of antibody / immunoglobulin frameworks or scaffolds can be used, so long as the resulting polypeptide contains at least one binding region that specifically binds to CD137. Such frameworks or scaffolds include the five major idiotypes of human immunoglobulins, antigen-binding fragments thereof, and immunoglobulins of other animal species, preferably with humanized versions.

[0102] In one embodiment, the present invention relates to a method for generating non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds that can be grafted with the CDRs of the present invention. Known or future non-immunoglobulin frameworks and scaffolds can be used as long as they contain binding regions specific to the target CD137 protein. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, Massachusetts), ankyrin (Molecular Partners AG, Zurich, Switzerland), lipocalin (Pieris Proteolab AG, Friessing, Germany), small modular immuno-pharmaceutical (Trubion Pharmaceuticals Inc., Seattle, Washington), maxibody (Avidia, Inc., Mountain View, California), protein A (Affibody AG, Sweden), and affilin (gamma crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

[0103] Suitably, the antibodies of the invention specifically bind to CD137 and are characterized by one or more of the following parameters: (i) binds to human CD137 with a dissociation constant (KD) of less than 50 nM, specifically less than 10 nM, more specifically less than 5 nM, as measured by surface plasmon resonance; (ii) 5 × 10 as measured by SPR -3 s -1 Less than or equal to 10 -3 s -1 Less than or equal to 5×10 -4 s -1 Less than or equal to 10 -4 s -1 The following K off Binds to human CD137 with kinetics; (iii) at least 10 as measured by SPR 4 M -1 s-1 More than, at least 10 5 M -1 s -1 More than or equal to 5×10 5 M -1 s -1 More than, at least 10 6 M -1 s -1 K on Binds to human CD137 with kinetics; (iv) optionally cross-competes with urelumab; (v) optionally cross-reacts with Macaca fascicularis (Cynomolgus) CD137; (vi) optionally, does not inhibit the interaction between CD137 and CD137L, particularly as measured by competitive ELISA.

[0104] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0105] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity," "binds to," "binds to," or "binding to" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody fragment and an antigen). The affinity of a molecule X for a partner Y is generally measured by the dissociation constant (K D) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific exemplary and illustrative embodiments for measuring binding affinity, i.e., binding strength, are described below.

[0106] As used herein, the term "K assoc ", "Ka" or "K on " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term "K dis ", "Kd" or "K off " is intended to refer to the dissociation rate of a particular antibody-antigen interaction. In one embodiment, the term "KD" as used herein is intended to refer to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). "KD" or "KD value" or "KD value" according to the present invention is intended to refer to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). D " or "K DThe "Affinity Value" is measured in one embodiment by using a surface plasmon resonance assay using a MASS-1 SPR instrument (Sierra Sensors). To measure affinity, an antibody specific for the Fc region of rabbit IgG (Bethyl Laboratories, Cat. No. A120-111A) is immobilized on a sensor chip (SPR-2 affinity sensor, high capacity amine, Sierra Sensors) using standard amine coupling methods. Rabbit monoclonal antibodies in the B cell supernatant are captured by the immobilized anti-rabbit IgG antibody. A minimum IgG concentration in the B cell supernatant is required to allow sufficient capture. After capturing the monoclonal antibodies, human CD137 ECD (Peprotech, Cat. No. 310-15-1MG) is injected into the flow cell at a concentration of 90 nM for 3 minutes and dissociation of the protein from the IgG captured on the sensor chip is allowed to proceed for 5 minutes. After each injection cycle, the surface is regenerated with two injections of 10 mM glycine-HCl. The apparent dissociation (kd) and association (ka) rate constants, and the apparent dissociation equilibrium constant (KD) were calculated using a one-to-one Langmuir binding model in the MASS-1 analysis software (Analyzer, Sierra Sensors), and the quality of the curve fits was evaluated using the relative Chi, a measure of the quality of the curve fit. 2 (Chi normalized to the extrapolated maximum binding level of the analyte 2 ) is monitored based on 2 The smaller the value of k, the more accurate the fit to the one-to-one Langmuir binding model. Results are considered valid if the response units (RU) of ligand binding are at least 2% of the RU of antibody capture. Samples with RU of ligand binding less than 2% of the RU of antibody capture are considered to not show specific binding of CD137 to the captured antibody. The equilibrium dissociation constant (KD) is k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0107] Advantageously, the affinity of the antibody of the present invention to CD137 may be comparable to or higher than the affinity of CD137L to CD137. Advantageously, the affinity of the antibody of the present invention to CD137 may be comparable to or higher than the affinity of urelumab to CD137. It will be understood that the higher affinity of the CD137 binding domain may be particularly suitable for use in antibodies, where said antibodies are monovalent to CD137. The binding affinity of an antibody may be determined, for example, by dissociation constant (KD). Stronger affinity is represented by a lower KD, and weaker affinity is represented by a higher KD.

[0108] Thus, in preferred embodiments, the antibodies of the invention may have a KD of between 5-50,000 pM, 5-40,000 pM, 5-30,000 pM, 5-20,000 pM, 5-10,000 pM, 5-9,000 pM, 5-8,000 pM, 5-7,000 pM, 5-6,000 pM, 5-5,000 pM, particularly as measured by SPR. In further embodiments, the antibodies of the invention bind to human CD137 with a KD of between 10 nM and 10 pM, preferably between 10 nM and 0.1 nM, more preferably between 5 nM and 1 nM, particularly as measured by SPR.

[0109] In a preferred embodiment, the antibody of the present invention may have a KD of less than about 50 nM, less than about 45 nM, less than about 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, especially when measured by SPR. Advantageously, the antibody of the present invention has a KD of less than 10 nM, especially when measured by SPR. Preferably, the antibody of the present invention binds to human CD137 with a KD of less than 5 nM, especially when measured by SPR.

[0110] Preferably, the antibody of the invention has a molecular weight of at least 10 as measured by surface plasmon resonance. 3 M -1s -1 More than, at least 10 4 M -1 s -1 More than or equal to 5×10 4 M -1 s -1 More than, at least 10 5 M -1 s -1 More than or equal to 5×10 5 M -1 s -1 More than, at least 10 6 M -1 s -1 More than K on Suitably, the antibodies of the invention bind to human CD137 with a rate of at least 10 as measured by SPR. 5 M -1 s -1 More specifically, at least 5×10 5 M -1 s -1 More than K on Has speed.

[0111] Preferably, the antibody of the invention has a molecular weight of at least 10 as measured by surface plasmon resonance. -3 s -1 Below, 3 x 10 -3 s -1 Below, 5 x 10 -3 s -1 Below, 10 -4 s -1 Below, 5 x 10 -4 s -1 The following K off Suitably, the antibodies of the invention bind to human CD137 with a rate of 5×10 as measured by SPR. -3 s -1 The following K off Has speed.

[0112] Preferably, the antibodies of the present invention have beneficial biophysical properties.

[0113] Suitably, the antibodies of the invention, when in scFv format, have a melting temperature (Tm) of at least 50°C, e.g., at least 55°C, preferably at least 60°C, more preferably at least 65°C, as determined by differential scanning fluorimetry, where the antibodies are formulated in 50 mM phosphate-citrate buffer at pH 6.4, 150 mM NaCl. DSF has been previously described (Egan, et al., MAbs, 9(1) (2017), 68-84; Niesen, et al., Nature Protocols, 2(9) (2007) 2212-2221). The midpoint of the thermal denaturation transition of the scFv construct is determined by differential scanning fluorimetry using the fluorescent dye SYPRO® Orange (see Wong & Raleigh, Protein Science 25 (2016) 1834-1840). Samples are formulated in pH 6.4 phosphate-citrate buffer at a final protein concentration of 50 μg / mL and contain a final concentration of 5×SYPRO® Orange in a total volume of 100 μl. 25 μl of prepared samples are added in triplicate to a white-walled AB gene PCR plate. The assay is performed on a qPCR machine used as a thermal cycler and fluorescence emission is detected using a custom dye calibration routine in the software. The PCR plate containing the test samples is subjected to a temperature ramp from 25° C. to 96° C. in 1° C. increments with a 30 second pause after each temperature increase. The total assay time is approximately 2 hours. Tm is calculated by the software GraphPad Prism using the mathematical second derivative method to calculate the inflection point of the curve. The reported Tm is the average of triplicate measurements.

[0114] The antibodies of the invention, particularly when expressed in scFv (single chain variable fragment) antibody format, are characterized by a loss of less than 7%, e.g., less than 6%, less than 5%, less than 3%, preferably less than 2%, of monomer content after storage for at least 2 weeks, particularly at least 4 weeks, when the antibodies of the invention have an initial concentration of 10 mg / ml, particularly when the antibodies of the invention are formulated in 50 mM phosphate-citrate buffer with 150 mM NaCl at pH 6.4. The loss of monomer content is determined by calculation of the area under the curve of the SE-HPLC chromatogram. SE-HPLC is a separation technique based on a solid stationary phase and a liquid mobile phase, as outlined in Chapter 621 of the United States Pharmacopeia. This method utilizes a hydrophobic stationary phase and an aqueous mobile phase to separate molecules based on their size and shape. The separation of molecules occurs between the void volume (V0) and total permeation (VT) of a particular column. Measurements by SE-HPLC are performed on a Chromaster HPLC system (Hitachi High-Technologies Corporation) equipped with automatic sample injection and a UV detector set at a detection wavelength of 280 nm. The instrument is controlled by the software EZChrom Elite (Agilent Technologies, version 3.3.2 SP2). This software also supports the analysis of the resulting chromatograms. Protein samples are removed by centrifugation and kept at a temperature of 4-6 °C in the autosampler before injection. For the analysis of scFv samples, a Shodex KW403-4F column (Showa Denko K.K., #F6989202) was used with a standardized buffered saline mobile phase (50 mM sodium phosphate pH 6.5, 300 mM sodium chloride) at a recommended flow rate of 0.35 mL / min. The target sample load per injection is 5 μg. Samples are detected by a UV detector at a wavelength of 280 nm and data are recorded with a suitable software suite. The resulting chromatograms are analyzed in the range V0 to VT, thus excluding matrix-related peaks with elution times greater than 10 min.

[0115] Furthermore, the antibodies of the invention, particularly when expressed in scFv (single chain variable fragment) antibody format, are characterized by a loss of less than 5%, preferably less than 3%, more preferably less than 1% monomer content after 5 successive freeze-thaw cycles when the antibodies of the invention have an initial concentration of 10 mg / ml, particularly where the antibodies of the invention are formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4.

[0116] The term "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide, or an epitope can be, for example, from two or more non-contiguous regions of a polypeptide or polypeptides.

[0117] In one embodiment, the antibody of the present invention cross-competes with urelumab for binding to CD137. Urelumab, also called BMS-663513, is a fully humanized IgG4 mAb from Bristol-Myers Squibb and is described in WO 2004 / 010947, U.S. Patent No. 6,887,673, and U.S. Patent No. 7,214,493, which are incorporated by reference in their entirety into this application. Advantageously, the antibody of the present invention may bind to the same or overlapping (e.g., structurally similar or spatially proximal) epitope as urelumab, according to non-limiting theory.

[0118] In one embodiment, the antibody of the present invention does not cross-compete for binding with utomirumab. The present invention provides an antibody that binds to an epitope different from utomirumab. Utomirumab, also called PF-05082566, is a fully human IgG2 mAb from Pfizer and is described in WO 2012 / 032433 and U.S. Patent No. 8,821,867, which are incorporated by reference in their entirety into this application. Advantageously, the antibody of the present invention may bind to an epitope different from utomirumab (e.g., structurally different or spatially distant), according to a non-limiting theory. The term "recognize" as used herein refers to an antibody that finds and interacts with (e.g., binds to) its conformational epitope.

[0119] The antibodies of the invention bind to the human CD137 extracellular domain at an epitope located in the distal part of the extracellular domain of CD137, particularly an epitope located within cysteine-rich domains 1-2 (CRD1-2), particularly within amino acid residues 24-86 of SEQ ID NO:32, provided that amino acid residue Asn42 of CD137 is not a critical residue for binding.

[0120] Thus, in a further aspect, the present disclosure also provides an antibody that binds to the same epitope as any of the exemplary antibodies of the disclosure, particularly any of the exemplary antibodies listed in Table 1. The present disclosure provides an isolated antibody that binds to the human CD137 extracellular domain at an epitope located in the distal portion of the extracellular domain of CD137, within amino acid residues 24-86 of SEQ ID NO:32, particularly within the cysteine-rich domain, with the proviso that amino acid residue Asn42 of CD137 is not a critical residue for CRD1 and / or CRD2 binding, more particularly, with the proviso that amino acid residue Asn42 of CD137 is not a critical residue for binding.

[0121] In a specific embodiment, the antibody binds to the human CD137 extracellular domain at an epitope characterized by a set of key residues determined according to Example 13, including residues Arg 41, Gln43, Cys45, Pro49, Ser52, and Ser80.

[0122] In the context of the present invention, the term "critical residues" relates to residues of an antigen that are part of the epitope to which the antibody-based molecule binds and that are important for the interaction between the antibody-based molecule and its epitope. In particular, critical residues are characterized by meeting one or more of the following criteria: (i) they are more than 50% buried when the antibody-based molecule binds to the epitope, (ii) they have specific side chain hydrogen bonding interactions, (iii) they are involved in a hydrogen bonding network, and / or (iv) they show important interactions with key binding residues of the antibody-based molecule. In certain embodiments, these criteria are determined according to the method disclosed in Example 13.

[0123] In a specific embodiment, the antibody binds to the human CD137 extracellular domain at an epitope characterized by a set of key residues determined according to Example 13, further comprising residues Ala33, Asn40, and Cys48.

[0124] In a specific embodiment, the antibody binds to the human CD137 extracellular domain at an epitope characterized by a set of key residues determined according to Example 13, further including residues Pro32, Gly34, Thr35, Ser46, Pro47, Pro50, Cys78, and Ser79.

[0125] In some embodiments, the isolated antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and / or the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, preferably wherein the antibody comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively. Suitably, the antibody has one or more biological properties as described above.

[0126] Thus, additional antibodies can be identified based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies of the invention in CD137 binding assays. In certain embodiments, the present disclosure provides isolated antibodies that compete or cross-compete with any of the exemplary antibodies of the disclosure, particularly any of the exemplary antibodies listed in Table 1, for binding to the same epitope on human CD137.

[0127] The terms "compete" or "cross-compete" and related terms are used interchangeably herein and refer to the ability of an antibody or other binding agent to interfere with the binding of another antibody or binding agent to CD137 in a standard competitive binding assay.

[0128] The ability or extent to which an antibody or other binding agent can interfere with the binding of another antibody or binding molecule to CD137, and therefore whether it can be said to cross-compete according to the invention, can be determined using standard competitive binding assays. One particularly suitable quantitative cross-competition assay measures the competition between a labeled (e.g. His-tagged, biotinylated or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof for their binding to a target, using a FACS or AlphaScreen-based approach. In general, a cross-competing antibody or fragment thereof is one that binds to a target, for example in a cross-competition assay, such that in the assay and in the presence of a second antibody or fragment thereof, the recorded displacement of an immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g. in a FACS-based competitive assay) of the maximum theoretical displacement by a potentially cross-blocking antibody or fragment thereof tested, present in a given amount (e.g. displacement by a cold (e.g. unlabeled) antibody or fragment thereof that needs to be cross-blocked). Preferably, cross-competing antibodies or fragments thereof have a recorded displacement that is between 10% and 100%, more preferably between 50% and 100%.

[0129] Suitably, the isolated antibody of the present invention is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, IgG antibodies, Fab, Fv, scFv, dsFv, scAb, STAB, and alternative scaffold-based binding domains, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectin, and binding sites engineered into the constant region of an antibody (e.g., F-star's Modular Antibody Technology™).

[0130] Preferably, the isolated antibody of the present invention is an Fv fragment. Preferably, the isolated antibody of the present invention is an scFv fragment. A "single chain Fv" or "scFv" or "sFv" antibody comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding (see, for example, Pluckthun, The pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315). In certain embodiments, the functional fragment is an scFv format that comprises a polypeptide linker between the VH and VL domains, where the linker comprises one or more units of four glycine amino acid residues and one serine amino acid residue (GGGGS). nwhere n=1, 2, 3, 4, 5, 6, 8 or 8, preferably n=4. In a particular embodiment, the functional fragment is in scFv format with a linker according to SEQ ID NO:28. In one embodiment, the isolated antibody of the invention that specifically binds to human CD137 comprises an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, preferably SEQ ID NO:29. In a further embodiment, the isolated antibody of the invention is a single-chain variable fragment (scFv) as set forth in SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, preferably SEQ ID NO:29. In one embodiment, the isolated antibody of the invention is a single-chain variable fragment (scFv) as set forth in SEQ ID NO:30. In one embodiment, the isolated antibody of the invention is a single-chain variable fragment (scFv) as set forth in SEQ ID NO: 31. In a preferred embodiment, the isolated antibody of the invention is a single-chain variable fragment (scFv) as set forth in SEQ ID NO: 35.

[0131] Suitably, the isolated antibody of the present invention is an IgG antibody isotype. The term "isotype" refers to the antibody class (e.g., IgM, IgE, IgG, e.g., IgG1 or IgG4) provided by the heavy chain constant region genes. Isotype also includes modified versions of one of these classes, where modifications have been made to alter Fc function, e.g., to enhance or reduce effector function or binding to Fc receptors. In one embodiment, the isolated antibody of the present invention is an IgG selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, preferably IgG4. Suitably, the isolated antibody of the invention is an IgG4 comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90%, identical to SEQ ID NO:13, and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90% identical to SEQ ID NO:25. In a more specific embodiment, the isolated antibody of the invention is an IgG4 comprising a heavy chain sequence comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90%, identical to SEQ ID NO:14, and a light chain sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90% identical to SEQ ID NO:26.Suitably, the isolated antibody of the invention is an IgG4 comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, a VH sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90%, identical to SEQ ID NO:15, and a VL sequence comprising an amino acid sequence that is at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90% identical to SEQ ID NO:27.

[0132] In another specific embodiment, the isolated antibody of the present invention is a multispecific molecule, in particular a multispecific molecule having at least one second functional molecule, e.g., a bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific molecule.

[0133] The term "multispecific molecule" or "multispecific antibody" as used herein refers to an antibody that binds to two or more different epitopes on at least two or more different targets (e.g., CD137 and another target different from CD137) or to two or more different epitopes on the same target. The term "multispecific molecule" includes bispecific, trispecific, tetraspecific, pentaspecific, and hexaspecific antibodies. The term "bispecific antibody" as used herein refers to an antibody that binds to two different epitopes on two different targets or on the same target. The term "trispecific antibody" as used herein refers to an antibody that binds to three different epitopes on three different targets or on the same target.

[0134] The antibodies of the invention, or antigen-binding regions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a multispecific molecule that binds at least two binding sites and / or different target molecules. The antibodies of the invention can in fact be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to two or more different binding sites and / or target molecules. To generate a multispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent linkage, etc.) to one or more other binding molecules, e.g., another antibody, an antibody fragment, a peptide, or a binding mimetic, resulting in a multispecific molecule.

[0135] Accordingly, the present invention includes multispecific molecules comprising at least one first binding specificity for CD137 and a second binding specificity for a second target epitope, e.g., the second target epitope is present on a separate target molecule different from CD137.

[0136] Bivalent CD137 antibodies have been shown to generally have a weak ability to induce signaling in the absence of exogenous clustering. To illustrate, the anti-CD137 antibody utomirumab can activate CD137 signaling only when crosslinked to an anti-human F(ab')2 secondary antibody or immobilized to tissue culture plastic (Fisher at al., Cancer Immunol Immunother 61:1721-1733(2012)). Studies of rodent agonistic antibodies against another member of the TNFRSF, CD40 (TNFRSF5), suggest that exogenous clustering can be achieved in part by interaction with Fcγ receptors (Li F, Ravetch JV, Science 333(6045):1030-10 (2011); White AL, et al., J Immunol 187(4):1754-1763 (2011)). However, interaction with Fcγ receptors may deplete CD137-expressing cells via effector mechanisms. Thus, current bivalent antibodies targeting CD137 are ineffective agonists or lead to depletion of CD137-positive cells. Advantageously, the second binding specificity of the multispecific molecule can provide additional cross-linking of the CD137-binding antibodies of the present invention. Thus, the present invention includes multispecific molecules comprising at least one first binding specificity for CD137 and a second binding specificity for a second target epitope. For example, the second target epitope is another epitope of CD137 that is different from the first target epitope. The multispecific molecule may further comprise a third binding specificity in addition to the first and second target epitopes.

[0137] In a further embodiment, the invention comprises multispecific molecules that are monovalent, bivalent or multivalent, preferably monovalent, with respect to CD137 specificity.

[0138] In another specific embodiment of the invention, the isolated antibody of the invention is a molecule that is monovalent, or multivalent, for example bivalent, trivalent, tetravalent, pentavalent, or hexavalent, with respect to CD137 specificity.

[0139] The term "monovalent molecule" or "monovalent antibody" as used herein refers to an antibody that binds to a single epitope of a target molecule, for example CD137.

[0140] The term "multivalent molecule" or "multivalent antibody" refers to a single binding molecule with multiple valencies, where "valency" is described as the number of antigen-binding moieties that bind to an epitope on the same target molecule. Thus, a single binding molecule can bind to multiple target molecules, or multiple binding sites on a target molecule that contains multiple copies of an epitope. Examples of multivalent antibodies include, but are not limited to, bivalent antibodies, trivalent antibodies, tetravalent antibodies, pentavalent antibodies, and the like. As used herein, the term "bivalent antibody" refers to an antibody having two antigen-binding moieties, each of which binds to the same epitope.

[0141] Suitably, the isolated antibody of the present invention is a multispecific molecule, e.g. a bispecific molecule, and / or a multivalent molecule, e.g. a monovalent for a CD137 specific molecule, a bivalent for a CD137 specific molecule, which may be, by way of non-limiting example, a single chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a cyclic dimeric scDb (CD-scDb), a bispecific T cell trigger (BiTE; tandem di-scFv), a tandem tri-scFv, a tribody (Fab-(scFv)2) or a bibody (Fab-(scFv)1), a Fab, a Fab-Fv2, a Morrison (IgG CH3-scFv fusion (Morrison L), or an IgG CL-scFv fusion (Morrison L), H), triabody, scDb-scFv, bispecific Fab2, di-miniantibody, tetrabody, scFv-Fc-scFv fusion, scFv-HSA-scFv fusion, di-diabody, DVD-Ig, COVD, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusion, e.g. bsAb (scFv bound to the C-terminus of the light chain), Bs1Ab (scFv bound to the N-terminus of the light chain), Bs2Ab (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the heavy chain), BsAb (scFv bound to the C-terminus of the light chain), BsAb (scFv bound to the N-terminus of the light chain), BsAb (scFv bound to the N-terminus of the light chain), BsAb (scFv bound to the C ... s3Ab (scFv attached to the C-terminus of the heavy chain), Ts1Ab (scFv attached to the N-terminus of both the heavy and light chains), Ts2Ab (dsscFv attached to the C-terminus of the heavy chain), bispecific antibodies based on heterodimeric Fc domains, e.g., Knob-into-Hole antibodies (KiHs); Fv, scFv, scDb, tandem-di-scFv, tandem tri-scFv, Fab-(scFv)2, Fab-(scFv)1, Fab, Fab-Fv2, COVD, MATCH (WO 2016 / 0202457; Egan T., et al., mAbs 9 (2017) 68-84), and formats based on DuoBodies (bispecific IgG prepared by Duobody technology) (MAbs. 2017 Feb / Mar;9(2):182-212. doi: 10.1080 / 19420862.2016.1268307).

[0142] The term "diabody" refers to an antibody fragment with two antigen-binding sites, which comprises a VH bound to a VL in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. In certain embodiments, the polypeptide linker is a unit of four glycine amino acid residues and one serine amino acid residue (GGGGS). n where n=1, or 2, preferably 1. Diabodies may be bivalent or bispecific. Diabodies are more fully described, for example, in EP 404097, WO 93 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

[0143] Bispecific scDbs, particularly bispecific monomeric scDbs, can be organized as VHA-L1-VLB-L2-VHB-L3-VLA, VHA-L1-VHB-L2-VLB-L3-VLA, VLA-L1-VLB-L2-VHB-L3-VHA, VLA-L1-VHB-L2-VLB-L3-VHA, VHB-L1-VLA-L2-VHA-L3-VLB, VHB-L1-VLA-L2-VHA-L3-VLB, VHB-L1-VHA-L2-VLA- It particularly comprises two variable heavy domains (VH) or fragments thereof and two variable light domains (VL) or fragments thereof linked in the order L3-VLB, VLB-L1-VLA-L2-VHA-L3-VHB, or VLB-L1-VHA-L2-VLA-L3-VHB, where the VLA and VHA domains together form an antigen-binding site for a first antigen and the VLB and VHB together form an antigen-binding site for a second antigen.

[0144] The linker L1 is specifically a peptide of 2 to 10 amino acids, more specifically 3 to 7 amino acids, and most specifically 5 amino acids, and the linker L3 is specifically a peptide of 1 to 10 amino acids, more specifically 2 to 7 amino acids, and most specifically 5 amino acids. In a specific embodiment, the linker L1 and / or L3 is a unit of four glycine amino acid residues and one serine amino acid residue (GGGGS). n where n=1, or 2, preferably 1.

[0145] The intermediate linker L2 is specifically a peptide of 10 to 40 amino acids, more specifically 15 to 30 amino acids, and most specifically 20 to 25 amino acids. In a specific embodiment, the linker L2 is one or more units of four glycine amino acid residues and one serine amino acid residue (GGGGS). n where n=1, 2, 3, 4, 5, 6, 7, or 8, preferably n=4.

[0146] In one embodiment of the invention, the isolated antibody is a multispecific and / or multivalent antibody in scDb-scFv format. The term "scDb-scFv" refers to an antibody format in which a single chain Fv (scFv) fragment is fused to a single chain diabody (scDb) by a flexible Gly-Ser linker. In one embodiment, said flexible Gly-Ser linker is a peptide of 2-40 amino acids, such as 2-35, 2-30, 2-25, 2-20, 2-15, 2-10 amino acids, specifically 10 amino acids. In a particular embodiment, said linker is a peptide of one or more units of four glycine amino acid residues and one serine amino acid residue (GGGGS). n where n=1, 2, 3, 4, 5, 6, 7, or 8, preferably n=2.

[0147] In one embodiment of the invention, the isolated antibodies of the MATCH format described in WO 2016 / 0202457; Egan T., et al., mAbs 9 (2017) 68-84 are multispecific and / or multivalent.

[0148] The multispecific and / or multivalent molecules of the invention can be produced using any convenient antibody production method known in the art (see, for example, Fischer, N. & Leger, O., Pathobiology 74 (2007) 3-14 for the generation of bispecific constructs; Hornig, N. & Farber-Schwarz, A., Methods Mol. Biol. 907 (2012) 713-727 for bispecific diabodies and tandem scFvs, and WO 99 / 57150). Specific examples of suitable methods for the preparation of the bispecific constructs of the invention further include, inter alia, the Genmab (see Labrijn et al., Proc. Natl. Acad. Sci. USA 110 (2013) 5145-5150) and Merus (see de Kruif et al., Biotechnol. Bioeng. 106 (2010) 741-750) technologies. Methods for the production of bispecific antibodies comprising functional antibody Fc portions are also known in the art (see, for example, Zhu et al., Cancer Lett. 86 (1994) 127-134; and Suresh et al., Methods Enzymol. 121 (1986) 210-228).

[0149] Other antibodies that can be used in the multispecific and multivalent molecules of the invention are murine, chimeric, and humanized monoclonal antibodies.

[0150] Multispecific molecules of the invention can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linkers include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al., 1984 J. Exp. Med. 160: 1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al., 1987 J. Immunol. 139: 2367-2375. Conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, 111).

[0151] When the binding specificities are antibodies, they can be linked by sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains, in certain embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, e.g., one, prior to conjugation.

[0152] Alternatively, two or more binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. The multispecific molecules of the invention can be single chain molecules containing one single chain antibody and a binding determinant, or single chain multispecific molecules containing two binding determinants. The multispecific molecule can comprise at least two single chain molecules. Methods for preparing multispecific molecules are described, for example, in U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

[0153] Binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0154] In a further aspect, the present invention provides a nucleic acid encoding the antibody of the present invention. The present invention also provides a nucleic acid sequence encoding the CDR, VH, VL, full-length heavy chain, and full-length light chain of an antibody that specifically binds to CD137 protein. Such a nucleic acid sequence can be optimized for expression in mammalian cells.

[0155] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to one or more deoxyribonucleic acids or ribonucleic acids and polymers thereof, either in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner as the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as described in more detail below, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0156] The present invention provides substantially purified nucleic acid molecules encoding polypeptides comprising segments or domains of the CD137-binding antibody chains described above. When expressed from an appropriate expression vector, the polypeptides encoded by these nucleic acid molecules are capable of exhibiting CD137 antigen-binding ability.

[0157] Also provided herein are polynucleotides that encode at least one, and usually all three, CDR regions from the heavy or light chain of a CD137 binding antibody listed in Table 1. Some other polynucleotides encode all or substantially all of the variable region sequences of the heavy and / or light chains of a CD137 binding antibody listed in Table 1. Due to the degeneracy of the code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence.

[0158] The polynucleotide sequence can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., as described in the Examples below) that encodes a CD137-binding antibody. Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109, 1979; the diethyl phosphoramidite method of Beaucage et al., Tetra. Lett., 22: 1859, 1981; and the solid support method of U.S. Pat. No. 4,458,066. Mutations can be introduced into polynucleotide sequences by PCR, for example, as described in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0159] Expression vectors and host cells for producing the above-described CD137-binding antibodies are also provided by the present invention.

[0160] The term "vector" is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can 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 the host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0161] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0162] The term "operatively linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically adjacent to or located in close proximity to the coding sequence whose transcription they enhance.

[0163] A variety of expression vectors can be used to express polynucleotides encoding CD137-binding antibody chains or binding fragments. Both viral-based and non-viral expression vectors can be used to produce antibodies in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors, and human artificial chromosomes, which usually carry expression cassettes for expressing proteins or RNA (see, for example, Harrington et al., NAT Genet. 15:345, 1997). For example, non-viral vectors useful for expressing CD137-binding polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invitrogen, San Diego, Calif.), MPS V vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40-based vectors, papilloma viruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV)-based vectors. See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807,1995; and Rosenfeld et al., Cell 68: 143,1992.

[0164] The choice of expression vector depends on the host cell in which the vector is to be expressed. Typically, the expression vector includes a promoter and other regulatory sequences (e.g., enhancers) operably linked to the polynucleotide encoding the CD137 binding antibody. In one embodiment, an inducible promoter is used to prevent expression of the inserted sequence except under inducible conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be expanded under non-inducing conditions without biasing the population towards coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may also be necessary or desirable for efficient expression of the CD137 binding antibody. These elements usually include an ATG initiation codon and adjacent ribosome binding sites or other sequences. Furthermore, the efficiency of expression can be enhanced by including enhancers appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125,1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0165] The expression vector may also provide a secretion signal sequence position to form a fusion protein with the polypeptide encoded by the inserted CD137 binding antibody sequence. In many cases, the inserted CD137 binding antibody sequence is linked to a signal sequence before being included in the vector. The vector used to receive the sequence encoding the light and heavy chain variable domains of the CD137 binding antibody may also encode a constant region or a portion thereof. Such vectors allow the expression of the variable region as a fusion protein with the constant region, thereby resulting in the production of an intact antibody and its antigen-binding fragments. Usually, such constant regions are human.

[0166] The term "recombinant host cell" (or simply "host cell") refers to 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 also to the progeny of such a cell. Because some degree of modification may occur in successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0167] Host cells for carrying and expressing CD137-binding antibody chains may be either prokaryotic or eukaryotic. Escherichia coli (E. coli) is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences (e.g., origins of replication) compatible with the host cell. In addition, there are a number of different well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or promoter systems derived from lambda phage. Promoters typically control expression, optionally with operator sequences, and have ribosome binding site sequences, etc., for initiating and completing transcription and translation. Other microbes, such as yeast, can also be used to express the CD137-binding polypeptides of the present invention. Insect cells in combination with baculovirus vectors can also be used.

[0168] In one embodiment, mammalian host cells are used to express and produce the CD137 binding polypeptides of the invention. For example, they may be either hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These include normal mortal or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, FROM GENES TO CLONES, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., Immunol. Rev. 89:49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell-type specific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0169] Methods for introducing an expression vector containing a polynucleotide sequence of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts (see generally Sambrook, et al., Molecular Cloning: A Laboratory Manual. 4 th (See, for example, The Journal of Clinical Chemistry, vol. 13, no. 1, 2012, Cold Spring Harbor, ed., Cold Spring Harbor 2012). Other methods include electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation-nucleic acid conjugates, naked DNA, artificial virions, fusion to herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. Stable expression is often desired for long-term, high-yield production of recombinant proteins. For example, cell lines stably expressing CD137-binding antibody chains or binding fragments can be prepared using the expression vectors of the invention containing viral origins of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, the cells can be grown for 1-2 days in enriched media before switching to selective media. The purpose of a selectable marker is to confer resistance to selection and its presence allows growth of cells that successfully express the introduced sequences in a selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type. Thus, the invention provides a method of producing an antibody of the invention, comprising culturing a host cell containing a nucleic acid or vector encoding an antibody of the invention, thereby expressing said antibody of the invention or a fragment thereof.

[0170] In a further aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the present invention and a pharma- ceutically acceptable carrier. The pharma-ceutically acceptable carrier enhances or stabilizes the composition or facilitates preparation of the composition. The pharma-ceutically acceptable carrier includes physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0171] The pharmaceutical composition of the present invention can be administered by various methods known in the art. The route and / or mode of administration varies depending on the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the target site. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compounds, i.e., antibodies, and multispecific molecules, can be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0172] The pharmaceutical composition of the present invention can be prepared according to methods well known and routinely practiced in the art. For example, see Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition is preferably manufactured under GMP conditions. Typically, a therapeutically effective amount or effective dose of CD137 binding antibody is used in the pharmaceutical composition of the present invention. The CD137 binding antibody is formulated into a pharma- ceutically acceptable dosage form by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dose. Dosage unit form, as used herein, refers to physically discrete units suited as single dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0173] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The dosage level selected will depend on factors such as the activity of the particular composition of the present invention, or its esters, salts, or amides, used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated.

[0174] The antibody is usually administered multiple times. The interval between single doses may be weekly, monthly, or yearly. The intervals may also be irregular, as indicated by measuring the patient's blood levels of the CD137-binding antibody. Alternatively, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will depend on the half-life of the antibody in the patient. In general, humanized antibodies exhibit a longer half-life than chimeric and non-human antibodies. The dose and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until the progression of the disease is reduced or terminated, preferably until the patient exhibits partial or complete remission of the symptoms of the disease. The patient may then be administered a prophylactic regime.

[0175] In one embodiment, the invention provides a pharmaceutical combination comprising an anti-CD137 antibody of the invention as defined herein and one or more additional therapeutic agents, such as one or more anti-cancer agents, cytotoxic or cytostatic agents, hormonal therapy, vaccines, and / or other immunotherapy. Advantageously, the anti-CD137 antibody of the invention may be used in combination with an inhibitor of an inhibitory (or immune checkpoint) molecule selected from PD-1, PDL1, PDL2, CTLA-4, TIM-3, LAG-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, and IDO (indoleamine-2,3 dioxygenase). Inhibition of the inhibitory molecule may be at the DNA, RNA, or protein level.

[0176] Surprisingly, it has been found that the anti-CD137 antibody of the present invention has a strong beneficial synergistic interaction and improved antiproliferative activity when used in combination with a PDL1 inhibitor.Therefore, the present invention provides a pharmaceutical combination comprising an anti-CD137 antibody of the present invention as defined herein and a PDL1 inhibitor, particularly for use in treating or preventing proliferative diseases.The present invention further relates to a pharmaceutical combination comprising an anti-CD137 antibody of the present invention as defined herein and a PDL1 inhibitor, particularly for simultaneous, separate or sequential use in treating or preventing proliferative diseases.

[0177] The term "combination" or "pharmaceutical combination" is defined herein to refer to a fixed combination in one dosage form, a non-fixed combination, or a kit of parts for co-administration, where the therapeutic agents, e.g., an anti-CD137 antibody and a PCL1 inhibitor of the present invention, can be administered together, independently simultaneously, or separately within a time interval that allows the combination partners to exhibit a coordinated, e.g., synergistic, effect.

[0178] The term "fixed combination" means that the therapeutic agents, eg, an anti-CD137 antibody of the invention and a PDL1 inhibitor, are administered to a patient simultaneously in the form of a single entity or dosage.

[0179] The term "non-fixed combination" means that therapeutic agents, e.g., both an anti-CD137 antibody and a PDL1 inhibitor of the present invention, are administered to a patient in separate entities or dosage forms simultaneously, concurrently, or sequentially without specific time limitations, where such administration provides therapeutically effective levels of the two therapeutic agents in the body of a subject, e.g., a mammal or human in need thereof.

[0180] The term "PDL1" specifically refers to human PDL1, UniProt ID number Q9NZQ7.

[0181] The term "blocker" or "inhibitor" or "antagonist" refers to an agent that inhibits or reduces the biological activity of a target molecule to which it binds. In some embodiments, the inhibitor substantially or completely inhibits the biological activity of the target molecule. A suitable PDL1 inhibitor targets, reduces, and / or inhibits the binding ability of PDL1 to its binding partner, thereby interfering with the function of PDL1. In particular, a suitable PDL1 inhibitor blocks the interaction of PDL1 with PD-1. In some embodiments, a suitable PDL1 inhibitor blocks the interaction of PDL1 with PD-1 and B7-1. Preferably, the PDL1 inhibitor utilized in the pharmaceutical combination of the present invention is an anti-PDL1 antibody.

[0182] The term "synergy" as used herein refers to the action of two therapeutic agents, such as (a) an anti-CD137 antibody of the present invention, and (b) a PDL1 inhibitor, to produce an effect, for example, of slowing the symptomatic progression of a proliferative disease, particularly cancer, or a condition thereof, that is greater than the simple addition of the effects of each therapeutic agent administered by itself.Synergy can be calculated using suitable methods, such as the Sigmoid-Emax formula (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity formula (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114:313-326(1926)), and the median-effect formula (Chou, TC and Talalay, P., Adv. Enzyme Regul. 22:27-55(1984)). Each of the above equations can be applied to experimental data to generate corresponding graphs, which can be used to evaluate the effect of drug combination.The corresponding graphs related to the above equations are concentration-effect curve, isobologram curve, and combination index curve, respectively.Synergism can be further shown by calculating the synergistic effect score of combination according to the method known by those skilled in the art.

[0183] As used herein, the term "co-administration" is defined to encompass the administration of selected therapeutic agents to a single patient, and is intended to include therapeutic regimens in which the therapeutic agents are not necessarily administered by the same route of administration or at the same time.

[0184] The term "combination preparation" is defined to refer in particular to a "kit of parts" in the sense that the therapeutic agents (a) and (b) defined above can be administered independently or by simultaneous or different time use of different fixed combinations of distinct amounts of therapeutic agents (a) and (b). The parts of the kit of parts can then be administered, for example, simultaneously or chronologically staggered, i.e., at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of therapeutic agent (a) to therapeutic agent (b) administered in the combination preparation can be varied, for example, to accommodate the needs of the treated patient subpopulation or the needs of a single patient.

[0185] As used herein, the term "jointly therapeutically active" or "joint therapeutic effect" means that the therapeutic agents can be given separately (in a chronologically staggered manner, particularly in a sequence-specific manner) at such time intervals that they still prefer to exhibit a beneficial interaction (preferably synergistic) (joint therapeutic effect) in the warm-blooded animal, particularly a human, being treated. Whether this is the case can be determined, inter alia, by following blood levels, showing that both therapeutic agents are present in the blood of the human being treated for at least a particular time interval.

[0186] A pharmaceutical combination of the invention comprises an anti-CD137 antibody of the invention as defined herein, in particular for use in the treatment or prevention of a proliferative disease. In a preferred embodiment, the pharmaceutical combination of the invention comprises an antibody of the invention, wherein said antibody is an IgG4 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 16, 17 and 18, respectively, a VH sequence comprising an amino acid sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, identical to SEQ ID NO: 13, and a VL sequence comprising an amino acid sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, identical to SEQ ID NO: 25. In another embodiment, the pharmaceutical combination of the present invention comprises an antibody of the present invention, wherein said antibody is an IgG4 comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, a VH sequence comprising an amino acid sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90 percent, identical to SEQ ID NO:14, and a VL sequence comprising an amino acid sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90 percent, identical to SEQ ID NO:26.In another embodiment, the pharmaceutical combination of the present invention comprises an antibody of the present invention, wherein said antibody is an IgG4 comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, a VH sequence comprising an amino acid sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90 percent, identical to SEQ ID NO:15, and a VL sequence comprising an amino acid sequence at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent, preferably at least 90 percent, identical to SEQ ID NO:27.

[0187] In one aspect the invention relates to an antibody of the invention, or a composition of the invention, or a combination of the invention, for use as a medicament.

[0188] In another aspect the invention relates to an antibody of the invention, or a composition of the invention, or a combination of the invention, for use in the manufacture of a medicament for use in the treatment of a proliferative disease, in particular cancer.

[0189] In one aspect, the invention relates to an antibody of the invention, or a composition of the invention, or a combination of the invention, for use in the treatment of a proliferative disease, in particular cancer.

[0190] In another aspect, the present invention relates to the use of an antibody of the present invention, or a composition of the present invention, or a combination of the present invention, for the treatment of a proliferative disease, in particular cancer, in a subject in need thereof.

[0191] In one aspect, the present invention provides a method of treating a proliferative disease, particularly cancer, in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antibody of the present invention, or a composition of the present invention, or a combination of the present invention.

[0192] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise noted, the terms "patient" or "subject" are used interchangeably herein.

[0193] As used herein, the terms "treatment," "treating," "treat," "treated," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in that it partially or completely cures a disease and / or side effects resulting from a disease, or slows the progression of a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, e.g., a human, and includes: (a) inhibiting the disease, i.e., arresting its onset; (b) relieving the disease, i.e., causing regression of the disease.

[0194] The term "therapeutically effective amount" or "efficacious amount" refers to the amount of a drug that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the drug, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0195] In one embodiment, the proliferative disease is cancer. The term "cancer" refers to a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or via the bloodstream and lymphatic system to other parts of the body. The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant, as well as malignant cancers and tumors. The term "cancer" is used herein to refer to a broad range of tumors, including all solid and hematological malignancies. Examples of such tumors include, but are not limited to, benign or particularly malignant tumors, solid tumors, brain cancer, kidney cancer, liver cancer, adrenal gland cancer, bladder cancer, breast cancer, gastric cancer (e.g., gastric tumors), esophageal cancer, ovarian cancer, cervical cancer, colon cancer, rectal cancer, prostate cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), vaginal cancer, thyroid cancer, melanoma (e.g., unresectable or metastatic melanoma), renal cell carcinoma, sarcoma, glioblastoma, multiple myeloma or gastrointestinal cancer, in particular colon cancer or colon Rectal adenoma, head and neck tumor, endometrial cancer, Cowden syndrome, Lhermitte-Duclos disease, Bannayan-Zonana syndrome, prostatic hyperplasia, epithelial neoplasms, preferably breast cancer or squamous cell carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia (e.g. Philadelphia chromosome positive chronic myeloid leukemia), acute lymphoblastic leukemia (e.g. Philadelphia chromosome positive acute lymphoblastic leukemia), non-Hodgkin's lymphoma, plasma cell myeloma, Hodgkin's lymphoma, leukemia, and any combination thereof. In a preferred embodiment, the cancer is lung cancer, preferably non-small cell lung cancer (NSCLC). In another embodiment, the cancer is colorectal cancer.

[0196] The antibodies of the invention, or the multispecific molecules of the invention, or the compositions of the invention, or the combinations of the invention inhibit the growth of solid tumors as well as liquid tumors. In a further embodiment, the proliferative tumor is a solid tumor. The term "solid tumor" refers in particular to breast cancer, ovarian cancer, colon cancer, rectal cancer, prostate cancer, stomach cancer (in particular gastric cancer), cervical cancer, lung cancer (e.g. non-small cell lung cancer and small cell lung cancer), and head and neck tumors. Furthermore, depending on the type of tumor and the particular combination used, a reduction in tumor volume can be obtained. The antibodies of the invention, or the multispecific molecules of the invention, or the compositions of the invention, or the combinations of the invention are also suitable for preventing the metastatic spread of tumors and the growth or development of micrometastases in subjects suffering from cancer.

[0197] The term "prevent" or "prevention" refers to the complete inhibition of the onset of a disease or any secondary effects of a disease. As used herein, the term "prevent" or "prevention" encompasses preventing a disease or condition from occurring in individuals who may be predisposed to the disease but have not yet been diagnosed as having it.

[0198] In a further aspect, the present invention relates to a kit comprising the antibody of the present invention described herein. Also within the present disclosure is a kit comprising the multispecific molecule of the present invention. Also within the present disclosure is a kit comprising the pharmaceutical composition of the present invention. The kit may comprise one or more other elements, such as: instructions for use; other reagents, such as labels, therapeutic agents, or agents useful for chelating or otherwise binding the antibody to a label or therapeutic agent or radioprotective composition; devices or other materials for preparing the antibody molecule for administration; a pharma- ceutically acceptable carrier; and devices or other materials for administration to a subject. In certain embodiments, the kit comprises an antibody of the present invention in a pharma- ceutical effective amount. In further embodiments, the kit comprises a pharma- ceutical effective amount of the antibody of the present invention in lyophilized form, and a diluent, and, optionally, instructions for use. The kit may further comprise a filter needle for reconstitution and a needle for injection.

[0199] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0200] [Table 2]

[0201] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0202] Throughout the text of this application, in the event of a conflict between the text of the specification (eg, Tables 1-3) and the Sequence Listing, the text of the specification shall control.

[0203] It is understood that certain features of the invention that are described in the context of separate embodiments for clarity can also be provided in combination in a single embodiment. Conversely, various features of the invention that are described in the context of a single embodiment for brevity can also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and disclosed herein as if all combinations were individually and explicitly disclosed. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and disclosed herein as if each such subcombination was individually and explicitly disclosed herein.

[0204] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0205] To the extent possible under the respective patent laws, all patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

[0206] The following examples illustrate the above invention, but are not intended to limit the scope of the invention in any way. Other test models known to those skilled in the art as such can also determine the beneficial effects of the claimed invention. EXAMPLES

[0207] A novel antibody against human CD137 Example 1: Production of rabbit antibodies against human CD137 Rabbits were immunized with recombinantly produced and purified human CD137 extracellular domain (Peprotech, Cat. No. 310-15-1MG). During immunization, the strength of the humoral immune response to the antigen was qualitatively assessed by determining the maximum dilution (titer) of serum from each rabbit that still produced detectable binding of polyclonal serum antibodies to the antigen. Serum antibody titers against the immobilized antigen (recombinant human CD137 ECD) were assessed using an enzyme-linked immunosorbent assay (ELISA).

[0208] Example 2: Hit Identification and Selection Among the hit identification methods, a flow cytometry-based sorting method was developed. It allows to specifically detect and separate high-affinity human CD137 ECD-binding B cells. To identify CD137-binding B cells, CD137 ECD was labeled with the fluorescent dye R-phycoerythrin (RPE). Because the CD137L binding site and the binding site of anti-CD137 antibodies on labeled CD137 could be blocked by the bulky RPE label, the accessibility of the epitope was confirmed by flow cytometry. CD137L ECD fused to the Fc portion of human IgG1, urelumab, rabbit polyclonal anti-human CD137 or goat polyclonal anti-human CD137 was captured on protein G beads, and the binding of R-PE-labeled CD137 was confirmed by flow cytometry. The fluorescence intensity is proportional to the amount of labeled CD137 bound to CD137L immobilized on the beads. Binding of CD137 to CD137L and anti-CD137 antibodies was observed, but no binding of RPE-tagged CD137 to infliximab was detected.

[0209] screening: B cells expressing CD137-specific antibodies (IgG) were isolated in a sorting campaign. The results obtained during the screening phase were based on assays performed using unpurified antibodies derived from culture supernatants of antibody-secreting cells (ASCs). Rabbit monoclonal antibodies in each cell culture supernatant were characterized by high-throughput ELISA for binding to recombinant human CD137 ECD. CD137-bound supernatants were further characterized for their binding kinetics to human and cynomolgus CD137. Furthermore, the neutralizing capacity of CD137 interaction for CD137L and urelumab was determined by competitive ELISA. Binding to membranous CD137 expressed in stably transduced Jurkat cells was also evaluated. Supernatants were analyzed for their murine CD137-binding capacity by direct ELISA.

[0210] direct ELISA ELISA plates were coated by adding 50 μl of PBS containing 250 ng / ml human CD137 (Peprotech, Cat. No. 310-15-1MG) overnight at 4 °C. The next day, plates were washed 3 times with 300 μl of wash buffer (PBS, 0.005% Tween 20) per well in overflow mode and 270 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added to each well for 1 h at RT without shaking. Plates were then washed 3 times with 300 μl of wash buffer in overflow mode, 50 μl of each supernatant was added and plates were incubated for 1.5 h at RT with gentle agitation. After washing 3 times in overflow mode with 300 μl of wash buffer, 50 μl of HRP-conjugated goat anti-rabbit IgG antibody diluted 1:5,000 in blocking buffer was added to each well. After 1 h incubation at RT on a nutating mixer, the plates were washed three times with 300 μl of wash buffer per well in overflow mode before adding 50 μl of TMB (3,3',5,5'-tetramethylbenzidine). After 5-10 min of development, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl per well and the plates were read at 450 nm using 690 nm as the reference wavelength.

[0211] Affinity to hCD137 by SPR The binding affinity of antibodies to human CD137 was measured by SPR using a MASS-1 SPR instrument (Sierra Sensors). For affinity screening, an antibody specific for the Fc region of rabbit IgG (Bethyl Laboratories, Cat. No. A120-111A) was immobilized on a sensor chip (SPR-2 affinity sensor, high capacity amine, Sierra Sensors) using a standard amine coupling method. Rabbit monoclonal antibodies in B cell supernatants were captured by immobilized anti-rabbit IgG antibodies. A minimum IgG concentration in the B cell supernatant is required to allow sufficient capture. After capturing the monoclonal antibodies, human CD137 ECD (Peprotech, Cat. No. 310-15-1MG) was injected into the flow cell at a concentration of 90 nM for 3 min, and dissociation of the protein from the IgG captured on the sensor chip was allowed to proceed for 5 min. After each injection cycle, the surface was regenerated with two injections of 10 mM glycine-HCl. The apparent dissociation (k d ) and meeting (k a ) and the apparent dissociation equilibrium constant (K D ) was calculated using a one-to-one Langmuir binding model in the MASS-1 analysis software (Analyzer, Sierra Sensors), and the quality of the curve fit was evaluated using the relative Chi 2 (Chi normalized to the extrapolated maximum binding level of the analyte 2 ) was monitored based on the 2 The smaller the value of , the more accurate the fit to the one-to-one Langmuir binding model. For most hits, the relative Chi 2 Values ​​were less than 15%. Results were considered valid if the response units (RU) of ligand binding were at least 2% of the RU of antibody capture. Samples with RU of ligand binding less than 2% of the RU of antibody capture were considered not to show specific binding of CD137 to the captured antibody.

[0212] CD137 / CD137L competitive ELISA ELISA plates were coated by adding 50 μl of PBS containing 50 ng / ml of CD137 Fc chimera (R&D Systems, Cat. No. 838-4B-100) overnight at 4°C. The next day, plates were washed 3 times with 450 μl of washing buffer (PBS, 0.005% Tween 20) per well in overflow mode and 300 μl of blocking buffer (PBS containing 1% BSA and 0.2% Tween 20) was added to each well for 1 h at RT on a nutating mixer. Next, positive control (neutralizing goat anti-CD137 antibody) was diluted in 100% negative supernatant and 50 μl of neutralizing antibody was added to the corresponding well of the binding plate. Additionally, 50 μl of the supernatant of the positive hits was transferred to the binding plate and incubated for 1 h at RT with shaking. The ELISA plate was then washed three times with 450 μl of wash buffer per well in overflow mode, and 50 μl of 20 ng / ml biotinylated recombinant human CD137 ligand (Acro Biosystem, Cat. No. 41L-H5257) diluted in blocking buffer was added to the wells. After 1 h of incubation with shaking, the ELISA plate was washed three times with 450 μl of wash buffer per well in overflow mode. Then, 50 μl of 10 ng / ml streptavidin-poly-HRP diluted in blocking buffer was added to each well of the ELISA plate. After 1 h of incubation at room temperature, the plate was washed three times with 450 μl of wash buffer, and 50 μl of TMB was added followed by development for 5–10 min. Finally, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl, and the plate was read at 450 nm using 690 nm as the reference wavelength.

[0213] Species specificity by SPR: Cynomolgus monkey CD137 Binding kinetics to cynomolgus CD137 was also determined for hits identified in the initial screening ELISA using the same SPR setup as described for binding to human CD137, but substituting cynomolgus CD137 ECD for human CD137 ECD (Acro Biosystem, catalog no. 41B-C52H4).

[0214] Urelumab competitive ELISA ELISA plates were coated by adding 50 μl of 2 μg / ml urelumab (manufactured by Evitria, Schlieren, Switzerland) in PBS overnight at 4 °C. The next day, plates were washed 3 times with 450 μl of washing buffer (PBS, 0.005% Tween 20) per well in overflow mode and 300 μl of blocking buffer (PBS with 1% BSA and 0.2% Tween 20) was added to each well for 1 h at RT on a nutating mixer. Urelumab was then diluted by adding 95% negative supernatant, pre-incubated with 7.5 ng / ml of 5% biotinylated human CD137ECD (Peprotech, cat. no. 310-15-1MG) for 1 h and added to the corresponding wells of the binding plate. Additionally, 50 μl of the positive hits' supernatants were also added and pre-incubated with 7.5 ng / ml 5% biotinylated human CD137ECD (Peprotech, Cat. No. 310-15-1MG) for 1 h, transferred to the binding plate and incubated for 1 h at room temperature with shaking. The ELISA plate was then washed three times with 450 μl of washing buffer per well in overflow mode. Then, 50 μl of 10 ng / ml streptavidin-poly-HRP diluted in blocking buffer was added to each well of the ELISA plate. After 1 h of incubation at room temperature, the plate was washed three times with 450 μl of washing buffer and developed for 5–10 min after adding 50 μl of TMB. Finally, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl, and the plate was read at 450 nm using 690 nm as the reference wavelength.

[0215] Cell-based binding assay by FC: human CD137 Jurkat wild type (control cells not expressing CD137) and Jurkat CD137 cells (clone C6,1) were harvested and cell numbers were determined. The cell suspension was centrifuged at 400 × g for 5 min and 40 μl (40,000 cells) of cell suspension diluted in PBS-EB (1 × DPBS, 2% BCS HI, 2 mM EDTA) was added to the designated wells of the binding plate. Supernatants from positive hits were transferred directly to the 96-well plate according to the plate layout. Positive control samples (urelumab) were diluted in PBS-EB and transferred to the plate. The final samples were 95% negative supernatants. After 1 h incubation at 4 °C, the plate was washed three times using 100 μl of PBS-EB. The cell pellet was then resuspended in 50 μl secondary antibody solution at a concentration of 2 μg / ml (for B cell clones: goat anti-rabbit IgG labeled with AF647; for urelumab: goat anti-human IgG labeled with PE) and incubated for 1 h at 4 °C. The cells were then washed again three times using 100 μl PBS-EB. The cell pellet was then resuspended in 50 μl PBS-EB and analyzed on a NovoCyte2060 flow cytometer device. The fluorescence intensity of PE and AF647 for 20,000 events was recorded for each sample and the geometric mean of the fluorescence intensity MFI was calculated. The data was first corrected for non-specific antibody binding (blank and Jurkat wild-type cell binding) and then normalized to the binding level obtained for urelumab.

[0216] Direct ELISA Mouse CD137 As a first step to identify mouse cross-reactive CD137 binders, a direct ELISA against mouse CD137 was performed. For this purpose, cell culture supernatants of B cell clones were screened by ELISA for the presence of antibodies against mouse CD137. ELISA plates were coated by adding 50 μl of PBS containing 250 ng / ml of mouse CD137 (Acro Biosystem, Cat. No. 41B-M52H7) overnight at 4°C. The next day, plates were washed 3 times with 300 μl of washing buffer (PBS, 0.005% Tween 20) per well in overflow mode and 270 μl of blocking buffer (PBS, 1% BSA, 0.2% Tween 20) was added to each well for 1 h at RT without shaking. Then, plates were washed 3 times with 300 μl of washing buffer in overflow mode, 50 μl of each supernatant was added and plates were incubated at RT for 1.5 h under gentle agitation. After washing three times in overflow mode with 300 μl of wash buffer, 50 μl of HRP-conjugated goat anti-rabbit IgG antibody diluted 1:5,000 in blocking buffer was added to each well. After 1 h of incubation at RT on a nutating mixer, the plate was washed three times with 300 μl of wash buffer per well in overflow mode before adding 50 μl of TMB. After 5–10 min of development, the enzymatic reaction was stopped by adding 50 μl of 1 M HCl per well, and the plate was read at 450 nm using 690 nm as the reference wavelength. In this assay, supernatants from 85 B cell clones produced signals clearly above background (>0.1 OD).

[0217] Species specificity by SPR: Mouse Binding kinetics to mouse CD137 was also determined using the same SPR setup as described for binding to human CD137, but in this case the human CD137 ECD was replaced by mouse CD137 ECD (Acro Biosystem, Cat. No. 41B-M52H7).

[0218] Selection of screening hits Based on the pharmacological properties of the monoclonal antibody in B cell supernatant, clone 38-27-A11 was selected for hit confirmation analysis. The pharmacological properties of the monoclonal antibody of clone 38-27-A11 in B cell supernatant are shown in Table 4. In the next step, the selected clone was used for RNA isolation and RT-PCR to amplify the sequence of the rabbit antibody light and heavy chain variable region (38-27-A11).

[0219] [Table 4]

[0220] [Table 5]

[0221] [Table 6]

[0222] Example 4: Pharmacological analysis of monoclonal antibody 38-27-A11 4.1 Affinity for human and cynomolgus CD137 The binding kinetics of purified monoclonal rabbit antibody 38-27-A11 to human and cynomolgus CD137 was measured by SPR measurements (Tables 5 and 6). The binding affinity of the antibody to human CD137 was measured using a Biacore T200 SPR instrument (GE Healthcare). For the experimental setup, an antibody specific for the Fc region of rabbit IgG (Bethyl Laboratories, Cat. No. A120-111A) was immobilized on a sensor chip (CM5 chip, GE Healthcare) using standard amine coupling methods. The rabbit monoclonal antibody was captured with immobilized anti-rabbit IgG antibody. Following monoclonal antibody capture, human CD137 (PeproTech, Cat. No. 310-15) or cynomolgus CD137 (Acro Biosystem, Cat. No. 41B-C52H4) was injected into the flow cell at concentrations ranging from 90 to 0.35 nM for 3 min, and dissociation of the protein from the IgG captured on the sensor chip was allowed to proceed for 12 min. The apparent dissociation (kd) and association (ka) rate constants, and the apparent dissociation equilibrium constant (KD) were calculated with the Biacore T200 software evaluation tool (GE Healthcare) using a one-to-one Langmuir binding model.

[0223] 4.2 Epitope binning of recombinant rabbit IgG 38-27-A11 To analyze the binding epitope of recombinant rabbit IgG 38-27-A11, epitope binning was performed by SPR using a MASS-1 instrument (Sierra Sensors). By using this approach, the binding epitope of rabbit IgG 38-27-A11 on CD137 was mapped against urelumab and utomirumab. A sandwich setup was utilized to investigate whether the antibodies block each other's binding to human CD137. Rabbit IgG 38-27-A11 and a competitor IgG were immobilized on a high-capacity amine sensor chip (HCA, Sierra Sensors). Then, 90 nM of antigen CD137 (PeproTech, Cat. No. 310-15) was injected and captured by rabbit IgG 38-27-A11, followed immediately by injection of 22.5 nM of secondary antibody (competitor IgG). The capture level of human CD137 with each rabbit IgG and the response level of the second binder were determined (response units, RU). The relative binding level (%) of proteins on the captured antigen was determined by calculating the theoretical maximum response (Rmax), which depends on the molecular weight of the proteins involved and the capture level. No binding of the injected antibodies on the captured CD137 should be observed if the molecules bind to the same or overlapping (e.g., structurally similar or spatially proximal) epitopes on CD137. As a result, if antibody binding is observed, the two antibody pairs bind to non-overlapping epitopes. The relative binding level (%) was determined for each antibody pair. By definition, a binding level below 10% indicates the same or overlapping (e.g., structurally similar or spatially proximal) epitopes on CD137, and above 30% indicates non-overlapping epitopes. IgG 38-27-A11 did not compete with utomirumab for binding to CD137, suggesting a non-overlapping epitope, but competed with urelumab for binding to CD137, suggesting the same or overlapping epitope (Figure 1)

[0224] 4.3 Activation of CD137 signaling by NF-kB reporter gene assay CD137 clustering, and the subsequent potency to activate CD137 signaling, was assessed in an NF-kB reporter gene assay. In this assay, activation of CD137 signaling in NF-kB Jurkat reporter cells was evaluated. The activity of CD137 signaling is reported by measuring luciferase expression, which is driven by CD137-induced NF-kB activation in the Jurkat reporter cell line. Furthermore, CD137 clustering, which is required for activation of the signaling pathway, is promoted via binding of bivalent anti-CD137 rabbit IgG.

[0225] In detail, 50,000 cells of NF-kB reporter gene Jurkat cells (Promega) expressing CD137 were seeded in 96-well white cell culture plates. Three-fold serial dilutions of rabbit IgG ranging from 9,000 to 1.37 ng / ml were prepared in assay buffer. To each dilution, 2.5-fold excess of crosslinking antibody (goat anti-rabbit IgG Fc-specific antibody, Bethyl, catalog number A120-111A) was added. To determine whether antibody binding without further clustering was sufficient to induce CD137 signaling, the highest concentration of each rabbit IgG was also measured without crosslinking agent. Urelumab was included in each plate as a positive control, and crosslinking of urelumab was performed by adding 1.25 excess of crosslinking agent (rabbit anti-human IgG Fc-specific antibody, Bethyl, catalog number A80-304A) to each dilution. As was done with rabbit IgG, the highest concentration of urelumab was also measured without the addition of crosslinking agent. The prepared serial dilutions of recombinant IgG and urelumab were added to the reporter gene cells and incubated for 6 hours at 37°C in a humidified cell culture incubator. Luciferase expression was detected by addition of luciferase reagent and read by a luminescence reader 6 hours after addition of anti-CD137 IgG. Data were analyzed by normalizing the relative luminescence units (RLU) of the test samples to the RLU measured for the highest concentration of urelumab using a crosslinker. The normalized data were plotted as a function of rabbit IgG concentration and fitted using a sigmoidal 4PL fit. Maximal activation of NF-kB signaling (relative to urelumab), EC 50 value, and relative EC 50 Values ​​(compared to urelumab) are reported (Table 7).

[0226] [Table 7]

[0227] Example 5: Humanization of rabbit IgG 38-27-A11 Based on the data obtained during hit screening, the CD137 binder 38-27-A11 was humanized by grafting CDRs into a VH3-based framework (Table 8). The full graft shows the grafting of CDRs and framework residues according to the AHo humanization protocol (CDR grafting and grafting of all rabbit residues that may contact the antigen (according to AHo) was limited to residues with more than 20% change in solvent accessibility upon interface formation in order to reduce the total number of mutations (rabbit framework residues)).

[0228] [Table 8]

[0229] Codon-optimized nucleotide sequences were designed and the corresponding genes were synthesized and cloned into mammalian expression vectors. Table 9 summarizes the production of scFv molecules. Expression of mammalian constructs was performed in CHO-S cells using the CHOgro transient transfection kit (Mirus). Cultures were harvested by centrifugation after 5-7 days of expression at 37 °C (cell viability <70%) and proteins were purified from the clarified culture supernatants by protein L affinity chromatography, followed, if necessary, by a polishing step by size exclusion chromatography. Standard analytical methods such as SE-HPLC, UV280, and SDS-PAGE were used for quality control of the produced material.

[0230] Example 6: Pharmacological characterization of human scFv 6.1 Affinity for human CD137 The affinity of humanized scFv PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03) for human CD137 was measured by SPR analysis on a T200 device (Biacore, GE Healthcare). In this experiment, Fc-tagged human CD137 (R&D Systems, catalog no. 838-4B-100) was captured using the Human Antibody Capture kit (catalog no. BR-1008-39) from GE Healthcare. After each analyte injection cycle, anti-human Fc-specific IgG was regenerated and new antigen was captured. The scFvs were injected as analytes using a dose-response multi-cycle kinetic assay against captured CD137, diluted in running buffer, with analyte concentrations ranging from 0.19 to 45 nM (3-fold dilution steps). Binding and dissociation times were set at 300 and 720 s, respectively. The resulting sensorgrams were fitted using a 1:1 binding model and the data are shown in Table 10.

[0231] 6.2 Cross-reactivity of species (binding to cynomolgus monkey CD137 by SPR) Cross-reactivity to cynomolgus CD137 was measured using cynomolgus Fc-tagged CD137 (R&D Systems, Cat. No. 9324-4B-100) in an assay similar to that used to measure binding to human CD137. Table 11 summarizes the affinities obtained with scFv PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03).

[0232] 6.3 Neutralization of CD137 / CD137L by competitive ELISA A competitive ELISA was used to show that anti-human CF137 scFv PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03) did not interfere with the binding of CD137 to CD137. A commercially available inhibitory polyclonal anti-CD137 goat antibody (Antibodies online, Cat. No. ABIN636609) was used as a reference. For the experimental setup, 50ng / ml of human CD137 (Fc-tagged, R&D Systems, Cat. No. 838-4B-100) was coated overnight onto an ELISA plate, and three-fold serial dilutions of the scFv starting from 50μg / ml were added to the ELISA plate. Biotinylated CD137L (in-house biotinylation of CD137L, Acro Biosystem, Cat. No. 41L-H5257) was then added, and bound ligand was detected by the addition of streptavidin-HRP. Finally, the HRP substrate TMB was added. After 5 min of development, the reaction was stopped with 1 M HCl. The absorbance was measured at 450 nm and at 690 nm as the reference wavelength. The data are shown in Table 12.

[0233] [Table 9]

[0234] [Table 10]

[0235] [Table 11]

[0236] 6.4 Binding to human CD137-expressing cells by flow cytometry Binding capacity to human CD137-expressing cells was measured for PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03). 50,000 CD137-expressing Jurkat cells (or as a reference cell line Jurkat cells lacking expression of CD137) were distributed into round-bottom non-tissue culture treated 96-well plates. Cells were washed twice with 100 μl PBS by centrifugation at 400 × g for 5 min. Cells were resuspended in 100 μl of 5-fold serial dilutions of the tested scFv and reference IgG urelumab, ranging from 10,000 to 0.64 ng / ml (for scFv: 381.19 to 0.02 nM), prepared in staining buffer (PBS, 2% heat-inactivated BCS, 2 mM EDTA). After 1 h incubation at 4 °C on a nutating mixer, cells were washed three times with 100 μl staining buffer and subjected to a centrifugation step at 400 × g for 5 min. Then, cells treated with scFv were resuspended in 100 μl staining buffer containing 0.5 μg / ml APC-labeled protein L, and cells treated with urelumab (human IgG4) were resuspended in 100 μl staining buffer containing 2 μg / ml APC-labeled goat anti-human IgG. After 1 h incubation of the plate on a nutating mixer at 4 °C, cells were washed three times with 100 μl staining buffer and resuspended in a final volume of 50 μl staining buffer. Finally, the APC signal of 20,000 events per well was analyzed by flow cytometry using a Novocyte flow cytometer system (ACEA Bioscience). Individual ECs from each plate were analyzed by flow cytometry using a Novocyte flow cytometer system (ACEA Bioscience). 50 Values ​​are the EC values ​​of the reference molecule urelumab taken along each plate. 50 Calibrated against (relative EC 50 :EC 50 , urelumab / EC50, test scFv). The data are summarized in Table 13 and Figure 1.

[0237] 6.5 Selectivity of CD137 vs. CD40 and OX40 by SPR In addition to cross-reactivity with cynomolgus CD137, selectivity of the anti-human CD137 scFvs PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03) for binding to human CD137 and not to other members of the TNFR superfamily such as CD40 or OX40 is desirable. Therefore, we tested the binding of PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03) to human CD40 and OC40. Binding of the scFvs human Fc-tagged CD40 (AcroBiosystems, Cat. No. CD0-H5253) and human Fc-tagged OX40 (Acro-Biosystems, Cat. No. OX0-H5255) was measured by SPR analysis on a T200 device (Biacore, GE Healthcare). In this experiment, Fc-tagged human CD40 and OX40 were captured using GE Healthcare's Human Antibody Capture kit (catalog no. BR-1008-39). After each analyte injection cycle, anti-human Fc-specific IgG was regenerated and new antigen was captured. scFv was injected as analyte using a high concentration of 180 nM analyte diluted in running buffer. Association and dissociation times were set at 300 and 720 seconds, respectively. The resulting sensorgrams were fitted using a 1:1 binding model. The data are summarized in Table 14.

[0238] [Table 12]

[0239] [Table 13]

[0240] [Table 14]

[0241] Example 7: Biophysical characterization of humanized scFv scFv PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03) were purified and concentrated to less than 10 mg / mL using centrifugal concentrator tubes (Table 15).

[0242] The scFvs were subjected to stability studies, including a 4-week stability study. In this study, the scFvs were formulated at 10 mg / ml in aqueous buffer (50 mM citrate phosphate buffer with 150 mM NaCl at pH 6.4) and stored at -80°C, 4°C, and 40°C for 4 weeks. At a minimum, the percentage of monomers and oligomers in the formulations was assessed by integrating the SE-HPLC peak areas after 1 week, 2 weeks, and at the end of each study. Table 16 compares the measurements obtained on d7 and the end point on d28 of the investigation. Additionally, the suitability of the scFv molecules was evaluated with respect to freeze-thaw (F / T) cycles (colloidal stability). In the F / T stability evaluation, the same analytical methods (SE-HPLC, UV-Vis) and parameters (% monomer content and % monomer loss) as in the storage stability study were applied to monitor the quality of the molecules over 5 F / T cycles. Table 17 shows the evolution of the monomer content in % over 5 repeated F / T cycles. None of the molecules lost more than 2% monomer content after repeated F / T cycles.

[0243] Thermal denaturation of the molecules was assessed using the fluorescent dye SYPRO orange. Samples of relevant excipient conditions were prepared and assays were performed on a qPCR machine. Fluorescence emission was detected using a custom dye calibration routine in the software. The PCR plate containing the test samples was ramped in temperature from 25°C to 96°C in 1°C increments. The midpoint of the unfolding transition (Tm) was calculated by the software GraphPad Prism using the mathematical second derivative method to calculate the inflection point of the curve. The reported Tm is the average of triplicate measurements. Table 18 shows the melting temperatures of the molecules formulated in common buffers (50 mM phosphate-citrate buffer at pH 6.4, 150 mM NaCl).

[0244] scFv PRO1359 (38-27-A11 sc02) and PRO1360 (38-27-A11 sc03) were subjected to a short-term pH stress stability study. In this study, the scFv molecules were formulated at 1 mg / ml in a series of aqueous buffer (phosphate-citrate) systems ranging from pH 3.5 to 7.5. After 2 weeks of storage at 4°C and 40°C in each buffer system, the % monomer content and % monomer loss were analyzed. A tabular summary of the monomer content, monomer loss, concentration, and concentration loss over the study period is shown in Table 19.

[0245] [Table 15]

[0246] [Table 16]

[0247] [Table 17]

[0248] [Table 18]

[0249] [Table 19]

[0250] Example 8: Epitope mapping by complex formation analysis of CD137 ECD variants in solution In this study, various CD137 ECD variants were designed based on annotated structural motifs, the cysteine-rich domain (CRD) and the membrane-proximal stalk (UniProtKB, Q07011; Figure 4A):

[0251] [ka] *The extracellular domain of CD137, spanning amino acids 24-186 (UniProt accession number: Q07011; see SEQ ID NO: 32 for the complete sequence of CD137), is composed of four cysteine-rich domains (CRD1-4) and the membrane proximal stalk. The underlined residues indicate the amino acid sequence in which the epitope of utomirumab is located (WO 2012 / 032433).

[0252] The motif combinations (Figure 4B) were N-terminally bound to a PreScission protease site (3C site) and a human hinge Fc domain. The membrane proximal motifs CRD4 and stalk were always exclusively incorporated together. A total of eight CD137 ECD variants and PRO1480 were included in the binding analysis (Figure 4, Table 20). Protein expression was performed in FreeStyleCHO-S cells using the transient CHOgro expression system (Mirus). Genes of interest were optimized for mammalian expression, synthesized, and cloned into a standard pcDNA3.1 vector. The signal sequence was derived from mouse heavy chain IgG. Expression cultures were grown in batch at 37 °C for 6-7 days (cell viability <70%) or at 37 °C for 1 day, followed by a temperature shift to 32 °C for 5-6 days. Culture supernatants were separated by centrifugation followed by 0.45 μm filtration. Target proteins were captured from clarified culture supernatants by protein L affinity chromatography for PRO1480 or protein A affinity chromatography for CD137 ECD variants followed by polishing size-exclusion chromatography. During binding experiments, PRO1480 was incubated with each CD137 ECD variant in equimolar ratios at a concentration of at least 500-fold the KD. Binding was assessed (yes or no) by retention time shift analysis by SE-HPLC of the complex compared to the individual proteins. The results are summarized in Table 21.

[0253] [Table 20-1] [Table 20-2]

[0254] [Table 21]

[0255] Multispecific molecules comprising the antibodies of the invention Exemplary multispecific molecules comprising antibodies of the invention are included in Table 3. PRO1480 and PRO1481 are derived from 38-27-A11 sc02 and 38-27-A11 sc03, respectively.

[0256] Example 9: Affinity for PDL1, CD137, HSA, and MSA Affinity for PDL1 was measured by SPR measurements using a Biacore T200 device (GE Healthcare). In this experiment, Fc-tagged PDL1 from various species was captured using the HumanAntibody Capture kit (catalog no. BR-1008-39) from GE Healthcare. After each analyte injection cycle, anti-human Fc-specific IgG was regenerated and new antigen was captured. In all formats, multispecific molecules were injected as analytes using a dose-response multi-cycle kinetic assay, with analyte concentrations ranging from 0.18 to 45 nM (2-fold dilution steps), diluted in running buffer. Association and dissociation times were set at 300 and 720 s, respectively. The apparent dissociation (kd) and association (ka) rate constants, and the apparent dissociation equilibrium constant (KD) were calculated using a one-to-one Langmuir binding model. The affinity of different species to CD137 was measured using the same setup as for PDL1, except that the CD137-Fc chimeric proteins of different species were captured by immobilized antibodies.

[0257] The Fc-containing formats were directly captured by an antibody specific for the Fc region of human IgG. Two-fold serial dilutions of PDL1 extracellular domain or CD137 extracellular domain ranging from 90 to 0.35 nM were tested for binding to IgG captured on the biosensor chip. After each injection cycle, the surface was regenerated by a single injection of 3 M MgCl2 solution.

[0258] The affinity of the molecules for different species of serum albumin (SA) was measured by SPR measurements using a Biacore T200 device (GE Healthcare). SA was directly coupled to a CM5 sensor chip (GE Healthcare) using amine coupling chemistry. After regeneration scouting and surface performance testing to find optimal assay conditions, dose-response was measured and the resulting binding curves were double-referenced (empty reference channel and zero analyte injection) and fitted using a 1:1 Langmuir model to obtain kinetic parameters. The assay was performed in 1x PBS-Tween buffer at pH 5.5.

[0259] The data obtained are summarized in Table 22. Measurement of the binding kinetics of the CD137-specific humanized construct derived from clone 38-27-A11 shows nearly identical affinity (compare the CDR grafts of clones 38-27-A11, PRO1480, and the STR graft PRO1481 in Table 22).

[0260] Example 10: Evaluation of the CD137 agonist effect of anti-PDL1xCD137 molecules using a cell-based assay of a transgenic NF-kB Jurkat reporter cell line expressing CD137 In this assay, we assessed the activation of CD137 signaling in Jurkat cells. The activity of CD137 signaling is reported by measuring luciferase expression, which is driven by CD137-induced NF-kB activation in a Jurkat reporter cell line. Luciferase expression directly correlates with CD137 activity. Furthermore, CD137 clustering, which is required for signal pathway activation, is promoted via the formation of an immune synapse between Jurkat cells and a PDL1-expressing cell line. Thus, PDL1 expression is required for CD137 clustering and activation in the reporter cell line.

[0261] HCC827 cells stimulated with 10ng / ml IFNy for 24 hours to increase PDL1 expression were seeded at 25,000 cells per well in 96-well culture plates. Serial dilutions of anti-PDL1 x CD137 molecules and competitor urelumab were then prepared and added to the cells. Jurkat reporter cells were then prepared in assay medium with or without 25mg / ml HSA and added at a cell density of 40,000 cells per well. Luciferase expression was detected by addition of luciferase reagent and read by a luminescence reader 6 hours or 24 hours after addition of Jurkat cells. Data were analyzed by normalizing the relative luminescence units (RLU) of the test samples to the RLU measured for PRO885 (Figure 5), yielding a value for relative activation of CD137 signaling. As shown in FIG. 5 and Table 23, scDb-scFv PRO1480 and PRO1481 derived from clone 38-27-A11 were able to stimulate CD137 signaling.

[0262] [Table 22]

[0263] [Table 23]

[0264] Example 11: Evaluation of the stimulatory effect of combined PDL1 blockade and CD137 stimulation in a cell-based assay using human PBMCs stimulated with the superantigen SEA In this experiment, the synergistic effect of PD-1 / PDL1 blockade and CD137 agonism was evaluated. In this assay, peripheral blood mononuclear cells (PBMCs) stimulated with the superantigen Staphylococcal enterotoxin A (SEA) were used to induce the expression of PDL1 in antigen-presenting cells (APCs) and CD137 in T cells, respectively. By applying anti-PDL1×CD137 molecules, two T cell regulatory signaling pathways were simultaneously targeted: inhibition of the inhibitory PD-1 / PDL1 pathway, and activation of the CD137 pathway via the formation of an immune synapse mediated by the trispecific anti-PDL1×CD137×HSA molecule (PRO1480). T cell activation, assessed by the secretion of interleukin 2 (IL-2), was compared to the effect mediated by PDL1 blockade mediated by a mixture of the reference molecules avelumab and urelumab.

[0265] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh human whole blood by density gradient centrifugation. PBMCs were then depleted of NK cells using anti-CD56 antibody and the MACS cell isolation kit (Miltenyi Biotec). 100,000 PBMCs per well were then added to a 96-well plate, followed by serial dilutions of PRO1480 or a combination of urelumab and avelumab in assay buffer containing SEA at a concentration of 10 ng / ml. After 96 h of incubation at 37 °C and 5% CO2, cell supernatants were harvested and human interleukin-2 (IL-2) levels in culture supernatants were quantified using BioLegend's IL-2 human ELISA MAX assay according to the kit instructions. IL-2 concentrations were interpolated and back-calculated from the IL-2 standard curve to obtain the EC 50 Values ​​were plotted against the avelumab / urelumab combination and PRO1480 concentration for calculation.

[0266] As shown in Figure 6, IL-2 was secreted by T cells following blockade of PD1 / PDL1 interaction and stimulation of CD137 by addition of PRO1480. PRO1480 showed higher T cell activation and better potency when compared to the combination of avelumab and urelumab. This finding indicates that the trispecific anti-PDL1 x CD137 x HSA scDb-scFv PRO1480 can induce stronger T cell stimulation when compared to the mixture of the reference molecules avelumab and urelumab.

[0267] Example 12: Epitope dependence of the stimulatory effect of simultaneous PDL1 blockade and CD137 stimulation Example 11 was repeated by comparing the stimulatory effect of two constructs with different anti-CD137 antibody fragments. As shown in FIG. 7, which shows data from a single representative example from a series of 3-4 individual experiments, IL-2 was secreted by T cells following simultaneous blockade of PD-1 / PDL1 interaction and stimulation of CD137 by addition of PRO1480 and PRO1186 (see Example 10). PRO1480 (see FIG. 9(A)), which targets membrane-distal tip epitopes of CRD1 and CRD2 of CD137 ECD, showed higher maximal T cell activation as indicated by higher IL-2 secretion and superior potency, when compared to PRO1186 (see FIG. 9(B)), which targets a membrane-proximal epitope of CRD4 of CD137 ECD (see FIG. 9(B)). This finding indicates that the trispecific anti-PDL1xCD137xHSA scDb-scFv PRO1480, which targets a membrane distal epitope, is able to induce stronger T cell costimulation when compared with molecules targeting non-distal epitopes.

[0268] Example 13: Epitope Determination Structural determination of PRO1359 alone and in complex with the ECD of 4-1BB The scFv (PRO1359) was transiently expressed in CHO cells and purified from the harvest using Capto-L resin (GE-Healthcare). The protein was polished to high monomer content using a Superdex 75 size-exclusion chromatography column (GE-Healthcare).

[0269] Residues 24-160 of 4-1BB (Uniprot:Q07011) were expressed with a C-terminal hingeless Fc tag containing an N-terminal secretion sequence and an IdeS cleavage site [Novarra, S., et al., A hingeless Fc fusion system for site-specific cleavage by IdeS. mAbs, 2016. 8(6): p.1118-1125]. The fusion protein was transiently expressed in CHO cells and captured from the cell supernatant using Protein A resin (GE Healthcare). The Fc tag was cleaved by IdeS at 37°C and removed using Protein A beads.

[0270] Crystallization of anti-4-1BB scFv PRO1359 Anti-4-1BB scFv was concentrated to 10 mg / ml in 25 mM Hepes, 100 mM NaCl, pH 6.7. Crystallization conditions were screened by sitting drop vapor diffusion with a 1:1 ratio of mother liquor to protein. Crystals were grown in 0.1 M Tris pH 8.5, 2 M NH4H2PO4 and cryoprotected by adding 100% ethylene glycol to the mother liquor to a final concentration of 20%.

[0271] Crystallization of the complex between anti-4-1BB scFv PRO1359 and the ECD of 4-1BB The complex was formed by mixing an equimolar ratio of both proteins and then purifying the complex on a Superdex 75 size-exclusion chromatography column in 50 mM Hepes, 100 mM NaCl, pH 6.7.

[0272] Fractions corresponding to the complex were concentrated to 10 mg / ml. Initial crystals were grown by sitting drop vapor diffusion at 20 °C in 0.1 M sodium acetate, pH 5.5, 22% PEG2000 MME, 0.17 M to 0.23 M calcium acetate, and equal volumes of mother liquor and complex. Crystals were crushed using SeedBeads (Hampton Research) and used as seeds in subsequent crystallization screens.

[0273] Final crystals were grown in 0.1M Tris acetate pH 8.5, 1M sodium formate, 25% PEG2000 MME with a mother liquor to protein to seed ratio of 1:1:0.125 at 20°C. Crystals were cryoprotected in 80mM Tris acetate pH 8.5, 0.8M sodium formate, 22.4% PEG2000 MME, 20% ethylene glycol and frozen in liquid nitrogen.

[0274] Diffraction experiments and structure solutions Native data sets of the scFv alone and in complex with 4-1BB were collected at the Swiss Light Source at the Paul-Scherrer Institute, Villigen, Switzerland. The scFv was crystallized in space group P65 and processed at 1.6 Å resolution using XDS. The complex was crystallized in space group I222 and processed at 2.2 Å resolution using XDS [Kabsch, W., XDS. Acta Crystallographica Section D, 2010. 66(2): p.125-132.].

[0275] The structure of the scFv was solved by molecular replacement with Phaser using an in-house scFv model [McCoy, AJ, et al., Phaser crystallographic software. Journal of Applied Crystallography, 2007. 40(4): p.658-674]. The complex structure was solved by molecular replacement using the scFv apo structure and a reduced model of 4-1BB (pdb code: 6BWV, chain D).

[0276] Refinement The structure was refined using Refmac [Murshudov, GN, AA Vagin, and EJ Dodson, Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Crystallographica Section D, 1997. 53(3): p.240-255.].

[0277] The apo structure was refined to 1.6 Å resolution, with residues 1–110 and 128–251 well defined by the electron density and final Rwork / Rfree values ​​of 15.8% / 18%. The complex structure was similarly refined to 2.2 Å resolution. For CD137, residues 24–158 are represented by the electron density, with residues 139–149 and 156–158 weakly defined. The scFv is well defined for residues 3–109 and 131–252. The complex was refined to Rwork / Rfree values ​​of 19.2% / 23.5%.

[0278] Interface Description CD137 epitope The binding interface will be analyzed using the PISA service of the European Bioinformatics Institute [Krissinel, E. and K. Henrick, Inference of Macromolecular Assemblies from Crystalline State. Journal of Molecular Biology, 2007. 372(3): p.774-797.]. The epitope of CD137 is located within the first and second cysteine-rich domains (CRDs). The accessible surface area buried upon binding of the scFv is 770Å. 2 (See Table 26). Important residues are shown in Table 24. The hydrogen bond network is summarized in Table 25.

[0279] [Table 24-1] [Table 24-2]

[0280] [Table 25] *When mutated to alanine, affinity was reduced by more than 1000-fold.

[0281] [Table 26] Some aspects of the invention are described below. 1. An isolated antibody having binding specificity for human CD137, comprising HCDR1, HCDR2, and HCDR3 sequences having at least 90% identity to the sequences of SEQ ID NOs:1, 2, and 3, respectively, LCDR1 and LCDR3 sequences having at least 90% identity to the sequences of SEQ ID NOs:16 and 18, respectively, and an LCDR2 sequence having at least 85% identity to the sequence of SEQ ID NO:17. 2. The antibody of item 1, comprising HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs: 16, 17, and 18, respectively. 3. The antibody according to item 1 or 2, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 15, preferably SEQ ID NOs: 13 and 15, more preferably SEQ ID NO: 13; and a light chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, and 27, preferably SEQ ID NOs: 25 and 27, more preferably SEQ ID NO: 25. 4. The antibody of item 3, comprising: (a) a VH sequence of SEQ ID NO: 13 and a VL sequence of SEQ ID NO: 25; (b) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26; or (c) a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO: 27. 5. The antibody according to any of items 1 to 4, wherein the antibody does not inhibit the interaction between CD137 and its ligand CD137L, in particular as measured by competitive ELISA. 6. The antibody (i) binds to human CD137 with a dissociation constant (KD) of less than 50 nM, particularly less than 10 nM, and more particularly less than 5 nM, as measured by surface plasmon resonance; (ii) optionally binds to cynomolgus CD137 with a KD of less than 50 nM, particularly less than 10 nM, more particularly less than 5 nM, as measured by surface plasmon resonance; (iii) optionally, does not bind to human CD40 and / or does not bind to human OX40, particularly as measured by SPR; (iv) in the case of an scFv format, has a melting temperature (Tm) of at least 50°C, e.g., at least 55°C, preferably at least 60°C, more preferably at least 64°C, as determined by differential scanning fluorimetry, particularly where the antibody is formulated in 50 mM phosphate-citrate buffer, 150 mM NaCl, pH 6.4; (v) in the case of an scFv format, the antibody of the invention, when at a starting concentration of 10 mg / ml, particularly where the antibody of the invention is formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4, has a loss of less than 7%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, preferably less than 2%, of monomer content after storage at 4° C. for at least 2 weeks, particularly at least 4 weeks; and / or (vi) in the case of an scFv format, the antibodies of the invention, when at a starting concentration of 10 mg / ml, have less than 5%, preferably less than 3%, and more preferably less than 1% loss of monomer content after five successive freeze-thaw cycles, particularly where the antibodies of the invention are formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4; 6. The antibody according to any one of items 1 to 5. 7. The antibody according to any of items 1 to 6, wherein the isolated antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, Fab, Fv, scFv, dsFv, scAb, STAB, and alternative scaffold-based binding domains, preferably Fv, or scFv, including but not limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites engineered into the constant region of the antibody (e.g., F-star's Modular Antibody Technology). 8. The antibody according to item 7, wherein the scFv has an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, preferably SEQ ID NO:29 and SEQ ID NO:31, more preferably SEQ ID NO:29. 9. An isolated antibody that binds to the human CD137 extracellular domain at an epitope located in the distal part of the extracellular domain of CD137, in particular within the cysteine-rich domains CRD1 and / or CRD2, more particularly within amino acid residues 24 to 86 of SEQ ID NO:32, provided that amino acid residue Asn42 of CD137 is not a critical residue for binding. 10. The isolated antibody described in item 9, wherein the antibody binds to the human CD137 extracellular domain at an epitope characterized by a set of key residues determined in accordance with Example 13, including residues Arg41, Gln43, Cys45, Pro49, Ser52, and Ser80. 11. The isolated antibody described in item 10, wherein the antibody binds to the human CD137 extracellular domain at an epitope characterized by a set of key residues determined in accordance with Example 13, further comprising residues Ala33, Asn40, and Cys48. 12. The isolated antibody according to item 11, wherein the antibody binds to the human CD137 extracellular domain at an epitope characterized by a set of key residues determined according to Example 13, further comprising residues Pro32, Gly34, Thr35, Ser46, Pro47, Pro50, Cys78, and Ser79. 13. The antibody according to any of items 1 to 12, wherein the antibody is a multispecific molecule, in particular a multispecific molecule having at least a second functional molecule. 14. A pharmaceutical composition comprising the antibody according to any one of items 1 to 13 and a pharma- ceutically acceptable carrier. 15. The antibody according to any of items 1 to 13, or the composition of item 14, for use as a medicament. 16. The antibody according to any of items 1 to 13, or the composition according to item 14, for use in the manufacture of a medicament for use in the treatment of cancer. 17. A nucleic acid encoding the antibody according to any one of items 1 to 13. 18. A method for producing the antibody according to any one of items 1 to 13, comprising the step of culturing a host cell containing a nucleic acid or vector encoding the antibody according to any one of items 1 to 13. 19. A kit comprising the antibody according to any one of items 1 to 13, or the composition according to item 14.

Claims

1. 1. An isolated antibody having binding specificity for human CD137 comprising a VH region comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs:1, 2, and 3, respectively, and a VL region comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NOs:16, 17, and 18, respectively, wherein said antibody binds to the human CD137 extracellular domain at an epitope located in a distal portion of the extracellular domain of CD137 within amino acid residues 24-86 of SEQ ID NO:32, wherein said epitope is characterized by residues Ala33, Asn40, Arg41, Gln43, Cys45, Cys48, Pro49, Ser52, and Ser80, and wherein said antibody does not inhibit the interaction between CD137 and its ligand CD137L as measured by competitive ELISA.

2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, and 15; and a light chain variable region comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, and 27.

3. The antibody of claim 2, comprising: (a) a VH sequence of SEQ ID NO: 13 and a VL sequence of SEQ ID NO: 25; (b) a VH sequence of SEQ ID NO: 14 and a VL sequence of SEQ ID NO: 26; or (c) a VH sequence of SEQ ID NO: 15 and a VL sequence of SEQ ID NO:

27.

4. The antibody is (i) binds to human CD137 with a dissociation constant (KD) of less than 50 nM, as measured by surface plasmon resonance; (ii) optionally binds to cynomolgus monkey CD137 with a K of less than 50 nM as measured by surface plasmon resonance; (iii) optionally, does not bind to human CD40 and / or does not bind to human OX40, as measured by SPR; (iv) in the case of an scFv format, has a melting temperature (Tm) of at least 50° C., as determined by differential scanning fluorimetry, where the antibody is formulated in 50 mM phosphate-citrate buffer, 150 mM NaCl, pH 6.4; (v) in the case of an scFv format, the antibody of the invention has a starting concentration of 10 mg / ml, where the antibody in the scFv format has less than 7% loss of monomer content after storage for at least 2 weeks at 4° C. when formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4; and / or (vi) in the case of an scFv format, the antibody of the invention has less than 5% loss of monomer content after five successive freeze-thaw cycles when at a starting concentration of 10 mg / ml, where the antibody in the scFv format is formulated in 50 mM phosphate-citrate buffer containing 150 mM NaCl at pH 6.4; The antibody according to any one of claims 1 to 3.

5. The antibody of any one of claims 1 to 4, wherein the isolated antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a Fab, an Fv, a scFv, a dsFv, and an scAb, and is preferably an Fv or an scFv.

6. The antibody of claim 5, wherein the antibody is an scFv having an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:

31.

7. 7. The isolated antibody of any one of claims 1 to 6, wherein the epitope further comprises residues Pro32, Gly34, Thr35, Ser46, Pro47, Pro50, Cys78, and Ser79.

8. The antibody according to any one of claims 1 to 7, which is a multispecific molecule having at least a second functional molecule.

9. A pharmaceutical composition comprising the antibody of any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

10. An antibody according to any one of claims 1 to 8, or a composition according to claim 9, for use as a medicament.

11. An antibody according to any one of claims 1 to 8, or a composition according to claim 9, for use in the manufacture of a medicament for use in the treatment of cancer.

12. A nucleic acid encoding the antibody according to any one of claims 1 to 8.

13. A method for producing an antibody according to any one of claims 1 to 8, comprising the steps of culturing a host cell comprising a nucleic acid or vector encoding the antibody according to any one of claims 1 to 8, and expressing the nucleic acid encoding the antibody according to any one of claims 1 to 8 contained in said nucleic acid or vector.

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