Novel combination therapy and uses thereof

Abstract

The present invention relates to a combination therapy for treating cancer in a subject, and methods for its use. The combination therapy comprises: (a) an antibody, or antigen-binding fragment thereof, that specifically binds to CD137; and (b) an additional immunotherapeutic agent, wherein the additional immunotherapeutic agent is a PD-1 inhibitor. The present invention also relates to pharmaceutical compositions comprising the combination therapy of the present invention, uses thereof, methods of use, and kits comprising the combination therapy of the present invention. The cancer may be a solid tumor.

Description

[Technical field]

[0001] The present invention relates to a combination therapy for treating cancer in a subject, and methods for its use. The combination therapy comprises: (a) an antibody, or antigen-binding fragment thereof, that specifically binds to CD137; and (b) an additional immunotherapeutic agent, wherein the additional immunotherapeutic agent is a PD-1 inhibitor. The present invention also relates to pharmaceutical compositions comprising the combination therapy of the present invention, uses thereof, methods of use, and kits comprising the combination therapy of the present invention. The cancer may be a solid tumor. [Background technology]

[0002] Cancer is the leading cause of premature death in developed countries. The goal of immunotherapy in cancer is to mount an effective immune response by the body against tumors, especially solid tumors. This can be achieved, for example, by breaking tolerance to tumor antigens, enhancing antitumor immune responses, and stimulating local cytokine responses at the tumor site. The main effector cells of long-lasting antitumor immune responses are activated tumor-specific effector T cells. Strong expansion of activated effector T cells can redirect the immune response against tumors. In this context, regulatory T cells (Tregs) play a role in inhibiting antitumor immunity. Thus, depleting, inhibiting, reversing, or inactivating Tregs can provide antitumor effects and reverse immune suppression in the tumor microenvironment. Furthermore, incomplete activation of effector T cells, for example, by dendritic cells, can cause T cell anergy, which results in an inefficient antitumor response, whereas sufficient induction by dendritic cells can result in strong expansion of activated effector T cells and redirect the immune response against tumors. In addition, natural killer (NK) cells play an important role in tumor immunology by attacking tumor cells expressing downregulated human leukocyte antigens (HLA) and by inducing antibody-dependent cellular cytotoxicity (ADCC). Therefore, stimulating NK cells can also reduce tumor growth.

[0003] CD137 (4-1BB, TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily and mediates the activation of activated CD4 + and CD8 + It is expressed on T cells, Tregs, DCs, monocytes, mast cells, and eosinophils. Activation of CD137 is mediated by the activation of CD8 + It plays a key role in T cell activation and survival (Lee et al., 2002; Pulle et al., 2006). It sustains and enhances, rather than initiates, effector function and preferentially supports Th1 cytokine production (Shuford et al., 1997). CD4 + In T cells, CD137 stimulation first leads to activation and then activation-induced cell death, explaining why CD137 agonist antibodies have shown therapeutic efficacy not only in tumor immunity but also in autoimmunity (Zhang, JCI, 2007; Sun, Trends Mol Med, 2003). CD137 also suppresses Treg function (So, Cytokine Growth Factor Rev, 2008). CD137 activation depends on receptor oligomerization (Rabu et al., 2005; Wyzgol et al., 2009).

[0004] CD137 agonistic antibodies have been shown to activate endothelial cells in the tumor environment, leading to upregulation of ICAM-1 and VCAM-1 and improved T cell recruitment (Palazon, Cancer Res, 2011).

[0005] CD137 is upregulated on NK cells activated by cytokines or CD16 in mice or humans, respectively (see, e.g., Melero, CCR 19(5)1044-53, 2013 and references cited therein). CD137 has been shown to activate NK cells and enhance ADCC in mice as well as humans (Kohrt et al., 2014), although there are reports suggesting opposing effects on NK cells in mice and humans, leading to NK cell activation in mice and inhibition in humans (Baessler, Blood, 2010).

[0006] Several studies have demonstrated the induction of tumor immunity by treatment with agonistic CD137 antibodies (Dubrot et al., 2010; Gauttier et al., 2014; Kim et al., 2001; McMillin et al., 2006; Melero et al., 1997; Miller et al., 2002; Sallin et al., 2014; Taraban et al., 2002; Uno et al., 2006; Vinay and Kwon, 2012; Wilcox et al., 2002). In addition, it interacts with several immunomodulatory agents in preclinical models (Curran et al., 2011; Gray et al., 2008; Guo et al., 2013; Kwong et al., 2013; Lee et al., 2004; Morales-Kastresana et al., 2013; Pan et al., 2002; St Rose et al., 2013; Uno et al., 2006; Wei et al., 2013; Westwood et al., 2010; Westwood et al., 2014a; Westwood et al., 2014b).

[0007] Urelumab is a potent 4-1BB agonist with demonstrated limited clinical efficacy (Chester et al. 2017, Chin et al. 2018). However, development of urelumab was hampered by hepatotoxicity at doses of 0.3 mg / kg or higher (including two fatal events at doses of 1 mg / kg or higher) (Segal et al. 2017). Thus, the maximum tolerated dose was set at 0.1 mg / kg (or a flat dose of 8 mg). In subsequent studies, no clear objective responses were observed with urelumab as monotherapy (Chester et al. 2017). The mechanism behind hepatotoxicity is not fully understood.

[0008] On the other hand, utomilumab is considered a weaker agonist than urelumab and has also shown limited clinical efficacy (Chin et al. 2018, Segal et al. 2018, Tolcher et al. 2017). Utomilumab showed an acceptable clinical safety profile up to 10 mg / kg with no dose-limiting toxicities (DLTs).

[0009] Utomilumab, unlike urelumab, relies on FcγR cross-linking to exert its agonistic effect. FcγRs in the blood are saturated by endogenous circulating human IgG, so that at approximately 10 g / L, FcγR cross-linking-dependent antibodies such as utomilumab must compete with IgG for binding to FcγR (Jolliff 1982). 10 g / L of endogenous IgG is the maximum serum concentration (C ) reached at the highest clinical dose of utomilumab (155 μg / mL at 10 mg / kg). max) is 60-fold greater than in the liver (Segal et al. 2018). The liver is a highly vascularized organ, and endogenous IgG concentrations in the liver have been shown to be similar to circulating levels (Eigenmann et al. 2017). Therefore, FcγR crosslinking-dependent 4-1BB activation may also be expected to be reduced in the liver due to competition with endogenous IgG. This reduced potential for FcγR crosslinking of utomilumab in the liver may explain the absence of hepatotoxicity detected with urelumab. 4-1BB activation by ALG.APV-527 is 5T4 crosslinking-dependent, and hepatotoxicity by APV-527 is not expected because 5T4 is not expressed in the liver.

[0010] Nine additional monospecific 4-1BB antibodies: ADG106, administered at doses ranging from 0.03 to 10 mg / kg and currently in phase I / II (Liu et al. 2017), as well as CTX-471, AGEN2373, LVGN6051 ATOR-1017, EU101 (IND / CTA), PE0116, STA551 and HOT1030 have entered clinical development between 2018 and 2021 and are currently being evaluated for safety in phase I trials.

[0011] The agonistic effect of CD137 antibodies is influenced by the isotype of the Fc region. Antibodies tested in the clinic are either IgG2 or IgG4. Like most TNFR family members, CD137 depends on cross-linking for activation (Wilson 2011, Cancer Cell). CD137L expressed on the membrane of APCs can induce significant multiple cross-linking of receptors. An antibody can only cross-link two CD137 receptors by itself, and further cross-linking (trans) through FcγRs expressed on other cells may be necessary to induce strong CD137-mediated signals. An exception to this may be IgG2 antibodies, which induce cross-linking-independent signaling by an unknown mechanism (White et al, 2015 Cancer Cell). T cells do not express FcγRs, and FcγR-mediated cross-linking in vivo is thought to be mediated by monocytes, macrophages, DCs, and potentially B cells and other cell types.

[0012] Another factor to consider is that FcγR receptor engagement can also trigger ADCC, antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) on antibody-coated cells (for simplicity, hereafter ADCC will include ADCP and CDC). Typically, human IgG1 is a strong inducer of NK / macrophage-dependent ADCC, depending on the nature of the target, cell type, and receptor density. IgG4 antibodies can also induce ADCC, but to a lesser extent than IgG1 (Wang 2015, Front Imm; Vidarson 2014 Front Imm).

[0013] Thus, the effect of CD137 agonist antibodies with different isotypes may be influenced by the balance between 1) induction of cross-linking resulting in stronger immune activation, and 2) induction of ADCC that may result in the killing of both effector T cells (mainly CD8 T cells) and Tregs. The net effect of 1) and 2) is likely to depend on the distribution of CD137-expressing cells, the likelihood of target cells binding to FcγR-expressing immune cells, receptor density and affinity, and the susceptibility of Teff vs. Tregs to ADCC. CD137 expression is high on both CD8 and Tregs in melanoma tumors (Quezada, presentation SITC 2015). IgG4 type allows FcγRI-mediated cross-linking by macrophages and monocytes, yet minimizes NK-mediated ADCC of effector CD8 T cells.

[0014] However, as outlined above, due to differences in expression and affinity between mouse and human FcRs, it is difficult to describe the comparison of different human Fcs in mouse models. Furthermore, although the in vivo functional consequences of antibodies that block CD137L binding to CD137 are currently under debate, it can be assumed that CD137 agonists that block CD137L and therefore do not allow simultaneous activation via CD137L and CD137 agonist antibodies have a low risk of inducing exaggerated activation and systemic toxicity.

[0015] Several studies have demonstrated the induction of tumor immunity by treatment with agonistic CD137 mAbs (Dubrot et al., 2010; Gauttier et al., 2014; Kim et al., 2001; McMillin et al., 2006; Melero et al., 1997; Miller et al., 2002; Sallin et al., 2014; Taraban et al., 2002; Uno et al., 2006; Vinay and Kwon, 2012; Wilcox et al., 2002). Two different antibodies, Lob12.3 and 3H3, are commonly used for in vivo studies in mice (Shuford 1997 J Exp Med).

[0016] The toxicity seen in the mouse model was detected after repeated administration in a time-dependent but not dose-dependent manner (Ascierto 2010 Semin Onc, Dubrot 2010 Can Imm, Niu 2007 JI). The toxicity includes dermal and hepatotoxicity: aspartate aminotransferase / alanine aminotransferase ratio (ASAT / ALAT) and cytokine release. This suggests that the toxicity requires CD137-mediated preactivation of immune cell populations (probably T cells) or depends on secondary effects caused by anti-drug antibody (ADA) responses potentially forming aggregates of CD137 antibodies that may result in enhanced cross-linking. The toxicity seen in mice is reversible and appears to be TNFα / CD8 cell dependent (Ascierto 2010 Sem Onc). Toxicity studies in monkeys showed that both single and repeated doses of up to 100 mg / kg, once weekly for 4 weeks, were tolerated without detectable skin or liver toxicity (Ascierto 2010,Semin Onc).

[0017] Prolonged and continuous activation via TNF receptor family members can lead to immune exhaustion. It may therefore be advantageous to administer such antibodies in a manner that allows for a period of rest for cells expressing the receptor. One approach to extend the period of rest in a particular dosing protocol is to shorten the half-life of the antibody, for example, by reducing binding to neonatal Fc receptor (FcRn). This can also reduce toxicity associated with the treatment, depending on the route of administration.

[0018] The programmed death 1 (PD-1) receptor is a negative regulator of antitumor T cell effector function when engaged by its ligand PD-L1, which is expressed on the cell surface within tumors (Ribas and Wolchok 2018). PD-1 is an immune checkpoint whose inhibitory function is mediated by the tyrosine phosphatase SHP-2, which dephosphorylates downstream signaling molecules of the T cell receptor (TCR). PD-1 has two ligands: programmed death ligand 1 (PD-L1, also known as CD274 or B7-H1), which is widely expressed by many somatic cells, mainly upon exposure to proinflammatory cytokines, and programmed death ligand 2 (PD-L2, also known as CD273 or B7-DC), which has more restricted expression in antigen-presenting cells. Inflammation-induced PD-L1 expression in the tumor microenvironment causes PD-1-mediated T cell exhaustion and inhibits antitumor cytotoxic T cell responses. PD-L1 is expressed on both tumor and myeloid cells. PD-1 resistance can be broadly subdivided into primary resistance or secondary (acquired) resistance (Kluger et al. 2020). Understanding the nature of PD-1 resistance is essential to select the right type of combination therapy (Yuan et al. 2021). [Prior art documents] [Non-patent literature]

[0019] [Non-Patent Document 1] Lee et al.,2002;Pulle et al.,2006 [Non-Patent Document 2] Shuford et al., 1997 [Non-Patent Document 3] Zhang,JCI,2007,Sun,Trends Mol Med,2003 [Non-Patent Document 4] So,Cytokine Growth Factor Rev,2008 [Non-Patent Document 5] Rabu et al.,2005;Wyzgol et al.,2009

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[0020] Thus, there remains a need for improved cancer therapies, in particular anti-CD137 antibodies suitable for use in the treatment of solid tumors and combination therapies thereof. [Means for solving the problem]

[0021] The inventors have surprisingly found that combination therapies comprising an anti-CD137 antibody, or an antigen-binding fragment thereof, and a PD-1 inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an antigen-binding fragment thereof) are surprisingly effective in treating cancer. Accordingly, a first aspect of the invention provides a combination therapy for use in the treatment or prevention of cancer in a subject, the combination therapy comprising (a) an antibody that specifically binds CD137, or an antigen-binding portion thereof, and (b) a further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor.

[0022] A second aspect of the invention provides an antibody, or antigen-binding portion thereof, that specifically binds CD137 for use in a method of treating cancer, wherein the antibody, or antigen-binding portion thereof, that specifically binds CD137 is for use in combination with a PD-1 inhibitor. Preferably, the cancer is a solid tumour.

[0023] A related third aspect of the invention provides the use of an antibody that specifically binds CD137, or an antigen-binding portion thereof, in the preparation of a medicament for treating a solid tumour, wherein the antibody that specifically binds CD137, or an antigen-binding portion thereof, is for use in combination with a PD-1 inhibitor.

[0024] A fourth aspect of the invention provides a pharmaceutical composition comprising (a) an antibody, or an antigen-binding portion thereof, that specifically binds to CD137; and (b) a further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor.

[0025] A fifth aspect of the present invention provides a kit for treating a solid tumor, comprising: (a) an antibody, or antigen-binding fragment thereof, that specifically binds to CD137; and (b) a PD-1 inhibitor.

[0026] A sixth aspect of the invention is a method for treating or preventing cancer, e.g., a solid tumor, in a subject, the method comprising administering to the subject a therapeutically effective amount, such as (a) administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that specifically binds to CD137, and (b) administering to the subject a therapeutically effective amount of a PD-1 inhibitor. In some embodiments, the method comprises simultaneously administering (a) an antibody, or antigen-binding portion thereof, that specifically binds to CD137, and (b) a PD-1 inhibitor. In other embodiments, the method comprises administering (a) an antibody, or antigen-binding portion thereof, that specifically binds to CD137 prior to administering (b) the PD-1 inhibitor. In other embodiments, the method comprises administering the PD-1 inhibitor prior to administering the antibody, or antigen-binding portion thereof, that specifically binds to CD137.

[0027] In a further aspect, the invention provides a PD-1 inhibitor for use in a method of treating cancer, wherein the PD-1 inhibitor is for use in combination with an antibody, or antigen-binding portion thereof, that specifically binds CD137. Preferably, the cancer is a solid tumour.

[0028] A related further aspect of the invention provides the use of a PD-1 inhibitor in the preparation of a medicament for treating a solid tumor, wherein the PD-1 inhibitor is for use in combination with an antibody, or antigen-binding portion thereof, that specifically binds CD137.

[0029] A further aspect of the invention relates to a method for the production of a medicament comprising administering to a subject an antibody or antigen-binding fragment thereof, comprising administering to said subject an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment being specifically bound to CD137 ... and (i) a PD-1 inhibitor, and / or (ii) a second isolated nucleic acid molecule (or a component peptide chain thereof) encoding an antibody that specifically binds to PD-1 or an antigen-binding fragment thereof.

[0030] A further aspect of the invention relates to a method for the preparation of a polypeptide comprising: a first isolated nucleic acid molecule encoding an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, or a component peptide chain thereof; (i) a PD-1 inhibitor, and / or (ii) a second isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, or a component peptide chain thereof.

[0031] A further aspect of the invention relates to a method for the preparation of a polypeptide comprising: a first isolated nucleic acid molecule encoding an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, or a component peptide chain thereof; (i) a PD-1 inhibitor, and / or (ii) a second isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, or a component peptide chain thereof.

[0032] A further aspect is a) an isolated nucleic acid molecule (or component peptide chains thereof) encoding an antibody, or antigen-binding fragment thereof, that specifically binds to CD137; b) PD-1 inhibitors, and / or c) an isolated nucleic acid molecule (or its component peptide chains) encoding an antibody, or an antigen-binding fragment thereof, that specifically binds to PD-1 or PD-L1; d) an antibody, or an antigen-binding portion thereof, that specifically binds to CD137; e) PD-1 inhibitors, f) an isolated nucleic acid molecule encoding an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, or a vector comprising its component peptide chains; g) an isolated nucleic acid molecule encoding an antibody that specifically binds to PD-1 or PD-L1, or an antigen-binding fragment thereof, or a vector comprising component peptide chains thereof; h) a host cell comprising an isolated nucleic acid molecule encoding an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, or a component peptide chain thereof; and / or i) an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, or a host cell comprising a component peptide chain thereof;

[0033] As outlined above, a first aspect of the invention provides a combination therapy for use in the treatment or prevention of cancer in a subject, the combination therapy comprising (a) an antibody that specifically binds CD137, or an antigen-binding portion thereof, and (b) a further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor.

[0034] In one embodiment, the cancer is a solid tumor. In one embodiment, the cancer and / or solid tumor is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), head and / or neck cancer, gastric cancer, esophageal cancer, renal cancer, urothelial cancer, melanoma, breast cancer, cervical cancer, prostate cancer, microsatellite instability high (MSI) cancer, DNA mismatch repair (dMMR) associated cancer, and / or high tumor mutation burden (TMB) colorectal cancer; renal cancer; pancreatic cancer; The cancer is selected from the group consisting of cancer; ovarian cancer; rhabdomyosarcoma; neuroblastoma; bone cancer; multiple myeloma; leukemia (e.g., acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g., melanoma), bladder cancer, glioblastoma, adenoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms' tumor, lung tumor, colon tumor, lymphoid tumor, breast tumor, and melanoma.

[0035] In a preferred embodiment, the cancer and / or solid tumor is lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), head and / or neck cancer, gastric cancer, esophageal cancer, renal cancer, urothelial cancer, melanoma, breast cancer, cervical cancer, prostate cancer, microsatellite instability high (MSI) cancer, DNA mismatch repair (dMMR) associated cancer, and / or high tumor mutational burden (TMB) cancer, preferably, the cancer and / or solid tumor is metastatic.

[0036] In one embodiment, the cancer and / or solid tumor is metastatic.

[0037] (a) Antibody that specifically binds to CD137 In one embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 ("antibody polypeptide") is a) has binding specificity for domain 2 of human CD137, b) is a CD137 agonist; and / or c) It is capable of inhibiting the binding of the reference antibody "1630 / 1631" to human CD137.

[0038] In some embodiments, the antibody or antigen-binding fragment that specifically binds to CD137 has binding specificity for domain 2 of human CD137, is a CD137 agonist, and can inhibit binding of the reference antibody "1630 / 1631" to human CD137.

[0039] In one embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 ("antibody polypeptide") is a) has binding specificity for domain 2 of CD137, b) is a CD137 agonist; and / or c) Capable of inhibiting binding of reference antibody "2674 / 2675" to human CD137. In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to CD137 has binding specificity for domain 2 of CD137, is a CD137 agonist, and is capable of inhibiting binding of reference antibody "2674 / 2675" to human CD137.

[0040] In one embodiment, the antibody or antigen-binding fragment that specifically binds to CD137 is capable of inhibiting the binding of reference antibodies "1630 / 1631" and / or "2674 / 2675" to human CD137.

[0041] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CD137 can inhibit binding of one or more reference antibodies to human CD137, for example, can inhibit binding of reference antibodies "1630 / 1631" and / or "2674 / 2675" to human CD137. Exemplary anti-CD137 antibodies are disclosed in WO2018 / 091740 to Alligator Bioscience AB, the disclosure of which is incorporated herein by reference. For example, such anti-CD137 antibodies are expressly disclosed on pages 7-8, 11-12, 16-17 and 19 of WO2018 / 091740, the disclosure of which is incorporated herein by reference.

[0042] "CD137" specifically includes the human CD137 protein, e.g., as set forth in GenBank Accession No. AAH06196.1 (the sequence of which is set forth in SEQ ID NO: 11 below). CD137 is also known in the scientific literature as 4-1BB and TNFRSF9. Human CD137, amino acid sequence:>gi|571321|gb|AAA53133.1|4-1BB[Homo sapiens] [ka] [SEQ ID NO: 11] "Domain 2" refers to that described above corresponding to amino acids 66-107 of human CD137 (see the bold underlined region of SEQ ID NO:11 above).

[0043] Thus, the combination therapy of the present invention comprises an antibody or antigen-binding fragment that specifically binds to CD137, i.e. has specificity for CD137. "Specificity" means that the antibody polypeptide is capable of binding to CD137 in vivo, i.e. under physiological conditions in which CD137 is present in the human body. Preferably, the antibody polypeptide does not bind to any other proteins in vivo. Such binding specificity can be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blot, flow cytometry, etc., using transfected cells expressing CD137.

[0044] The antibody or antigen-binding fragment that specifically binds to CD137 is preferably in the range of 10x10 -9 Less than M or 7 x 10 -9 M or less, more preferably 4 or 2×10 -9 M, most preferably less than 1.2 × 10 -9The antibody polypeptide binds to human CD137 with a Kd value that is less than M. Advantageously, the antibody polypeptide can selectively bind to CD137, i.e. binds to CD137 at least 10 times more strongly than any other protein. The anti-CD137 antibody preferably specifically binds to CD137, i.e. binds to CD137 but does not bind to other molecules (e.g., OX40 and / or CD40) or binds with a lower affinity (e.g., 10-fold lower affinity). Thus, it binds to CD137 with a higher binding affinity than it binds to another molecule. Thus, typically, the Kd of the antibody to human CD137 will be less than 2-fold, preferably 5-fold, more preferably 10-fold, than the Kd to other non-target molecules, such as mouse CD137, other TNFR superfamily members, or any other unrelated or associated substances in the environment. More preferably, the Kd will be less than 50-fold, even more preferably less than 100-fold, and even more preferably less than 200-fold.

[0045] Methods for measuring the overall affinity (KD) of an interaction (such as the interaction between an antibody and a ligand) as well as the on-rate (ka) and off-rate (kd) are well known in the art. Exemplary in vitro methods are described in the accompanying examples. It is also conceivable to use a method based on flow cytometry (Sklar et al., Annu Rev Biophys Biomol Struct, (31), 97-119, 2002).

[0046] The term CD137 used herein typically refers to human CD137. The antibody may have some binding affinity to CD137 from other mammals, such as CD137 from non-human primates, for example Macaca fascicularis (cynomolgus monkey). The antibody preferably does not bind to mouse CD137 and / or does not bind to other human TNFR superfamily members, for example human OX40 or CD40.

[0047] In one embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may have affinity for CD137 in its native state, particularly CD137 localized on the surface of a cell.

[0048] "Localized on the surface of a cell" means that CD137 is associated with a cell such that one or more regions of CD137 are present on the outer surface of the cell. For example, CD137 may be inserted into the cell plasma membrane (i.e., oriented as a transmembrane protein) with one or more regions presented on the outer cell surface. This may occur during the process of expression of CD137 by the cell. Thus, in one embodiment, "localized on the surface of a cell" may mean "expressed on the surface of a cell". Alternatively, CD137 may be present on the outside of the cell with covalent and / or ionic interactions that localize it to a specific region or regions of the cell surface.

[0049] In one embodiment, the antibodies and antigen-binding fragments thereof that specifically bind to CD137 as defined herein are CD137 agonists. For example, they may be capable of inducing the release of interferon gamma from CD8+ T cells. The agonist activity of anti-CD137 antibodies may be assessed in a T cell assay based on primary CD8+ T cells (see Examples).

[0050] Thus, an antibody or antigen-binding fragment that specifically binds to CD137 can modulate the activity of a cell expressing CD137, the modulation being an increase or decrease in the activity of the cell. The cell is typically a T cell. The antibody can increase the activity of CD4+ or CD8+ effector cells, or can decrease the activity of or deplete regulatory T cells (T reg). In either case, the net effect of the antibody is an increase in the activity of effector T cells, particularly CD4+, CD8+, or NK effector T cells. Methods for determining changes in the activity of effector T cells are well known and have been described above.

[0051] An antibody or antigen-binding fragment that specifically binds to CD137 preferably causes an increase in activity in CD8+ T cells in vivo, optionally the increase in activity being increased proliferation, IFN-γ production and / or IL-2 production by the T cells, the increase being preferably at least 2-fold, more preferably at least 10-fold, even more preferably at least 25-fold higher than the change in activity caused by an isotype control antibody measured in the same assay.

[0052] As outlined above, antibodies, or antigen-binding fragments thereof, are provided that are capable of inhibiting the binding of one or more reference antibodies to human CD137. The reference antibodies described herein are reference antibody 1630 / 1631 and reference antibody 2674 / 2675.

[0053] Exemplary anti-CD137 antibodies are disclosed in WO2018 / 091740 to Alligator Bioscience AB, the disclosure of which is incorporated herein by reference.

[0054] The reference antibody "1630 / 1631" refers to an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOs: 17 and 18, respectively. 1630 / 1631-Full sequence heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFGYSYMSWVRQAPGKGLEWVSSIGSGSSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYSSPGIDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO:17] 1630 / 1631-Full sequence light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTWVPFTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 18]

[0055] The reference antibody "2674 / 2675" refers to an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOs: 29 and 30, respectively. Antibody 2674 / 2675 is also known as ATOR-1017, and these terms are fully interchangeable. 2674 / 2675-Full sequence heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFNFGYSYMSWVRQAPGKGLEWVSSIGSTSSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYSSPGIDYWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO:29] 2674 / 2675-Full sequence light chain DIQMTQSPSSLSASVGDRVTITCRASQSIGSTLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTWVPFTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO:30]

[0056] As discussed below, reference antibody "1630 / 1631" binds to domain 2 of CD137. Reference antibody 2674 / 2675 also binds to domain 2 of CD137. It should therefore be understood that an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, in the combination therapy of the present invention also binds to domain 2 of CD137. In some embodiments, an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, binds to domain 2 of CD137.

[0057] "Capable of inhibiting the binding of reference antibody "1630 / 1631" to human CD137" means that the presence of the antibody polypeptide of the combination therapy of the invention inhibits, in whole or in part, the binding of "1630 / 1631" to human CD137. Similarly, "capable of inhibiting the binding of reference antibody "2674 / 2675" to human CD137" means that the presence of the antibody polypeptide of the combination therapy of the invention inhibits, in whole or in part, the binding of "2674 / 2675" to human CD137. Thus, the anti-CD137 antibody or fragment thereof used in the combination therapy of the invention may compete with "reference antibody" 1630 / 1631 and / or "reference antibody" 2674 / 2675 for binding to human CD137. Such competitive binding inhibition can be determined using assays and methods well known in the art, for example, using a BIAcore chip with immobilized CD137 and incubating with and without the antibody polypeptide being tested in the presence of reference antibody "1630 / 1631" or "2674 / 2675". Alternatively, a pair-wise mapping approach can be used in which the reference antibody "1630 / 1631" or "2674 / 2675" is immobilized on the surface of the BIAcore chip, the CD137 antigen is allowed to bind to the immobilized antibody, and a secondary antibody is then tested for simultaneous CD137 binding ability (see, for example, "BIAcore Assay Handbook", GE Healthcare Life Sciences, 29-0194-00 AA 05 / 2012, the disclosure of which is incorporated herein by reference).

[0058] As a further alternative, competitive binding inhibition can be determined using flow cytometry.For example, to determine whether a test antibody can inhibit the binding of 1630 / 1631 or 2674 / 2675 reference antibody to cell surface antigen, cells expressing the antigen can be pre-incubated with test antibody for 20 minutes before washing the cells and incubating with reference 1630 / 1631 or 2674 / 2675 antibody conjugated to a fluorophore that can be detected by flow cytometry.If pre-incubation with test antibody reduces the detection rate of reference 1630 / 1631 or 2674 / 2675 antibody in flow cytometry, the test antibody inhibits the binding of reference antibody to cell surface antigen.If the tested antibody shows high affinity for CD137, a shorter period of pre-incubation can be used (or no pre-incubation at all).

[0059] In a further alternative, competitive binding inhibition can be determined using ELISA.

[0060] In some embodiments, antibodies and antigen-binding fragments that specifically bind to CD137 of the combination therapy of the present invention are defined by reference to the variable regions of the reference antibodies 1630 / 1631 and 2674 / 2675.

[0061] Reference antibodies designated "1630 / 1631" include: (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 EVQLLESGGGLVQPGGSLRLSCAASGFTFGYSYMSWVRQAPGKGLEWVSSIGSGSSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYSSPGIDYWGQGTLVTVSS [SEQ ID NO:1] and (b) a light chain variable region having the amino acid sequence of SEQ ID NO:2 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTWVPFTFGQGTKLEIK [SEQ ID NO:2]

[0062] Reference antibodies designated "2674 / 2675" include: (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19 EVQLLESGGGLVQPGGSLRLSCAASGFNFGYSYMSWVRQAPGKGLEWVSSIGSTSSHTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYSSPGIDYWGQGTLVTVSS [SEQ ID NO: 19] and (b) a light chain variable region having the amino acid sequence of SEQ ID NO: 20 DIQMTQSPSSLSASVGDRVTITCRASQSIGSTLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTWVPFTFGQGTKLEIK [SEQ ID NO:20]

[0063] As used herein, the term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding "D" stereoisomers (as compared to the naturally occurring "l"), omega-amino acids and other naturally occurring amino acids, non-conventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derived amino acids (see below).

[0064] When an amino acid is specifically recited, such as "alanine," "Ala," or "A," the term refers to both l-alanine and d-alanine unless expressly stated otherwise. Other non-conventional amino acids may also be suitable components for the polypeptides of the invention, so long as the desired functional properties are retained by the polypeptide. For the peptides shown, where appropriate, each encoded amino acid residue is represented by a single letter designation that corresponds to the common name of the conventional amino acid.

[0065] In one embodiment, an antibody polypeptide as defined herein comprises or consists of l-amino acids.

[0066] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences and longer polypeptides and proteins. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, as well as amino acid analogs and peptidomimetics.

[0067] Those skilled in the art will appreciate that the binding specificity of an antibody or antigen-binding fragment thereof is conferred by the presence of complementarity determining regions (CDRs) within the variable regions of the constituent heavy and light chains, such as the CDRs described herein.

[0068] The skilled artisan will further appreciate that any intact IgG antibody comprising the above variable regions may be used as a reference antibody to identify antibody polypeptides of the combination therapy of the invention that competitively inhibit the binding of 1630 / 1631 or 2674 / 2675 to CD137. However, preferably, the reference antibody 1630 / 1631 consists of the heavy and light chains defined in SEQ ID NOs: 17 and 18, respectively, and the reference antibody 2674 / 2675 consists of the heavy and light chains defined in SEQ ID NOs: 29 and 30, respectively.

[0069] Competitive binding typically occurs because the test antibody binds to, or at least binds very close to, the epitope of the antigen that binds the reference antibody (in this case 1630 / 1631 or 2674 / 2675). However, one skilled in the art will understand that competitive binding can also occur due to steric hindrance. Thus, the test antibody may bind to an epitope different from that bound by the reference antibody, but may still be of sufficient size or configuration to prevent binding of the reference antibody to the antigen.

[0070] The antibodies and antigen-binding fragments that bind to CD137 and are part of the combination therapy of the present invention were identified after screening of anti-CD137 antibodies based on exhibiting properties that make them particularly suitable as diagnostic and therapeutic agents for cancer.

[0071] Thus, in one embodiment, an antibody or antigen-binding fragment that specifically binds to CD137 exhibits one or more of the following properties: a) the ability to stimulate CD137 and activate T cells and other immune cells via a cross-linking dependent mechanism (e.g., to induce the release of interferon gamma from CD8+ T cells, see Examples); and / or b) Cross-reactivity with cynomolgus CD137 (see Examples).

[0072] For example, an antibody or antigen-binding fragment that specifically binds to CD137 may exhibit both of the above properties. The antibody may be or may comprise a variant or fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that the variant or fragment retains specificity for CD137 and, in some embodiments, retains at least one of the above functional characteristics (a)-(b).

[0073] As mentioned above, antibodies that specifically bind to CD137 and comprise part of the combination therapy of the present invention may have a cross-linking dependent mechanism. A "cross-linking dependent mechanism" includes an Fc cross-linking dependent mechanism, in which an antibody must bind to both CD137 and an Fc receptor to stimulate CD137. Thus, in some embodiments, an antibody must be able to bind to both CD137 and an Fc receptor.

[0074] In one embodiment, an antibody or antigen binding domain that specifically binds to CD137 is capable of binding to an Fc receptor. In one embodiment, an antibody or antigen binding domain is capable of simultaneously binding to CD137 and an Fc receptor. In a preferred embodiment, the ability of an antibody and / or its antigen binding domain to specifically bind to CD137 and activate T cells is dependent on binding to both CD137 and an Fc receptor.

[0075] In a preferred embodiment, the targeted Fc receptor is FcγR. Examples of FcγR include FcγRI, FcγRIIA and FcγRIIB. Thus, in one embodiment, the FcγR can be FcγRIIA. FcγRIIA includes both R131 and H131 allotypes of FcγRIIA. Thus, in one embodiment, the targeted FcγR is the R131 allotype of FcγRIIA.

[0076] In alternative embodiments, antibodies that specifically bind to CD137 may be Fc cross-linking independent, such that they can stimulate CD137 in the absence of binding to Fc receptors.

[0077] Thus, the exemplary antibodies 2674 / 2675 and 1630 / 1631 are FcγR cross-linking dependent agonist antibodies targeting the costimulatory CD137 receptor. Thus, they are only active in tissues or tumors containing cells expressing CD137 and FcγR. "Tumors containing cells expressing CD137 and FcγR" include tumors or tumor-draining lymph nodes containing tumor cells and / or tumor-infiltrating immune cells (such as monocytes, macrophages, dendritic cells, NK cells, T cells, B cells and granulocytes) expressing CD137 and FcγR. It should be understood that CD137 and FcγR can be expressed on separate cells within a tumor and / or can be co-expressed on the same cell. Thus, the reference antibodies 2674 / 2675 and 1630 / 1631 will provide tumor-directed immune activation in indications associated with cells expressing both CD137 and FcγR in the tumor microenvironment. This is in contrast to FcγR-independent CD137 agonists (e.g., urelumab) that can induce systemic immune activation. The tumor-localizing effect of antibodies 2674 / 2675 and 1630 / 1631 is primarily dependent on the number of tumor-infiltrating macrophages / myeloid cells expressing different FcγRs.

[0078] IgG4 is known to bind with high affinity to FcγRI and moderate / low affinity to FcγRIIa and FcγRIIb. FcγRI and FcγRIIa are expressed on monocytes, while FcγRIIb is expressed at high density on B cells. Cross-linking of antibodies 2674 / 2675 and 1630 / 1631 occurs preferentially within tumors and in adjacent draining lymph nodes. In systemic blood, where serum IgG levels are high, the availability of free, unblocked FcγR is thought to be too low for effective cross-linking to occur. Thus, the risk of systemic immune activation is thought to be low, which improves the risk-benefit profile compared to other CD137 mAbs.

[0079] The patient selection and biomarker rationale for treatment with antibodies that specifically bind CD137, such as 2674 / 2675 and 1630 / 1631, and that form part of the combination therapy of the invention may be guided by the tumor type that has infiltrating cells that express CD137 and FcγR. Thus, the combination therapy of the invention may be for use in patients that have been selected on the basis of having a tumor that contains cells that express CD137 and FcγR (i.e., as a companion diagnostic test).

[0080] "Infiltrating cells" include tumor-infiltrating immune cells such as monocytes, macrophages, dendritic cells, NK cells, T cells, B cells, and granulocytes.

[0081] Advantageously, an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, can induce tumor immunity. Tumor immunity can be demonstrated using methods well known in the art, for example, by re-challenging mice cured of a given tumor by CD317 antibody therapy with the same tumor, and / or by re-challenging mice cured of a given tumor by the combination therapy of the present invention with the same tumor. If tumor immunity has been induced by antibody therapy and / or combination therapy, the tumor is rejected upon re-challenge.

[0082] In one embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137, upon binding to a cell expressing CD137, a) Antibody-dependent cellular cytotoxicity (ADCC) b) Antibody-dependent cellular phagocytosis (ADCP) and / or c) are substantially unable to induce complement-dependent cytotoxicity (CDC).

[0083] The antibody may be or may comprise a variant or fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137 and is unable to induce one or more of (a)-(c) upon binding to a cell expressing CD137.

[0084] Methods for determining the level of ADCC-mediated lysis or apoptosis in a cell sample are well known in the art. For example, chromium-51 release assay, europium release assay, or sulfur-35 release assay may be used. In such assays, a previously labeled target cell line expressing an antigen is incubated with the antibody to be tested. After washing, effector cells (typically expressing the Fc receptor CD16) are incubated with the antibody-labeled target cells. Target cell lysis is then measured by the release of intracellular label by scintillation counter or spectrophotometry. As an alternative to the labeling with radioisotopes required in such assays, methods can be used in which lysis is detected by measuring the release of an enzyme naturally present in the target cells. This can be achieved by detection of the product of an enzyme-catalyzed reaction (e.g., bioluminescence detection). In such assays, no previous labeling of the cells is required. A typical cellular enzyme detected in such assays is GAPDH.

[0085] Methods for determining the level of ADCP in a cell sample are well known in the art. For example, cancer cells expressing tumor antigens can be incubated in the presence of titrations of mAb and human leukemia monocytic cell line THP-1. Both effector and target cells can be fluorescently labeled, and cell phagocytosis can be measured by flow cytometry. Microscopy or imaging cytometry can also be used to confirm phagocytosis.

[0086] Methods for determining the level of CDC in a cell sample are well known in the art. For example, serum (typically human serum) containing components of the complement system may be mixed with target cells bound by the antibody to be detected, and cell death may then be determined by a suitable method. Cell death may be determined by preloading the target cells with a radioactive compound. When the cells die, the radioactive compound is released from the cells. Thus, the effectiveness of the antibody in mediating cell death may be determined by the radioactivity level. Non-radioactive CDC assays may also be used, which may use fluorescence or luminescence measurements to determine the release of abundant cellular components such as GAPDH.

[0087] In one embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can bind to an epitope on the extracellular domain of CD137 that at least partially overlaps with an epitope on CD137 to which reference antibodies 1630 / 1631 and / or 2674 / 2675 can bind. Thus, the antibody or antigen-binding fragment may be capable of binding to an epitope located in / within domain 2 of CD137 (i.e., amino acids 66-107 of human CD137).

[0088] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises or consists of an intact antibody (e.g., an IgG1, IgG2, IgG3 or IgG4 antibody). In a preferred embodiment, the antibody is an IgG4 antibody.

[0089] In alternative embodiments, antibodies or antigen-binding fragments thereof that specifically bind to CD137 include Fv fragments (e.g., single chain Fvs and disulfide-linked Fvs), Fab-like fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments), and domain antibodies (e.g., single V H Variable domain or V L In particular, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be an scFv.

[0090] In further embodiments, as discussed above, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises or consists of an antibody mimetic selected from the group comprising or consisting of an affibody, tetranectin (CTLD), adnectin (monobody), anticalin, DARPin (ankyrin), avimer, iMab, microbody, peptide aptamer, Kunitz domain and affilin.

[0091] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CD137 is a) a heavy chain CDR1 sequence having the consensus sequence G, F, T / N, F, G, Y, S, Y; b) a heavy chain CDR2 sequence having the consensus sequences I, G, S, G / T, S, S, Y / H, T, and c) a heavy chain CDR3 sequence having the sequence ARVYSSPGIDY.

[0092] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CD137 is a) a light chain CDR1 sequence having the consensus sequence Q, S, I, S / G, S, Y / T; b) a light chain CDR2 sequence having the consensus sequence A / G, A, S, and c) a light chain CDR3 sequence having the sequence QQYYTWVPFT.

[0093] In a preferred embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 comprises a heavy chain variable region comprising the following CDRs: a) CDRs of GFTFGYSY [SEQ ID NO: 3] or an amino acid sequence containing up to 3 amino acid mutations, e.g. 1, 2 or 3 mutations compared to SEQ ID NO: 3; b) the CDRs of IGSGSSYT [SEQ ID NO: 4] or an amino acid sequence containing up to 3 amino acid mutations, e.g. 1, 2 or 3 mutations, compared to SEQ ID NO: 4; and c) CDR of ARVYSSPGIDY [SEQ ID NO: 5] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 5, for example 1, 2 or 3 mutations.

[0094] Thus, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can comprise a heavy chain variable region that comprises one, two, or all three of the CDRs of SEQ ID NOs:3, 4, and 5.

[0095] For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region having the amino acid sequence of the corresponding region of the 1630 / 1631 reference antibody, i.e., SEQ ID NO:1.

[0096] In an alternative preferred embodiment, the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention comprises a heavy chain variable region comprising the following CDRs: a) CDRs of GFNFGYSY [SEQ ID NO: 21] or an amino acid sequence containing up to 3 amino acid mutations, e.g. 1, 2 or 3 mutations compared to SEQ ID NO: 21; b) the CDRs of IGSTSSHT [SEQ ID NO: 22] or an amino acid sequence containing up to three amino acid mutations, e.g., one, two or three mutations, compared to SEQ ID NO: 22; and c) CDR of ARVYSSPGIDY [SEQ ID NO: 23] or an amino acid sequence containing up to 3 amino acid mutations, such as 1, 2 or 3 mutations compared to SEQ ID NO: 23.

[0097] Thus, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can comprise a heavy chain variable region that comprises one, two, or all three of the CDRs of SEQ ID NOs:21, 22, and 23.

[0098] For example, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may comprise a heavy chain variable region having the amino acid sequence of the corresponding region of the 2674 / 2675 reference antibody, i.e., SEQ ID NO:19.

[0099] However, it should be understood that low levels of variation within the CDR sequences (typically only 1, 2, or 3 amino acids) can be tolerated without loss of specificity of the antibody or antigen-binding fragment for CD137 (1630 / 1631 or 2674 / 2675 for either embodiment).

[0100] For example, in alternative embodiments, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may comprise a heavy chain variable region comprising the CDRs defined above, wherein the H1 and H2 CDRs are mutated versions of SEQ ID NOs: 3 and 4, respectively, and the H3 CDR is SEQ ID NO: 5.

[0101] In further alternative embodiments, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may comprise a heavy chain variable region comprising the CDRs defined above, wherein the H1 and H2 CDRs are mutated versions of SEQ ID NOs: 21 and 22, respectively, and the H3 CDR is SEQ ID NO: 23.

[0102] In some embodiments, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0103] Percent sequence identity can be determined, for example, by the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357, the disclosure of which is incorporated herein by reference) at the Expasy organization site (http: / / www.ch.embnet.org / software / LALIGN_form.html) using the following parameters: global alignment option, scoring matrix BLOSUM62, opening gap penalty -14, extension gap penalty -4. Alternatively, the percent sequence identity between two polypeptides may be determined using a suitable computer program, for example the GAP program of the University of Wisconsin Genetic Computing Group, with the understanding that the percent sequence identity is calculated relative to the polypeptides whose sequences are optimally aligned.

[0104] Alternatively, the alignment can be performed using the Clustal W program (as described in Thompson et al., 1994, Nucl. Acid Res. 22:4673-4680, incorporated herein by reference). The parameters used can be as follows: -Fast pairwise alignment parameters: K-tuple (word) size; 1, window size; 5, gap penalty; 3, number of upper diagonals; 5. Scoring method: x%. -Multiple alignment parameters: Gap opening penalty; 10, Gap extension penalty; 0.05. -Scoring matrix: BLOSUM.

[0105] Alternatively, the BESTFIT program may be used to determine local sequence alignments.

[0106] In a further preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 according to the first aspect of the invention comprises a light chain variable region comprising the following CDRs: a) CDRs of QSISSY [SEQ ID NO: 6] or an amino acid sequence containing up to 3 amino acid mutations, e.g. 1, 2 or 3 mutations compared to SEQ ID NO: 6; b) AAS [SEQ ID NO: 7] or the CDRs of an amino acid sequence containing up to two amino acid mutations, e.g., one or two mutations, compared to SEQ ID NO: 7; and c) QQYYTWVPFT [SEQ ID NO: 8] or a CDR of an amino acid sequence containing up to 3 amino acid mutations, such as 1, 2 or 3 mutations compared to SEQ ID NO: 8.

[0107] Thus, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can comprise a light chain variable region comprising the CDRs of SEQ ID NOs:6, 7 and 8.

[0108] For example, an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, may comprise a light chain variable region having the corresponding region of the 1630 / 1631 reference antibody, i.e., the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0109] In an alternative embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs defined above, wherein the CDRs of L1 and L2 are mutated versions of SEQ ID NOs: 6 and 7, respectively, and the CDR of L3 is SEQ ID NO: 8.

[0110] In a further preferred embodiment, the antibody, or antigen-binding fragment thereof, which specifically binds CD137 and which may comprise part of the combination therapy of the first aspect of the invention comprises a light chain variable region comprising the following CDRs: a) the CDRs of QSIGST [SEQ ID NO: 24] or an amino acid sequence containing up to three amino acid mutations, e.g., one, two or three mutations, compared to SEQ ID NO: 24; b) CDRs of GAS [SEQ ID NO: 25] or an amino acid sequence containing up to two amino acid mutations, e.g., one or two mutations, compared to SEQ ID NO: 25; and c) QQYYTWVPFT [SEQ ID NO: 26], or a CDR of an amino acid sequence containing up to three amino acid mutations, such as one, two or three mutations, compared to SEQ ID NO: 26.

[0111] Thus, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can comprise a light chain variable region comprising the CDRs of SEQ ID NOs:24, 25 and 26.

[0112] For example, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may comprise a light chain variable region having the amino acid sequence of the corresponding region of the 2674 / 2675 reference antibody, i.e., SEQ ID NO:20.

[0113] In an alternative embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs defined above, wherein the CDRs of L1 and L2 are mutated versions of SEQ ID NOs: 24 and 25, respectively, and the CDR of L3 is SEQ ID NO: 26.

[0114] The anti-CD137 antibodies used in the combination therapies and methods of the invention may be antibodies that comprise one, two, or all three of the CDR sequences of SEQ ID NOs: 3-5, and / or one, two, or all three of the CDR sequences of SEQ ID NOs: 6-8. The antibodies may comprise all six CDR sequences of SEQ ID NOs: 3-8.

[0115] The antibody may comprise or consist of the light chain variable region sequence of SEQ ID NO:2 and / or the heavy chain variable region sequence of SEQ ID NO:1, or an amino acid sequence having at least 60% sequence identity thereto, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 and / or SEQ ID NO:2.

[0116] The antibody may be an antibody that comprises, or may bind to the same epitope as, the light chain variable region sequence of SEQ ID NO: 2 and the heavy chain variable region sequence of SEQ ID NO: 1. In addition, the antibody may comprise the light chain constant region sequence of SEQ ID NO: 16 and / or the heavy chain constant region sequence of SEQ ID NO: 13, or an amino acid sequence having at least 60% sequence identity thereto, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 and / or SEQ ID NO: 13.

[0117] The anti-CD137 antibodies used in the combination therapies and methods of the invention may be antibodies that comprise one, two, or all three of the CDR sequences of SEQ ID NOs: 21-23, and / or one, two, or all three of the CDR sequences of SEQ ID NOs: 24-26. The antibodies may comprise all six CDR sequences of SEQ ID NOs: 21-26.

[0118] The antibody may comprise a light chain variable region sequence of SEQ ID NO:20 and / or a heavy chain variable region sequence of SEQ ID NO:19, or an amino acid sequence having at least 60% sequence identity thereto, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs:20 and / or 19.

[0119] The antibody may be an antibody comprising the light chain variable region sequence of SEQ ID NO: 20 and the heavy chain variable region sequence of SEQ ID NO: 19, or may bind to the same epitope as thereof. In addition, the antibody may comprise the light chain constant region sequence of SEQ ID NO: 16 and / or the heavy chain constant region sequence of SEQ ID NO: 13, or an amino acid sequence having at least 60% sequence identity thereto, e.g., at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 16 and / or 13.

[0120] It will be understood by those skilled in the art that for human therapy, human or humanized antibodies are preferably used. Humanized forms of non-human (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments, preferably with minimal portions derived from the non-human antibody. Humanized antibodies include antibodies in which the complementarity determining regions of a human antibody (the recipient antibody) are replaced by residues from the complementarity determining regions of a non-human species (the donor antibody) such as mouse, rat, or rabbit that have the desired function. In some cases, Fv framework residues of the human antibody are replaced with corresponding non-human residues. Humanized antibodies may also contain residues that are not present in the recipient antibody or in the introduced complementarity determining regions or framework sequences. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the complementarity determining regions corresponding to those of a non-human antibody and all or substantially all of the framework regions corresponding to those of the relevant human consensus sequence. A humanized antibody optimally will comprise at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, e.g., Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596, the disclosures of which are incorporated herein by reference).

[0121] Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues, often referred to as introduced residues, are usually taken from the imported variable domain. Humanization can be performed essentially as described by replacing human complementarity determining regions with corresponding rodent complementarity determining regions (see, for example, Jones et al., 1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-15361; US ​​4,816,567, the disclosures of which are incorporated herein by reference). Thus, such humanized antibodies are chimeric antibodies, in which substantially less than an intact human variable domain is replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are considered to be typical human antibodies in which some complementarity determining region residues and possibly framework residues have been replaced by residues from analogous sites in rodent antibodies. Chimeric antibodies are discussed by Neuberger et al. th International Biotechnology Symposium Part 2,792-799).

[0122] Human antibodies can also be identified using a variety of techniques known in the art, including phage display libraries (see, e.g., Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol. 147:86-95, the disclosures of which are incorporated herein by reference).

[0123] One of skill in the art will appreciate that a humanized antibody, or antigen-binding fragment, that specifically binds to CD137 may further comprise a heavy chain constant region or a portion thereof (see below).

[0124] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises the CH1, CH2, and / or CH3 regions of an IgG heavy chain (such as an IgG1, IgG2, IgG3, or IgG4 heavy chain). Thus, an antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise part or all of the constant region from an IgG4 heavy chain. For example, an antibody or antigen-binding fragment thereof that specifically binds to CD137 may be a Fab fragment that is comprised of the CH1 and CL constant regions in combination with any of the heavy and light variable regions, respectively, as defined above.

[0125] Similarly, an antibody or antigen-binding fragment that specifically binds to CD137 as defined above may further comprise a light chain constant region or a part thereof (see below). For example, the antibody polypeptide may comprise a CL region from a kappa or lambda light chain.

[0126] In one embodiment, the antibody or antigen-binding fragment that specifically binds to CD137 and is included in the combination therapy of the present invention comprises an antibody Fc region. Those skilled in the art will understand that the Fc portion can be derived from an IgG antibody, or from an antibody of a different class (such as IgM, IgA, IgD or IgE). In one embodiment, the Fc region is derived from an IgG1, IgG2, IgG3 or IgG4 antibody. However, advantageously, the Fc region is derived from an IgG4 antibody.

[0127] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or artificial (e.g., containing one or more point mutations relative to a naturally occurring Fc region). A variant Fc region typically binds with altered affinity to an Fc receptor, such as FcγR and / or the neonatal Fc receptor (FcRn), and improves the function and / or half-life of the polypeptide. The biological function and / or half-life may be either extended or decreased compared to the half-life of a polypeptide comprising a native Fc region. Examples of such biological functions that may be modulated by the presence of a variant Fc region include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or apoptosis.

[0128] Thus, the Fc region can be naturally occurring (e.g., part of an endogenously produced human antibody) or can be artificial (e.g., contains one or more point mutations relative to a naturally occurring human Fc region).

[0129] As is well known in the art, the Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0130] The Fc region of therapeutic monoclonal antibodies and Fc fusion proteins can be engineered to generate molecules with the desired pharmacological activity (Strohl, 2009, Curr Opin Biotechnol 20(6):685-91, the disclosure of which is incorporated herein by reference).

[0131] (a) Engineered Fc region to extend half-life One approach to improving the efficacy of antibody drugs is to increase the serum persistence of the antibody, allowing for higher circulating levels and less frequent and less expensive administration.

[0132] The half-life of IgG depends on its pH-dependent binding to the neonatal receptor FcRn, which is expressed on the surface of endothelial cells and binds IgG in a pH-dependent manner, protecting it from degradation.

[0133] Some antibodies that selectively bind to FcRn at pH 6.0 but not at pH 7.4 exhibit extended half-lives in various animal models.

[0134] Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L (Hinton et al., 2004, J Biol Chem. 279(8):6213-6, the disclosure of which is incorporated herein by reference) and M252Y / S254T / T256E+H433K / N434F (Vaccaro et al., 2005, Nat. Biotechnol. 23(10):1283-8, the disclosure of which is incorporated herein by reference), have been shown to increase the binding affinity to FcRn and the in vivo half-life of IgG1.

[0135] (b) Engineered Fc regions to alter effector function Depending on the application of an antibody drug or Fc fusion protein, it may be desirable to decrease or increase the effector function (such as ADCC).

[0136] For antibodies that target cell surface molecules, particularly those on immune cells, deprivation of effector function may be required for certain clinical applications.

[0137] The four human IgG isotypes bind activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the first component of complement (C1q) with different affinities, resulting in very different effector functions (Bruhns et al., 2009, Blood. 113(16):3716-25, the disclosure of which is incorporated herein by reference).

[0138] FcγRI binding affinity of IgG4 versus IgG2 Bruhns et al. conducted a series of experiments evaluating the specificity and affinity of known human FcγRs and their polymorphic variants for different human IgG subclasses (Bruhns et al., 2009, Blood. 113(16):3716-25, the disclosure of which is incorporated herein by reference). In this study, it was clearly demonstrated that IgG2 had no detectable affinity for FcγRI, whereas IgG1, IgG3 and IgG4 all exhibited nanomolar range binding affinity for FcγRI (Bruhns et al., 2009, Blood. 113(16):3716-25, Lu et al., 2015, Proc Natl Acad Sci US A. 112(3):833-8, the disclosure of which is incorporated herein by reference). An overview of the relative binding affinities between the major human FcγRs and their variants and IgG isotypes is summarized in Table 1. (Stewart et al. 2014, J Immunother. 2(29), the disclosure of which is incorporated herein by reference. [Table 1]

[0139] However, cell activation affects the affinity of FcγRI for IgG immune complexes, and the data generated by surface plasmon resonance in the Bruhns paper may not accurately reproduce what occurs at sites of inflammation. A review by Hogarth et al. (Hogarth et al. 2012, Nat Rev Drug Discov 11(4):311-31, the disclosure of which is incorporated herein by reference) provides an overview of this work and others that have focused on FcγR binding to IgG.

[0140] FcγRI expression in myeloid cell subsets Human FcγRs are expressed primarily by cells of the myeloid lineage, as demonstrated in numerous studies of circulating myeloid cell subsets. + CD16- Classical monocytes, identified as FcγRII (CD32), display high levels of FcγRI, intermediate levels of FcγRIII, and low levels of FcγRIII (CD16) (Almeida et al. 2001, 100(3):325-38; Cheeseman et al. 2016, PLoS One 11(5):e0154656, the disclosures of which are incorporated herein by reference). However, CD14 - CD16 + Non-classical monocytes of the human genome display high levels of FcγRIII, intermediate levels of FcγRII, and low levels of FcγRI (Almeida et al. 2001). A summary and compilation of several published microarray data sets showing the expression of human FcγR genes in different myeloid cell subsets supports these observations (Guilliams et al. 2014, Nat Rev Immunol. 14(2):94-108, the disclosures of which are incorporated herein by reference).

[0141] Once inside the tissue, monocytes differentiate into macrophages and, depending on environmental factors, these macrophages acquire a specific phenotype. In a study by Roussel et al. (Roussel et al. 2017, J Leukoc Biol. 102(2):437-447, the disclosure of which is incorporated herein by reference), peripheral blood monocytes were polarized into different macrophage lineages by using various inflammatory stimuli and the expression profile of these cells was evaluated. In this case, monocytes stimulated with IFN-γ specifically resulted in highly elevated expression of CD64. Circulating CD14 + Similar observations were made in SLE patients where increased CD64 expression was detected on monocytes, which correlated with the expression of interferon-stimulated genes (Li et al. 2010, Arthritis Res Ther 12(3):R90, the disclosure of which is incorporated herein by reference).

[0142] Myeloid cell infiltration in various human tumors Various myeloid cell subsets, such as inflammatory monocytes, monocytic myeloid-derived suppressor cells (MDSCs) and macrophages, have been shown in numerous studies to accumulate in cancer patients (Solito et al. 2014, Ann NY Acad Sci 1319:47-65., Hu et al. 2016, Clin Transl Oncol. 18(3):251-8, the disclosure of which is incorporated herein by reference). Recent attempts have aimed to propose strategies to standardize the characterization of these cells (Bronte et al. 2016, Nat Commun. 7:12150, the disclosure of which is incorporated herein by reference), but the phenotypic definition of many of these cell populations can still be found in the literature (Elliott et al. 2017, Front Immunol. 8:86, the disclosure of which is incorporated herein by reference). Most commonly, these cells are defined by the expression of markers CD11b, CD14, CD33, and low expression of HLA-DR (monocytic MDSCs) (Bronte et al. 2016). In addition, tumor-associated macrophages (TAMs) are commonly identified by the expression of CD64 and CD68 (M1 polarizing, anti-tumorigenic), or CD163 and CD206 (M2 polarizing, anti-tumorigenic) (Elliott et al. 2017).

[0143] A recent review by Elliott et al. summarizes the numerous phenotypes used to identify myeloid cell subsets in cancer patients. Most of these studies have focused their analysis on circulating cells, with myeloid CD11b + Increased frequencies of CD14 cells have been observed in the blood of patients with, for example, bladder, breast, colorectal, hepatocellular, pancreatic, prostate and renal cell cancer (Solito et al. 2014, Elliott et al. 2017). Other studies have also attempted to characterize the level of infiltration of these cells into tumor tissue. In colorectal tumors, high frequencies of CD14 + CD169 +These cells also expressed CD163 and CD206, suggesting that they were M2-polarized TAMs (Li et al. 2015, PLoS One 10(10):e0141817, the disclosure of which is incorporated herein by reference). Another study in colorectal cancer patients also found increased numbers of CD11b TAMs compared to healthy individuals. + CD33 + HLA-DR - Cells were detected (Zhang et al. 2013, PLoS One 8(2):e57114, the disclosure of which is incorporated herein by reference).

[0144] Similarly, CD11b + Myeloid cells have also been identified in bladder tumors, where they accounted for 10-20% of all nucleated cells (Eruslanov et al. 2012, Int J Cancer 130(5):1109-19, the disclosure of which is incorporated herein by reference). Furthermore, high frequencies of CD11b + Cells were observed in pancreatic cancer and expressed CD45 + More than 60% of cells are CD11b + CD15 + CD33 + (Porembka et al. 2012, Cancer Immunol Immunother 61(9):1373-85, the disclosure of which is incorporated herein by reference). Also, one study showed that the predominant myeloid cell population in non-small cell lung cancer was CD11b + CD15 + CD66b + They concluded that these cells are a neutrophil-like population. Interestingly, once these cells migrate from the blood to tumor tissue, they show an altered expression profile, including upregulated FcγRI (Eruslanov et al. 2014, J Clin Invest. 124(12):5466-80, the disclosure of which is incorporated herein by reference).

[0145] Expression of FcγRI in tumor-infiltrating cells Although numerous studies have confirmed the high infiltration of myeloid cells in human tumors, no studies have explored in detail the expression of FcγR on these cells. However, several publications have demonstrated the presence of FcγRI-expressing cells in tumor tissues.

[0146] A study by Morimura et al. (Morimura et al. 1990, Acta Neuropathol. 80(3):287-94, the disclosure of which is incorporated herein by reference) evaluated gliomas from 12 human samples by immunocytochemistry and compared these to peritumoral control tissue. The study demonstrated a high presence of macrophages (using markers CD163, RM3 / 1) in gliomas compared to peritumoral tissue, as well as an increase in FcγRI and FcγRII (CD32). A more recent study by Griesinger et al. (Griesinger et al. 2013, J Immunol. 191(9):4880-8, the disclosure of which is incorporated herein by reference) confirmed these observations by performing a flow cytometric analysis of various pediatric brain tumor types, where a high frequency of CD45 + CD11b + Myeloid cells were observed in tissues from patients with pilocytic astrocytoma and ependymoma, and these cells also expressed high levels of FcγRI.

[0147] In addition to brain tumors, FcγRI expression has also been shown for other types of tumors. Grugan et al. (Grugan et al. 2012, J Immunol. 189(11):5457-66, the disclosure of which is incorporated herein by reference) reported that CD11b expression in human breast tumor tissue was significantly increased in human breast tumor tissue. + CD14 + We demonstrated the presence of CD45 cells, which express high levels of FcγRI and FcγRIIa, as well as FcγRIIb and FcγRIII. In gastrointestinal stromal tumors that display FcγRI expression, + CD11b + CD14 + CD68 +TAMs have been identified (Cavnar et al. 2013, J Exp Med. 210(13):2873-86, the disclosure of which is incorporated herein by reference). CD45 + CD11b + FcγRI + FcγRI cells have also been identified in colorectal cancer patients, and these cells showed higher expression of FcγRI in tumor tissue compared to healthy control tissue (Norton et al. 2016, Clin Transl Immunology. 5(4):e76, the disclosure of which is incorporated herein by reference). FcγRI expression has also been demonstrated for melanoma metastases (Hansen et al. 2006, Acta Oncol 45(4):400-5, the disclosure of which is incorporated herein by reference).

[0148] Binding of IgG to FcγRs or C1q depends on residues located in the hinge region and CH2 domain. Two regions of the CH2 domain are important for FcγR and C1q binding and have unique sequences in IgG2 and IgG4. Substitution of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 with human IgG1 has been shown to greatly reduce ADCC and CDC (Armour et al., 1999, Eur J Immunol. 29(8):2613-24; Shields et al., 2001, J Biol Chem. 276(9):6591-604, the disclosures of which are incorporated herein by reference). Furthermore, Idusogie et al. demonstrated that alanine substitutions at different positions, including K322, significantly reduced complement activation (Idusogie et al., 2000, J Immunol. 164(8):4178-84, the disclosure of which is incorporated herein by reference). Similarly, mutations in the CH2 domain of mouse IgG2A have been shown to reduce binding to FcγRI and C1q (Steurer et al., 1995. J Immunol. 155(3):1165-74, the disclosure of which is incorporated herein by reference).

[0149] A number of mutations have been made in the CH2 domain of human IgG1 and their effects on ADCC and CDC have been tested in vitro (see references cited above). In particular, an alanine substitution at position 333 has been reported to increase both ADCC and CDC (Shields et al., 2001, supra; Steurer et al., 1995, supra). Lazar et al. described a triple mutant (S239D / I332E / A330L) with higher affinity for FcγRIIIa and lower affinity for FcγRIIb, resulting in enhanced ADCC (Lazar et al., 2006, PNAS 103(11):4005-4010, the disclosure of which is incorporated herein by reference). The same mutations were used to generate antibodies with increased ADCC (see Ryan et al., 2007, Mol. Cancer Ther. 6:3009-3018, the disclosure of which is incorporated herein by reference). Richards et al. investigated a slightly different triple mutant (S239D / I332E / G236A) with improved FcγRIIIa affinity and FcγRIIa / FcγRIIb ratio that mediated enhanced phagocytosis of target cells by macrophages (Richards et al. al., 2008. Mol Cancer Ther. 7(8):2517-27, the disclosure of which is incorporated herein by reference.

[0150] IgG4 antibodies, due to their lack of effector functions, represent the preferred IgG subclass for receptor modulation without cell depletion. IgG4 molecules can exchange half molecules in a dynamic process called Fab-arm exchange. This phenomenon can also occur in vivo between therapeutic antibodies and endogenous IgG4.

[0151] The S228P mutation has been shown to disrupt this recombination process, allowing the design of less unpredictable therapeutic IgG4 antibodies (Labrijn et al., 2009, Nat Biotechnol. 27(8):767-71, the disclosure of which is incorporated herein by reference).

[0152] In further embodiments, the effector function of the Fc region may be altered by modification of carbohydrate moieties within the CH2 domain therein, for example by altering the relative levels of fucose, galactose, bisecting N-acetylglucosamine and / or sialic acid during production (see Jefferis, 2009, Nat Rev Drug Discov. 8(3):226-34 and Raju, 2008, Curr Opin Immunol., 20(4):471-8, the disclosures of which are incorporated herein by reference).

[0153] Thus, it is known that therapeutic antibodies lacking or having few fucose residues in the Fc region can exhibit enhanced ADCC activity in humans (see, e.g., Peipp et al., 2008, Blood 112(6):2390-9; Yamane-Ohnuki & Satoh, 2009, MAbs 1(3):230-26; Iida et al., 2009, BMC Cancer 9;58, the disclosures of which are incorporated herein by reference). Low-fucose antibody polypeptides can be produced by expression in cells cultured in medium containing an inhibitor of mannosidase, such as kinfunesin (see Example I below).

[0154] Other methods for altering the glycosylation of antibodies to the low-fucose form include the use of the bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase in cells unable to metabolize rhamnose (e.g., using GlymaxX® technology from ProBioGen AG, Berlin, Germany).

[0155] Another method for producing low-fucose antibodies is by inhibition or depletion of alpha-(1,6)-fucosyltransferase in antibody-producing cells (eg, using Potelligent® CHOK1SV technology from Lonza Ltd, Basel, Switzerland).

[0156] An exemplary heavy chain constant region amino acid sequence that may be combined with any VH region sequence disclosed herein (to form a complete heavy chain) is the IgG1 heavy chain constant region sequence reproduced below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 12]

[0157] Other heavy chain constant region sequences are known in the art and can also be combined with any of the VH regions disclosed herein. For example, as mentioned above, a preferred constant region is a modified IgG4 constant region such as that reproduced herein. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 13]

[0158] This modified IgG4 sequence results in stabilization of the IgG4 core hinge, making it more stable and preventing Fab arm exchange.

[0159] Another preferred constant region is a modified IgG4 constant region, such as that reproduced below. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSLGK [SEQ ID NO: 14]

[0160] This modified IgG4 sequence exhibits reduced FcRn binding and therefore reduced serum half-life compared to wild-type IgG4. In addition, it exhibits stabilization of the IgG4 core hinge, making it more stable and preventing Fab arm exchange.

[0161] Wild-type IgG4 constant regions, such as those reproduced herein, are also suitable for use in the polypeptides of the invention. ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 15]

[0162] An exemplary light chain constant region amino acid sequence that can be combined with any VL region sequence disclosed herein (to form a complete light chain) is the kappa chain constant region sequence reproduced below. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 16]

[0163] Other light chain constant region sequences are known in the art and can also be combined with any of the VL regions disclosed herein.

[0164] In an exemplary embodiment of the invention, an antibody or antigen-binding fragment that specifically binds to CD137 may comprise the IgG4 constant regions of SEQ ID NOs: 13 and 16, respectively.

[0165] Thus, an exemplary antibody, or antigen-binding fragment thereof, that specifically binds to CD137 is (a) a heavy chain comprising a variable region of SEQ ID NO: 1 together with a constant region of SEQ ID NO: 13; (b) a light chain comprising the variable region of SEQ ID NO:2 together with the constant region of SEQ ID NO:16.

[0166] For example, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can be an intact IgG4 molecule comprising, or consisting of, two heavy chains having the amino acid sequence of SEQ ID NO:17 and two light chains having the amino acid sequence of SEQ ID NO:18.

[0167] Alternative exemplary antibodies or antigen-binding fragments that specifically bind to CD137 include: (a) a heavy chain comprising a variable region of SEQ ID NO: 19 together with a constant region of SEQ ID NO: 13; (b) a light chain comprising a variable region of SEQ ID NO:20 together with a constant region of SEQ ID NO:16.

[0168] For example, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 can be an intact IgG4 molecule comprising, or consisting of, two heavy chains having the amino acid sequence of SEQ ID NO:29 and two light chains having the amino acid sequence of SEQ ID NO:30.

[0169] In one embodiment, an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 is or comprises a "fusion" polypeptide.

[0170] It should be understood that in addition to being fused to a moiety to improve pharmacokinetic properties, the antibody or antigen-binding fragment thereof that specifically binds to CD137 forming part of the combination therapy of the present invention may also be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A to facilitate purification of the polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be fused to an oligo-histidine tag such as His6, or an epitope recognized by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of the above-mentioned antibodies, or antigen-binding fragments thereof, are also within the scope of the present invention. It should be understood that fusions (or variants, derivatives or fusions thereof) that retain or improve desirable properties such as IL-1R binding properties or in vivo half-life are preferred.

[0171] Thus, the fusion may comprise an amino acid sequence as detailed above, together with a further moiety that confers desirable characteristics to the aforementioned polypeptides included in the combination therapy of the present invention. For example, the moiety may be useful in detecting or isolating the polypeptide, or in promoting cellular uptake of the polypeptide. The moiety may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, such as a small fluorophore or a green fluorescent protein (GFP) fluorophore, as is well known to those skilled in the art. The moiety may be, for example, an immunogenic tag, such as a Myc tag, as is well known to those skilled in the art, or a lipophilic molecule or polypeptide domain that can promote cellular uptake of the polypeptide, as is well known to those skilled in the art.

[0172] Those skilled in the art will understand that the antibodies, or antigen-binding fragments thereof, that specifically bind to CD137 included in the combination therapy of the present invention may comprise or consist of one or more amino acids that have been modified or derivatized.

[0173] Chemical derivatives of one or more amino acids can be achieved by reaction with a functional side chain. Such derivatized molecules include, for example, molecules in which free amino groups are derivatized to form amine hydrochloride, p-toluenesulfonyl, carboxybenzoxy, t-butyloxycarbonyl, chloroacetyl, or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides that contain naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be replaced by proline, 5-hydroxylysine can be replaced by lysine, 3-methylhistidine can be replaced by histidine, homoserine can be replaced by serine, and ornithine can be replaced by lysine. Derivatives also include peptides that contain one or more additions or deletions, so long as the required activity is maintained. Other modifications included are terminal modifications such as amidation, amino terminal acylation (eg, acetylation or thioglycolic acid amidation), terminal carboxylamidation (eg, with ammonia or methylamine).

[0174] It will further be appreciated by those skilled in the art that peptidomimetic compounds may also be useful. The term "peptidomimetic" refers to a compound that mimics the conformation and desirable characteristics of a particular peptide as a therapeutic agent.

[0175] For example, an antibody or antigen-binding fragment thereof that specifically binds to CD137 may include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages, but also molecules in which peptide bonds are reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, such as those described in Meziere et al. (1997) J.Immunol.159,3230-3237, which is incorporated herein by reference. This approach involves making pseudopeptides that contain changes involving the backbone rather than the orientation of the side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptide may be a peptidomimetic compound in which one or more amino acid residues are linked by -y(CH2NH)- bonds instead of traditional amide bonds.

[0176] In a further alternative, the peptide bonds may be fully distributed, provided that appropriate linker moieties are used which preserve the spacing between the carbon atoms of the amino acid residues, and which may advantageously have substantially the same charge distribution and substantially the same planarity as the peptide bonds.

[0177] It will also be appreciated that the above-mentioned antibodies, or antigen-binding fragments thereof, may be conveniently blocked at their N-terminus or C-terminus to help reduce susceptibility to exoproteolytic digestion.

[0178] Various uncoded or modified amino acids, such as D-amino acids and N-methyl amino acids, have also been used to modify mammalian peptides. In addition, the predicted bioactive conformation may be stabilized by covalent modifications such as cyclization or by the incorporation of lactam or other types of bridges, see, e.g., Veber et al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem. Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

[0179] Typically, an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, is a "naked" antibody polypeptide, i.e., does not include any additional functional moieties, such as a cytotoxic moiety or a detectable moiety. For example, if the therapeutic effect is mediated by a direct action included in the combination therapy of the invention on immune cells, e.g., to reduce inflammation, it may be advantageous for the antibody to lack any cytotoxic activity.

[0180] However, in alternative embodiments, antibodies that specifically bind to CD137, or antigen-binding fragments thereof, may be enhanced with functional moieties, for example to facilitate their intended use as diagnostic (e.g., in vivo imaging) agents or therapeutic agents. Thus, in one embodiment, antibodies that specifically bind to CD137, or antigen-binding fragments thereof, are directly or indirectly linked to a therapeutic moiety. Suitable therapeutic moieties are those that are capable of reducing or inhibiting the growth of, or in particular killing, cancer cells (or associated stem or progenitor cells). For example, the therapeutic agent may be a radioisotope (e.g., 90 Y, 177 Lu, 99 Tc m etc.) or a cytotoxic moiety such as a cytotoxic drug (eg, antimetabolite, toxin, cytostatic agent).

[0181] Alternatively, the cytotoxic moiety may comprise or consist of one or more moieties suitable for use in activation therapy, such as photon activation therapy, neutron activation therapy, neutron induced Auger electron therapy, synchrotron radiation therapy, or low energy X-ray photon activation therapy.

[0182] Optionally, the antibody, or antigen-binding fragment thereof, that specifically binds to CD137 may further comprise a detectable moiety. For example, the detectable moiety is 99m Tc, 111 In, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I and 201 Optionally, the agent may comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of: Tl. 86 Y / 90 Y or 124 I / 211 Alternatively, an antibody or antigen-binding fragment thereof that specifically binds to CD137 may contain a radioisotope capable of acting simultaneously in a multimodal manner as a detectable moiety and as a cytotoxic moiety to provide so-called "multimodality theragnostics." Thus, the binding moieties may be attached to nanoparticles that have multi-imaging (e.g., SPECT, PET, MRI, optical, or ultrasound) capabilities along with therapeutic capabilities with a cytotoxic drug such as a radionuclide or chemotherapeutic drug.

[0183] The therapeutic and / or detectable moieties (e.g., radioisotopes, cytotoxic moieties) may be directly or indirectly linked to the antibody or fragment thereof. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succimidyl benzoate; dodecaborate), chelating moieties of both macrocycles and acyclic chelators, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid, and the like. derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), derivatives of 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanato-benzyl)-3,6,9-triacetic acid (PCTA), derivatives of 5-S-(4-aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(acetic acid) (DO3A), and other chelating moieties.

[0184] One preferred linker is, for example, 177 Lu-DTPA-DTPA, as used in [antibody polypeptide]. Further preferred linkers are, for example: 89 Zr-DFO - Deferoxamine, DFO, used in [antibody polypeptide].

[0185] However, one of skill in the art will appreciate that many medical applications of the combination therapies of the invention comprising an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 do not require the presence of a cytotoxic or diagnostic moiety.

[0186] As discussed above, methods for the production of the antibody polypeptides included in the combination therapy of the present invention are well known in the art.

[0187] Advantageously, the antibody or antigen-binding fragment thereof that specifically binds to CD137 is or comprises a recombinant polypeptide. Suitable methods for the production of such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic host cells (see, for example, Green & Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor, New York, the relevant disclosures of which are incorporated herein by reference).

[0188] An antibody that specifically binds to CD137 may be a polyclonal antibody, but it is preferred that the antibody is a monoclonal antibody, or that an antigen-binding fragment, variant, fusion or derivative thereof is derived from a monoclonal antibody.

[0189] Suitable monoclonal antibodies may be prepared by known techniques, such as those disclosed in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Hurrell (CRC Press, 1982). Polyclonal antibodies may be produced that are either multispecific or monospecific. It is preferred that they are monospecific.

[0190] The antibody polypeptides included in the combination therapy of the present invention can also be produced using commercially available in vitro translation systems such as rabbit reticulocyte lysate or wheat germ lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system can be coupled to a transcription system such as the TNT transcription translation system (Promega). This system has the advantage of producing suitable mRNA transcripts from the encoding DNA polynucleotide in the same reaction as translation.

[0191] Those of skill in the art will appreciate that antibodies, or antigen-binding fragments thereof, that specifically bind to CD137 can alternatively be artificially synthesized, for example, using well-known liquid or solid phase synthesis techniques (e.g., t-Boc or Fmoc solid phase peptide synthesis).

[0192] (b) Further immunotherapeutic agents (PD-1 inhibitors) The combination therapy of the present invention includes a further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor. The PD-1 inhibitor may be effective in the treatment of cancer and / or may specifically bind to PD-1 or PD-L1. It will be appreciated that the therapeutic benefit of the further immunotherapeutic agent may be mediated by attenuating the function of the inhibitory immune checkpoint molecule PD-1.

[0193] Thus, in one embodiment of the present invention, PD-1 inhibitors are effective immunotherapeutic agents for the treatment of cancer.

[0194] The term "immunotherapeutic agent" is intended to include any molecule, peptide, antibody, or other agent capable of stimulating the host immune system to produce an immune response against a tumor or cancer in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein. In one embodiment, the immunotherapeutic agent is an antibody, such as an anti-PD-1 antibody capable of specifically binding to PD-1 or an anti-PD-L1 antibody capable of specifically binding to PD-L1, or an antigen-binding fragment thereof.

[0195] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxicity. In addition, the term immune response includes immune responses that are indirectly mediated by T cell activation, e.g., antibody production (humoral response), and activation of cytokine-responsive cells, e.g., macrophages.

[0196] Immune checkpoint molecules include proteins on the cell surface of immune cells such as CD4+ and / or CD8+ T cells, dendritic cells, NK cells, and macrophages, as well as proteins on certain tumor cells that regulate the immune response. Those skilled in the art will appreciate that PD-1 is an inhibitory immune checkpoint molecule.

[0197] Blocking or neutralizing one or more immune checkpoint molecules, such as PD-1, can block or otherwise neutralize inhibitory signaling, thereby upregulating immune responses to more effectively treat cancer. Exemplary agents useful for blocking inhibitory immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, or fragments thereof, that can bind to and / or either inactivate or inhibit inhibitory immune checkpoint proteins; and RNA interference, antisense, nucleic acid aptamers, etc., or fragments thereof, that can downregulate the expression and / or activity of inhibitory immune checkpoint nucleic acids. Exemplary agents for upregulating immune responses include antibodies against one or more inhibitory immune checkpoint proteins that block the interaction between the protein and its native receptor(s), inactivated forms of one or more immune checkpoint inhibitor proteins (e.g., dominant negative polypeptides), small molecules or peptides that block the interaction between one or more inhibitory immune checkpoint proteins and their native receptor(s), fusion proteins that bind to their native receptor(s) (e.g., the extracellular portion of an immune checkpoint inhibitor protein fused to the Fc portion of an antibody or immunoglobulin), nucleic acid molecules that block inhibitory immune checkpoint nucleic acid transcription or translation, and the like. Such agents can directly block the interaction between one or more inhibitory immune checkpoints and their native receptor(s) (e.g., antibodies), blocking inhibitory signaling and upregulating the immune response. Alternatively, agents can indirectly block the interaction between one or more inhibitory immune checkpoint proteins and their native receptor(s), blocking inhibitory signaling and upregulating the immune response. For example, soluble aspects of immune checkpoint protein ligands, such as stabilized extracellular domains, can bind to their receptors and indirectly reduce the effective concentration of the receptor for binding to the appropriate ligand. In one embodiment, anti-PD-1 and / or anti-PD-L1 antibodies, alone or in combination, are used to inhibit immune checkpoints.

[0198] Thus, in one embodiment, the additional immunotherapeutic agent is a PD-1 inhibitor, which binds to and inhibits the function of an inhibitory immune checkpoint molecule.

[0199] A "PD-1 inhibitor" (or a "PD-1 pathway inhibitor") includes an entity capable of inhibiting the PD-1 pathway.

[0200] PD-1 functions as a negative regulator of T cell activation when it engages with its ligands PD-L1 or PD-L2. PD-L1 is expressed by many solid tumors, including melanomas in particular. Thus, these tumors can downregulate immune-mediated antitumor effects through activation of the inhibitory PD-1 receptor on T cells. By blocking the interaction between PD-1 and PD-L1, a checkpoint in the immune response can be removed, leading to enhanced antitumor T cell responses. This interaction can be blocked by antibodies specific for PD-1 or PD-L1 or any other suitable agent. Such antibodies and agents can be generally referred to as PD-1 inhibitors. The additional immunotherapeutic agent in step (b) of the method of the present invention is a PD-1 inhibitor.

[0201] Thus, PD-1 inhibitors block the interaction between PD-1 (programmed cell death protein 1) and its ligand PD-L1 (programmed death-ligand 1). Such PD-1 inhibitors can therefore act on either or both PD-L1 and PD-1. Thus, the term PD-1 inhibitor includes both PD-1 and PD-L1 inhibitors. PD-1 inhibitors block the activity of PD-1 and PD-L1 immune checkpoint proteins.

[0202] "PD-1" specifically includes the human PD-1 protein, e.g., as set forth in GenBank Accession No. NP005009.2, the sequence of which is set forth in SEQ ID NO: 35 below. PD-1 is also known in the scientific literature as PD1, CD279, PDCD1, and SLEB2. [ka] [SEQ ID NO:35]

[0203] "PD-L1" specifically includes the human PD-L1 protein, for example as set forth in GenBank Accession No. AAI13735.1, the sequence of which is set forth in SEQ ID NO:36 below. PD-L1 is also known in the scientific literature as CD274, B7-H1, B7-H, PDCD1L1 and PDCD1LG1. [ka] [SEQ ID NO:36]

[0204] Thus, the combination therapy of the present invention includes a PD-1 inhibitor that specifically binds to PD-1 or PD-L1, i.e., has specificity for PD-1 or PD-L1. "Specificity" means that the inhibitor is capable of binding to PD-1 or PD-L1 in vivo, i.e., under physiological conditions in which PD-1 or PD-L1 are present in the human body. Preferably, the PD-1 inhibitor does not bind to any other proteins (other than PD-1 or PD-L1) in vivo. Such binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blot, flow cytometry, etc., using transfected cells expressing PD-1 or PD-L1.

[0205] The PD-1 inhibitor that specifically binds to PD-1 or PD-L1 is preferably at least 10×10 -9 Less than M or 7 x 10 -9 M or less, more preferably 4 or 2×10 -9 M, most preferably less than 1.2 × 10 -9The PD-1 inhibitor binds to human PD-1 or PD-L1 with a Kd value that is less than M. Advantageously, the PD-1 inhibitor can selectively bind to PD-1 or PD-L1, i.e., binds at least 10 times more strongly to PD-1 or PD-L1 than to any other protein. The PD-1 inhibitor preferably binds specifically to PD-1 or PD-L1, i.e., binds to PD-1 or PD-L1 but does not bind to or binds with a lower affinity (e.g., 10-fold lower affinity) to other molecules (e.g., OX40 and / or CD40). Thus, it binds to PD-1 or PD-L1 with a higher binding affinity than it binds to another molecule. Thus, typically, the Kd of an antibody against human PD-1 or PD-L1 will be less than 2-fold, preferably 5-fold, more preferably 10-fold, than the Kd against other non-target molecules, such as mouse PD-1 or PD-L1, other immune checkpoint molecules, or any other unrelated or associated substances in the environment. More preferably, the Kd will be less than 50-fold, even more preferably less than 100-fold, and even more preferably less than 200-fold.

[0206] Methods for measuring the overall affinity (KD) of an interaction (such as the interaction between an antibody and a ligand) as well as the on-rate (ka) and off-rate (kd) are well known in the art. Exemplary in vitro methods are described in the accompanying examples. It is also conceivable to use a flow cytometry-based method (Sklar et al., Annu Rev Biophys Biomol Struct, (31), 97-119, 2002).

[0207] The terms PD-1 and PD-L1 as used herein typically refer to human PD-1 and PD-L1. The inhibitors may have some binding affinity for PD-1 and PD-L1 from other mammals, such as PD-1 and PD-L1 from non-human primates, e.g., Macaca fascicularis (cynomolgus monkey). The antibodies preferably do not bind to mouse PD-1 or PD-L1 and / or do not bind to other immune checkpoint molecules.

[0208] In one embodiment, the PD-1 inhibitor that specifically binds to PD-1 or PD-L1 may have affinity for PD-1 or PD-L1 in its native state, e.g., PD-1 or PD-L1 localized on the surface of a cell. In one embodiment, the PD-1 inhibitor blocks PD-1 PD-L1 interaction. For example, the PD-1 inhibitor may bind to PD-1 or PD-L1 in a manner that inhibits the ability of PD-L1 to bind to PD-1, thereby blocking the PD-1 / PD-L1 interaction.

[0209] "Localized on the surface of a cell" means that PD-1 or PD-L1 is associated with a cell such that one or more regions of PD-1 are present on the outer surface of the cell. For example, PD-1 may be inserted into the cell plasma membrane (i.e., oriented as a transmembrane protein) with one or more regions displayed on the extracellular surface. This may occur during the course of expression of PD-1 by the cell. Thus, in one embodiment, "localized on the surface of a cell" may mean "expressed on the surface of a cell." Alternatively, PD-1 may be present on the outside of the cell with covalent and / or ionic interactions that localize it to a particular region or regions on the cell surface.

[0210] In one embodiment, the PD-1 inhibitors described herein can induce anti-tumor immunity through immune checkpoint blockade. The PD-1 inhibitors bind to PD-1 or PD-L1 in a manner that inhibits PD-L1 from binding to PD-1, i.e., block the PD-1 / PD-L1 interaction. The PD-1 inhibitors can enhance T cell responses, e.g., enhance or restore T cell effector function. In one embodiment, the PD-1 inhibitors can promote infiltration of tumor-reactive CD8+ T cells into established tumors.

[0211] Thus, PD-1 inhibitors can modulate the activity of cells expressing PD-1 or PD-L1, the modulation being an increase or decrease in the activity of the cells. The cells are typically T cells. The inhibitors can increase the activity of CD4+ or CD8+ effector cells, or can decrease the activity of or deplete regulatory T cells (T regs). In either case, the net effect of the antibody is an increase in the activity of effector T cells, particularly CD4+, CD8+, or NK effector T cells. Methods for determining changes in the activity of effector T cells are well known and have been described above.

[0212] The PD-1 inhibitor preferably causes an increase in activity in T cells (preferably CD8+ T cells) in vitro, optionally the increase in activity being an increase in proliferation, IFN-γ production and / or IL-2 production by the T cells, the increase being preferably at least 2-fold, more preferably at least 10-fold, even more preferably at least 25-fold higher than the change in activity caused by an isotype control antibody measured in the same assay.

[0213] In one embodiment, a PD-1 inhibitor can improve the effectiveness of another immunotherapy.

[0214] In one embodiment, the PD-1 inhibitor blocks the programmed death 1 (PD-1) receptor for its ligand PD-L1, which is expressed on the surface of cells in the tumor (Ribas and Wolchok 2018). PD-1 is an immune checkpoint whose inhibitory function is mediated by the tyrosine phosphatase SHP-2, which dephosphorylates downstream signaling molecules of T cell receptor (TCR) signaling molecules. In a preferred embodiment, the PD-1 inhibitor reactivates PD-1-expressing T cells, preferably by blocking the inhibitory signaling mediated by the tyrosine phosphatase SHP-2, which dephosphorylates downstream signaling molecules of T cell receptor (TCR) signaling molecules.

[0215] The PD-1 inhibitor may be an anti-PD-1 antibody or an antigen-binding fragment thereof capable of inhibiting PD-1 function (e.g., pembrolizumab (also known as lambrolizumab), nivolumab, pidilizumab, cemiplimab, AMP-224, PDR-001, MEDI-0680 (also known as AMP-514), JTX-4014 (pimivalimab), spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarimab, and INCMGA00012 (retifanlimab).

[0216] Alternatively, the PD-1 inhibitor may comprise or consist of an anti-PD-L1 antibody, or an antigen-binding fragment thereof, capable of inhibiting PD-1 function (e.g., atezolizumab (Tecentriq™, MPDL3280A), durvalumab (MEDI-4736), avelumab, MDX-1105, KN035 (embafolimab) and CK-301 (cosibelimab)).

[0217] Alternatively, the PD-1 inhibitor may be a small molecule or peptide-based inhibitor of PD-1 or PD-L1. For example, the PD-1 inhibitor may be a small molecule inhibitor of PD-L1, such as CA-170. Alternatively, the PD-1 inhibitor may be a peptide inhibitor of PD-L1, such as AUNP12 or BMS-986189.

[0218] In one embodiment, the PD-1 inhibitor binds to an epitope that blocks the PD-1 PD-L1 interaction.

[0219] In one embodiment, the PD-1 inhibitor is capable of inhibiting binding of the reference antibody pembrolizumab or nivolumab to human PD-1.

[0220] In another embodiment, the PD-1 inhibitor is capable of inhibiting the binding of the reference antibody atezolizumab to human PD-L1.

[0221] "Can inhibit the binding of the reference antibody pembrolizumab to human PD-1" means that the presence of the PD-1 inhibitor of the combination therapy of the present invention inhibits, in whole or in part, the binding of pembrolizumab to human PD-1. Similarly, "can inhibit the binding of the reference antibody nivolumab to human PD-1" means that the presence of the antibody polypeptide of the combination therapy of the present invention inhibits, in whole or in part, the binding of nivolumab to human PD-1. Thus, the PD-1 inhibitor used in the combination therapy of the present invention can compete with the "reference antibody" pembrolizumab and / or the "reference antibody" nivolumab for binding to PD-1. Such competitive binding inhibition can be determined using assays and methods well known in the art, for example, by using a BIAcore chip with immobilized PD-1, by incubating with and without the antibody polypeptide to be tested in the presence of the reference antibodies pembrolizumab and nivolumab. Alternatively, a pair-wise mapping approach can be used in which the reference antibodies pembrolizumab or nivolumab are immobilized on the surface of a BIAcore chip, PD-1 antigen is allowed to bind to the immobilized antibody, and a secondary antibody is then tested for simultaneous PD-1 binding ability (see, for example, 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05 / 2012, the disclosure of which is incorporated herein by reference).

[0222] "Capable of inhibiting binding of the reference antibody atezolizumab to human PD-L1" means that the presence of the antibody polypeptide of the combination therapy of the invention inhibits, in whole or in part, binding of atezolizumab to human PD-L1. Thus, the PD-1 inhibitors used in the combination therapy of the invention may compete with the "reference antibody" atezolizumab for binding to PD-L1. Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using a BIAcore chip with immobilized PD-L1, by incubating with and without the antibody polypeptide to be tested in the presence of the reference antibody atezolizumab. Alternatively, a pair-wise mapping approach can be used in which the reference antibody atezolizumab is immobilized on the surface of a BIAcore chip, PD-L1 antigen is allowed to bind to the immobilized antibody, and a secondary antibody is then tested for simultaneous PD-L1 binding ability (see, for example, 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05 / 2012, the disclosure of which is incorporated herein by reference).

[0223] In a further alternative, competitive binding inhibition can be determined using flow cytometry. For example, to determine whether a test antibody can inhibit the binding of a pembrolizumab or nivolumab (or atezolizumab) reference antibody to a cell surface antigen, cells expressing the antigen can be pre-incubated with the test antibody for 20 minutes before washing the cells and incubating with a reference pembrolizumab or nivolumab (or atezolizumab) antibody conjugated to a fluorophore that can be detected by flow cytometry. If pre-incubation with the test antibody reduces the detection rate of the reference pembrolizumab or nivolumab (or atezolizumab) antibody in flow cytometry, the test antibody inhibits the binding of the reference antibody to the cell surface antigen. If the tested antibody exhibits high affinity for PD-1 (or PD-L1), a shorter period of pre-incubation can be used (or no pre-incubation at all).

[0224] "Pembrolizumab" means an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOs: 33 and 34, respectively. Pembrolizumab heavy chain sequence (SEQ ID NO:33): [ka] Pembrolizumab light chain sequence (SEQ ID NO:34): [ka]

[0225] "Nivolumab" means an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOs: 31 and 32, respectively. Heavy chain sequence of Nivolumab (SEQ ID NO:31): [ka] Nivolumab light chain sequence (SEQ ID NO:32) [ka] Pidilizumab heavy chain sequence (SEQ ID NO:37) [ka] Pidilizumab light chain sequence (SEQ ID NO:38) [ka] Cemiplimab heavy chain sequence (SEQ ID NO:39) [ka] Cemiplimab light chain sequence (SEQ ID NO: 40) [ka] Spartalizumab heavy chain sequence (SEQ ID NO: 41) [ka] Spartalizumab light chain sequence (SEQ ID NO: 42) [ka] Camrelizumab heavy chain sequence (SEQ ID NO: 43) [ka] Camrelizumab light chain sequence (SEQ ID NO: 44) [ka] Heavy chain sequence of tislelizumab (SEQ ID NO: 45) [ka] Tislelizumab light chain sequence (SEQ ID NO: 46) [ka] Heavy chain sequence of toripalimab (SEQ ID NO:47) [ka] Light chain sequence of toripalimab (SEQ ID NO:48) [ka] Dostarlimab heavy chain sequence (SEQ ID NO: 49) [ka] Dostarlimab light chain sequence (SEQ ID NO:50) [ka] Heavy chain sequence of INCMGA00012 (SEQ ID NO:51) [ka] Light chain sequence of INCMGA00012 (SEQ ID NO:52) [ka]

[0226] "Atezolizumab" means an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOs: 53 and 54, respectively. Atezolizumab heavy chain sequence (SEQ ID NO:53) [ka] Atezolizumab light chain sequence (SEQ ID NO:54) [ka] Durvalumab heavy chain sequence (SEQ ID NO:55) [ka] Durvalumab light chain sequence (SEQ ID NO:56) [ka] Avelumab heavy chain sequence (SEQ ID NO:57) [ka] Avelumab light chain sequence (SEQ ID NO:58) [ka] CK-301 (cosibelimab) heavy chain sequence (SEQ ID NO:59) [ka] CK-301 (cosibelimab) light chain sequence (SEQ ID NO:60) [ka] Heavy chain sequence of JTX-4014 (SEQ ID NO:61) [ka] Light chain sequence of JTX-4014 (SEQ ID NO:62) [ka]

[0227] Such PD-1 inhibitors are also described in US8354509B2 and US8779105B2, the PD-1 inhibitors (particularly anti-PD-1 antibodies) of US8354509B2 and US8779105B2 being incorporated herein by reference.

[0228] In one embodiment, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, and the antibody or antigen-binding fragment included in the combination therapy of the present invention comprises an antibody Fc region. Those skilled in the art will appreciate that the Fc portion may be derived from an IgG antibody, or from an antibody of a different class (such as IgM, IgA, IgD or IgE). In one embodiment, the Fc region is derived from an IgG1, IgG2, IgG3 or IgG4 antibody. Advantageously, however, the Fc region is derived from an IgG4 antibody.

[0229] The Fc region may be naturally occurring (e.g., part of an endogenously produced antibody) or artificial (e.g., containing one or more point mutations relative to a naturally occurring Fc region). A variant Fc region typically binds with altered affinity to an Fc receptor, such as FcγR and / or the neonatal Fc receptor (FcRn), and improves the function and / or half-life of the polypeptide. The biological function and / or half-life may be either extended or decreased compared to the half-life of a polypeptide comprising a native Fc region. Examples of such biological functions that may be modulated by the presence of a variant Fc region include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and / or apoptosis.

[0230] Thus, the Fc region can be naturally occurring (e.g., part of an endogenously produced human antibody) or can be artificial (e.g., contains one or more point mutations relative to a naturally occurring human Fc region).

[0231] As is well known in the art, the Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0232] The Fc region may be engineered as described above for the CD137 antibodies of the combination therapy of the present invention.

[0233] Antibodies and Inhibitors The following definitions apply to either or both of the CD137 and PD-1 inhibitors of the present invention, where the PD-1 inhibitor is an antibody (either an antibody specific for PD-1 or an antibody specific for PD-L1).

[0234] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, 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. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with antigens. 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). The constant region of an 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.

[0235] "Antibody or antigen-binding fragment thereof" includes not only substantially intact antibody molecules, but also chimeric antibodies, humanized antibodies, isolated human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen-binding fragments and derivatives thereof. Suitable antigen-binding fragments and derivatives include, but are not necessarily limited to, Fv fragments (e.g., single chain Fvs and disulfide-linked Fvs), Fab-like fragments (e.g., Fab fragments, Fab' fragments and F(ab)2 fragments), single variable domains (e.g., V H and V L These include domain antibodies (dAbs, including single and double formats [i.e. dAb-linker-dAb]) and domain antibodies (dAbs, including single and double formats [i.e. dAb-linker-dAb]). The advantages of using antibody fragments rather than whole antibodies are several fold. The smaller size of the fragments may lead to improved pharmacological properties, such as improved penetration into solid tissues. Furthermore, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli and thus easily produced in large quantities.

[0236] For example, the antigen-binding fragment may be an scFv molecule, i.e.,H and V L It may also include molecules in which partner domains are linked via a flexible oligopeptide.

[0237] The heavy chain can be of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM and IgE.

[0238] Light chains include kappa and lambda chains.

[0239] Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs (including, e.g., monospecific and bispecific), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0240] Of particular relevance are antibodies and antigen-binding fragments thereof that have been "isolated" such that they are present in a physical environment different from that in which they may occur in nature, or that have been modified so that they differ in amino acid sequence from naturally occurring antibodies.

[0241] The term "antibody or antigen-binding fragment thereof" is also intended to encompass antibody mimetics (e.g., non-antibody scaffold structures that allow for the introduction of variability at specific positions while retaining high stability). Those skilled in the art of biochemistry will be familiar with many such molecules, as discussed in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3):245-255, the disclosure of which is incorporated herein by reference. Exemplary antibody mimetics include affibodies (also called trinectins; Nygren, 2008, FEBS J, 275, 2668-2676), CTLDs (also called tetranectins; Innovations Pharmac. Technol. (2006), 27-30), adnectins (also called monobodies; Meth. Mol. Biol., 352 (2007), 95-109), anticalins (Drug Discovery Today (2005), 10, 23-33), DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582), avimers (Nat. Biotechnol. (2005), 23, 1556-1561), microbodies (FEBS J, (2007), 274, 86-95), peptide aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797), Kunitz domains (J. Pharmacol. Exp. Ther. (2006) 318, 803-809), affilins (Trends. Biotechnol. (2005), 23, 514-522), and affimers (Avacta Life Sciences, Wetherby, UK).

[0242] Those skilled in the art will further appreciate that the present invention also encompasses combination therapies, whether existing now or in the future, that include modified versions of antibodies and antigen-binding fragments thereof, e.g., modified by the covalent attachment of polyethylene glycol or another suitable polymer (see below).

[0243] The antibody may be a polyclonal or a monoclonal antibody. The antibody may be produced by any suitable method.

[0244] Methods for producing antibodies and antibody fragments are well known in the art. For example, antibodies can be produced via any one of several methods using inducing in vivo production of antibody molecules, screening immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter et al., 1991, Nature 349:293-299, the disclosures of which are incorporated herein by reference), or production of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma method, the human B cell hybridoma method, and the Epstein-Barr virus (EBV) hybridoma method (Kohler et al., 1975. Nature 256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984. Mol. Cell. Biol. 62:109-120, the disclosures of which are incorporated herein by reference).

[0245] Suitable methods for the production of monoclonal antibodies are also disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988, the disclosure of which is incorporated herein by reference) and "Monoclonal Hybridoma Antibodies: Techniques and Applications", JGR Hurrell (CRC Press, 1982, the disclosure of which is incorporated herein by reference).

[0246] Similarly, antibody fragments can be obtained using methods well known in the art (e.g., Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York, the disclosure of which is incorporated herein by reference). For example, antibody fragments according to the invention can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems). Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

[0247] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD137, PD-1, or PD-L1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementarity determining regions (CDRs). Single chain antibodies, such as ScFv, and heavy chain antibodies, such as VHH and camelid antibodies, are also intended to be encompassed within the scope of the term "antigen-binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

[0248] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of a molecule (e.g., an antibody molecule) to bind or not bind to a target. Binding affinity can be quantified by determining the dissociation constant (Kd) of the antibody and its target. Similarly, the specificity of the binding of an antibody to its target can be defined in terms of the comparative dissociation constant (Kd) of the antibody for its target compared to the dissociation constant for the antibody and another non-target molecule.

[0249] Typically, the Kd of an antibody for a target will be less than 2-fold, preferably less than 5-fold, and more preferably less than 10-fold, its Kd for other non-target molecules, such as unrelated substances or accompanying substances in the environment. More preferably, the Kd will be less than 50-fold, even more preferably less than 100-fold, and even more preferably less than 200-fold.

[0250] The value of this dissociation constant can be determined directly by well-known methods, and even for complex mixtures, it can be calculated by methods such as those described in Caceci et al. (Byte 9:340-362, 1984). For example, Kd can be established using a double filter nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays for evaluating the binding ability of a ligand, such as an antibody, to a target are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding rate (e.g., binding affinity) of an antibody can also be evaluated by standard assays known in the art, such as, for example, Biacore™ system analysis.

[0251] Competitive binding assays may be performed in which the binding of an antibody to a target is compared to the binding of the target by another known ligand of the target, such as another antibody. The concentration at which 50% inhibition occurs is known as Ki. Under ideal conditions, Ki is equivalent to Kd. The Ki value is never less than Kd, so the measured Ki value can be conveniently substituted to determine an upper limit for Kd.

[0252] An anti-CD137 antibody used in the combination therapies and methods of the invention is preferably capable of binding to its target with an affinity at least 2-fold, 10-fold, 50-fold, 100-fold or more than its affinity to bind to another non-target molecule.

[0253] A PD-1 inhibitor (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) used in the combination therapies and methods of the invention is preferably able to bind to its target with an affinity at least 2-fold, 10-fold, 50-fold, 100-fold or more than its affinity to bind to another non-target molecule.

[0254] The antibody for use in the methods of the invention may be a human antibody. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline sequences of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Such antibodies are typically referred to as chimeric or humanized.

[0255] The human antibodies for use in the methods of the invention are typically human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy and light chain transgenes fused to an immortalized cell. Human antibodies can also be prepared by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus. The term "human antibody derivative" refers to any modified form of a human antibody, e.g., a conjugate of the antibody with another agent or antibody.

[0256] Alternatively, the antibody for use in the methods of the present invention may be a humanized antibody.

[0257] The term "humanized" refers to antibody molecules, typically prepared using recombinant techniques, that have an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete non-human antibody variable domain fused to a human constant domain, or only the complementarity determining regions (CDRs) of such a variable domain grafted into appropriate human framework regions in a human variable domain. The framework residues of such humanized molecules may be wild-type (e.g., fully human) or they may be modified to contain one or more amino acid substitutions not found in the human antibody whose sequence has served as the basis for humanization. Humanization reduces or eliminates the possibility that the constant regions of the molecule can function as immunogens in human individuals, but the possibility of an immune response to the foreign variable regions remains (LoBuglio, AF et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions to make them as close to human form as possible. It is known that the variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) that change in response to the antigen in question and determine the binding capacity, flanked by four framework regions (FRs) that are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When a non-human antibody is prepared for a particular antigen, the variable region can be "reshaped" or "humanized" by grafting CDRs from the non-human antibody onto the FRs present in the modified human antibody. The application of this approach to various antibodies is described in Sato, K. et al. (1993) Cancer Res 53:851-856; Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al.(1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534 - 1536, Kettleborough, C.A. et al. (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR - Grafting: The Importance Of Framework Residues On Loop Conformation," Protein Engineering 4:773 - 3783, Maeda, H. et al. (1991) "Construction Of Reshaped Human Antibodies With HIV - Neutralizing Activity," Human Antibodies Hybridoma 2:124 - 134, Gorman, S.D. et al. (1991) "Reshaping A Therapeutic CD4 Antibody," Proc. Natl. Acad. Sci. (U.S.A.) 88:4181 - 4185, Tempest, P.R. et al. (1991) "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo," Bio / Technology 9:266 - 271, Co, M.S. et al. (1991) "Humanized Antibodies For Antiviral Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 88:2869 - 2873, Carter, P. et al. (1992) "Humanization Of An Anti - p185her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 89:4285 - 4289, and Co, M.S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen," J.Immunol. 148:1149-1154. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6) that are modified relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanize antigens is well known (see, e.g., U.S. Patent Nos. 5,225,539, 5,530,101, 5,585,089, 5,859,205, 6,407,213, 6,881,557).

[0258] The antibody may be or comprise a variant or fragment of one of the specific antibodies disclosed herein, provided that said variant or fragment retains specificity for its target. For example, the antibody may be or comprise a variant or fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137. Alternatively, or in addition, the antibody may be or comprise a variant or fragment of one of the specific anti-PD-1 or PD-L1 antibodies disclosed herein, provided that said variant or fragment retains specificity for PD-1 or PD-L1.

[0259] The fragment is preferably an antigen-binding portion of the above-mentioned antibody. Fragments can be generated by truncation, e.g., removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. In this manner, up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N-terminus and / or C-terminus. Fragments can also be generated by one or more internal deletions.

[0260] Variants may include one or more substitutions, deletions, or additions with respect to the sequence of a particular anti-CD137 antibody or other antibody (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) disclosed herein. Variants may include 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, or more amino acid substitutions and / or deletions from the particular sequences disclosed herein. "Deletion" variants may include the deletion of individual amino acids, the deletion of small groups of amino acids, e.g., 2, 3, 4, or 5 amino acids, or the deletion of larger regions of amino acids, e.g., the deletion of a particular amino acid domain or other feature. "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids, making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid.

[0261] Some properties of the 20 main amino acids that can be used to select suitable substitutes are as follows: [Table 2]

[0262] Preferred "variants" include those in which an amino acid that appears in the sequence instead of a naturally occurring amino acid is a structural analog thereof. The amino acids used in the sequence may also be derivatized or modified, e.g., labeled, so long as the function of the antibody is not significantly adversely affected.

[0263] Variants may be prepared by modification during synthesis or after production of the antibody, or if the antibody is in recombinant form, may be prepared using known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0264] Preferably, the variant antibody has an amino acid sequence that has greater than 60%, or greater than 70% (e.g., 75% or 80%), preferably greater than 85%, for example greater than 90% or 95% amino acid identity to the VL or VH domain of an antibody disclosed herein. This level of amino acid identity may be found over the entire length of the sequence of the relevant SEQ ID NO, or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

[0265] "Sequence identity" in the context of amino acid sequences refers to sequences having the recited value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

[0266] Pairwise alignment parameters-method: exact, matrix: PAM, gap open penalty: 10.00, gap extension penalty; 0.10;

[0267] Multiple alignment parameters - matrix: PAM, gap open penalty: 10.00, % identity for delay: 30, end gap penalty: on, gap separation distance: 0, negative matrix: none, gap extension penalty: 0.20, residue specific gap penalty: on, hydrophilic gap penalty: on, hydrophilic residues: GPSNDQEKR. Sequence identity at specific residues is intended to include identical residues that have only been derivatized.

[0268] An anti-CD137 antibody or PD-1 inhibitor for use in the combination therapy and methods of the invention may bind to the same epitope as a particular antibody disclosed herein (e.g., an anti-CD137 antibody may bind to domain 2 of CD137). This is because such an antibody is more likely to mimic the action of the disclosed antibody. Whether an antibody binds to the same epitope as another antibody may be determined by routine methods. For example, the binding of each antibody to a target may use a competitive binding assay. Methods for performing competitive binding assays are well known in the art. For example, they may involve contacting an antibody and a target molecule together under conditions that allow the antibody to bind to the target molecule. The antibody / target complex may then be contacted with a second (test) antibody, and the extent to which the test antibody can displace the first antibody from the antibody / target complex may be evaluated. Such evaluation may use any suitable technique, including, for example, surface plasmon resonance, ELISA, or flow cytometry. The ability of the test antibody to inhibit binding of the first antibody to the target indicates that the test antibody is able to compete with said first antibody for binding to the target and thus binds to the same epitope or region on the target as the first antibody, and thus can mimic the action of the first antibody.

[0269] Any antibody referred to herein may be provided in isolated form or, optionally, linked (directly or indirectly) to another moiety, which may be a therapeutic molecule such as a cytotoxic moiety or a drug.

[0270] Therapeutic molecules can be directly connected to the antibodies of the present invention, for example, by chemical conjugation. Methods for conjugating molecules to antibodies are known in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) can be used to conjugate various drugs, including doxorubicin, to antibodies or peptides. The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), is particularly useful for conjugating functional moieties to binding moieties.

[0271] Other methods for conjugating a moiety to an antibody can also be used. For example, sodium periodate oxidation followed by reductive alkylation with a suitable reagent can be used, as can glutaraldehyde cross-linking. However, it is recognized that regardless of how the method for producing the conjugate of the present invention is selected, a decision must be made that the antibody maintains its targeting ability and the functional moiety maintains its relevant function.

[0272] A cytotoxic moiety may be directly and / or indirectly cytotoxic. "Directly cytotoxic" means that the moiety is a moiety that is itself cytotoxic. "Indirectly cytotoxic" means that the moiety is not itself cytotoxic but can induce cytotoxicity, for example, by its action on or a further action on a further molecule. A cytotoxic moiety may be cytotoxic only intracellularly and preferably not cytotoxic extracellularly.

[0273] The antibody or antigen-binding fragment is linked to a cytotoxic moiety that is a directly cytotoxic chemotherapeutic agent. Optionally, the cytotoxic moiety is a directly cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art.

[0274] Cytotoxic chemotherapeutic agents, such as anticancer drugs, include alkylating agents, including nitrogen mustards, such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; ethylenimines and methylmelamines, such as hexamethylmelamine, thiotepa; alkylsulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin; and decarbazine. triazenes such as tetracycline (DTIC, dimethyltriazenoimidazole-carboxamide); antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil, 5-FU), floxouridine (fluodeoxyuridine, FUdR) and cytarabine (cytosine arabinoside); and mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), and pentostatin (2'-deoxycoformycin). Natural products, including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenome. Miscellaneous agents include platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenediones such as mitoxantrone and the anthracyclines; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); and adrenal cortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogues / derivatives; and hormone agonists / antagonists such as flutamide and tamoxifen.

[0275] The cytotoxic moiety can be a cytotoxic peptide or a polypeptide moiety that leads to cell death. Cytotoxic peptides and polypeptide moieties are well known in the art, including, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor, and the like. Methods for linking them to targeting moieties, such as antibodies, are also known in the art. Other ribosome-inactivating proteins are described as cytotoxic agents in WO96 / 06641. Pseudomonas exotoxin can also be used as a cytotoxic polypeptide. Certain cytokines, such as TNFα and IL-2, can also be useful as cytotoxic agents.

[0276] Certain radioactive atoms may also be cytotoxic when delivered in sufficient doses. Thus, a cytotoxic moiety may comprise a radioactive atom that delivers sufficient radioactivity to a target site during use to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186, rhenium-188 or yttrium-90, or any other isotope that releases sufficient energy to destroy adjacent cells, organelles or nucleic acids. Preferably, the isotope and density of the radioactive atom in the agent of the present invention are such that a dose of more than 4000cGy (preferably at least 6000, 8000 or 10000cGy) is delivered to the target site, preferably the cells and their organelles at the target site, particularly the nucleus.

[0277] The radioactive atom may be attached to the antibody, antigen-binding fragment, variant, fusion, or derivative thereof in a known manner. For example, EDTA or another chelating agent may be attached to the binding moiety or may be used to attach 111In or 90Y. Tyrosine residues may be directly labeled with 125I or 131I.

[0278] The cytotoxic moiety may be a suitable indirectly cytotoxic polypeptide. The indirectly cytotoxic polypeptide may be a polypeptide that has enzymatic activity and can convert a non-toxic and / or relatively non-toxic prodrug into a cytotoxic drug. With antibodies, this type of system is often referred to as ADEPT (antibody-directed enzyme prodrug therapy). The system requires that the antibody places the enzyme moiety at the desired site in the patient's body, and after allowing time for the enzyme to localize at the site, a prodrug that is a substrate for the enzyme is administered, the end product of catalysis being a cytotoxic compound. The goal of this approach is to maximize the concentration of the drug at the desired site and minimize the concentration of the drug in normal tissues. The cytotoxic moiety may be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.

[0279] The enzyme and prodrug of the system using the targeted enzyme described herein can be any of those proposed before.The cytotoxic agent can be any existing anticancer drug, such as alkylating agent; an agent that intercalates into DNA; an agent that inhibits any important enzyme, such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinase or topoisomerase; or an agent that causes cell death by interacting with any other cellular component.Etoposide is an example of a topoisomerase inhibitor.

[0280] Reported prodrug systems include those listed in Table 2. [Table 3]

[0281] Suitable enzymes for forming part of the enzyme moiety include exopeptidases such as carboxypeptidase G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidase A and B (for MTX-based prodrugs), and aminopeptidases (for 2-α-aminosyl MTC prodrugs); endopeptidases such as thrombolysin (for thrombin prodrugs); hydrolases such as phosphatases (e.g. alkaline phosphatase) or sulfatases (e.g. arylsulfatase) (for phosphorylated or sulfated prodrugs); amidases such as penicillin amidase and aryl acylamidase; lactamases such as β-lactamase; glycosidases, e.g. For example, β-glucuronosidase (for β-glucuronomide anthracyclines), α-galactosidase (for amygdalin) and β-galactosidase (for β-galactose anthracyclines); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for ganciclovir); reductases, such as nitroreductase (for CB1954 and analogues); azoreductase (for azobenzene mustard) and DT-diaphorase (for CB1954); oxidases, such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemase, catalytic antibodies and cyclodextrins.

[0282] Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug: typically, it has less than 10% of the toxicity, preferably less than 1% of the toxicity, as measured in a suitable in vitro cytotoxicity test.

[0283] The moiety capable of converting a prodrug into a cytotoxic drug is likely to be active in isolation from the remainder of the agent of the invention, but need only be active (a) when combined with the remainder of the agent of the invention, and (b) when the agent of the invention is attached to, adjacent to, or internalized within a target cell.

[0284] When each moiety is a polypeptide, the two moieties can be linked together by any of the conventional methods for crosslinking polypeptides. For example, the antibody or antigen-binding fragment may be enriched in thiol groups, and the further moiety is reacted with a bifunctional agent capable of reacting with those thiol groups, such as N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Amide and thioether bonds, such as those achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulfide bonds.

[0285] The cytotoxic moiety may be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogs, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamides, 3-aminobenzodiamides, etanixadol, pimonidazole and mizonidazole. Gene delivery to cells can also render them radiosensitized, for example the delivery of the p53 gene or cyclin D. The further moiety may be a moiety that becomes cytotoxic upon irradiation or releases a cytotoxic moiety. For example, boron-10 isotopes release alpha particles that are cytotoxic when properly irradiated. Similarly, the cytotoxic moiety may be one that is useful in photodynamic therapy, such as photofrin.

[0286] Methods and Combination Therapies The present invention provides a combination therapy for use in treating cancer (e.g., a solid tumor) in a subject, the combination therapy comprising: (a) an antibody that specifically binds CD137, or an antigen-binding portion thereof; and (b) an additional immunotherapeutic agent, where the additional immunotherapeutic agent is a PD-1 inhibitor.

[0287] The antibody or antigen-binding portion thereof that specifically binds to CD137 and the PD-1 inhibitor are as described above.

[0288] Those skilled in the art will appreciate that the presence of two active agents (as detailed above) may provide a synergistic benefit in the treatment of a solid tumor in a subject. "Synergistic" includes that the therapeutic effect of the combination of the two agents (e.g., as determined by reference to the growth rate or size of the tumor) is greater than the additive therapeutic effect of the two agents administered by themselves. Such synergy may be identified by testing the active agents alone and in combination in relevant cell line models of solid tumors.

[0289] The terms "combination therapy" or "combination treatment" or "combined" as used herein refer to any form of simultaneous or sequential treatment with at least two different therapeutic agents.

[0290] According to certain embodiments, the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor are administered simultaneously, either in the same composition or in separate compositions. According to other embodiments, the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor are administered sequentially, i.e., the anti-CD137 antibody, or antigen-binding fragment thereof, is administered either before or after administration of the PD-1 inhibitor. In some embodiments, the administration of the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor is concurrent, i.e., the administration period of the anti-CD137 antibody, or antigen-binding fragment thereof, and the administration period of the PD-1 inhibitor overlap with each other. In some embodiments, the administration of the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor is non-concurrent or sequential. For example, in some embodiments, the administration of the anti-CD137 antibody, or antigen-binding fragment thereof, is terminated before the PD-1 inhibitor is administered. In some embodiments, administration of the PD-1 inhibitor is terminated before the anti-CD137 antibody, or antigen-binding fragment thereof, is administered.

[0291] According to certain exemplary embodiments, the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor are administered as a single therapeutic composition, which, according to some embodiments, further comprises a therapeutically acceptable diluent or carrier.

[0292] The combination of systemic PD-1 inhibitors with anti-CD137 antibodies is not attractive simply because of the expression of PD-L1 by tumors. CD137 stimulation causes activation of tumor-infiltrating T cells, enabling them to kill tumor cells. However, CD137-mediated T cell activation also causes upregulation of PD-1 on T cells (and indirectly causes upregulation of PD-L1 on tumor cells). Conversely, PD-1 inhibition causes reactivation (or removal of inhibition) of tumor-specific T cells. T cells reactivated by PD-1 inhibitors express CD137. CD137 stimulation of CD137-expressing T cells increases their ability to kill tumor cells and prevent them from becoming exhausted (or rescue them from exhaustion).

[0293] Thus, the further immunotherapeutic agent may be a PD-1 inhibitor, preferably an antibody or other agent which specifically binds to at least one of PD-1 or PD-L1 (as already described above).

[0294] Where the further immunotherapeutic agent is an antibody or a bispecific molecule comprising an antibody, it will be understood that all of the general considerations discussed above with respect to the definition of an antibody, antigen-binding fragments of an antibody, optional conjugation to additional therapeutic moieties, etc. also apply to antibodies that are further immunotherapeutic agents. Similarly, it will be understood that the definition of target specificity / affinity and methods for determining specificity / affinity discussed above for anti-CD137 antibodies apply equally to antibodies that are further immunotherapeutic agents, except that the specific target of the agent is read instead of CD137. Variants and fragments of antibodies that are further immunotherapeutic agents may also be defined in the same manner as variants and fragments of anti-CD137 antibodies.

[0295] The present invention provides methods for treating cancer, preferably a solid tumor, in a subject. The tumor is typically malignant and may be metastatic.

[0296] In one embodiment, the combination therapy of the present invention may be used to treat a patient or subject suffering from or at risk of suffering from cancer.

[0297] "Treatment" includes both therapeutic and prophylactic treatment of a patient. The term "prophylactic" is used to encompass the use of an agent or formulation thereof described herein to prevent or reduce the likelihood of cancer, or the spread, dissemination, or metastasis of cancer cells in a patient or subject. The term "prophylactic" also encompasses the use of an agent or formulation thereof described herein to prevent the recurrence of cancer in a patient who has previously been treated for a neoplastic disorder.

[0298] The cancer may be associated with the formation of solid tumors or may be a blood cancer. Types of cancer that may be treated include carcinoma, sarcoma, lymphoma, leukemia, blastoma and germ cell tumors.

[0299] The cancer may be selected from the group consisting of prostate cancer; breast cancer; colorectal cancer; renal cancer; pancreatic cancer; ovarian cancer; lung cancer; cervical cancer; rhabdomyosarcoma; neuroblastoma; bone cancer; multiple myeloma; leukemia (e.g., acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g., melanoma), bladder cancer, and glioblastoma.

[0300] In one embodiment, the cancer may be selected from the list of cancers in Table 4 or Table 5 below (taken from WO2018 / 091740). [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2] [Table 5-3]

[0301] Typically, the therapeutic agents in the combination therapy of the invention may be administered in parenteral form, for example, by injection into the bloodstream or at or near the site of a tumor. Typically, the therapeutic agents in the combination therapy of the invention are administered intravenously.

[0302] In one embodiment, the combination therapy and / or methods of the invention may be used to treat patients who have been pre-screened and identified as having tumors containing cells that express CD137 and an FcγR, e.g., FcγRI, FcγRIIA, FcγRIIB, or a combination thereof.

[0303] It is further understood that the combination therapy of the present invention may be used as the sole treatment for cancer in a patient or as part of an additional combination treatment (the additional treatment may be a pharmaceutical agent, radiation therapy, and / or surgery).

[0304] Cancer can be a solid tumor. Solid tumors are classically defined by the tissue they originate from, such as breast, colon, etc. However, because immunotherapy acts on the immune system, not on the tumor itself, the immune status of the tumor can be more predictive of response than the origin of the tumor. In the supporting study presented herein, the MC38 colon cancer model is evaluated in more detail, which is generally immunogenic and responds to PD-1 therapy alone.

[0305] In one embodiment of the present invention, the tumor is immunogenic. Such tumors are characterized by infiltration of immune cells, such as T cells and cells of myeloid origin. It has been demonstrated that infiltration of CD8 T cells, i.e. a more immunogenic tumor profile, correlates with a better prognosis after therapy, for example in colon cancer (Galon et al., 2014, J. Pathol. 232(2):199-209).

[0306] In an alternative embodiment of the invention, the tumor is non-immunogenic or poorly immunogenic. Poorly immunogenic tumors are often characterized by low or no MHCI expression and low numbers of infiltrating immune cells, such as T cells and cells of myeloid origin (Lechner et al., 2013, J Immunotherapy 36(9):477-89). The tumor may be an adenoma, adenocarcinoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms' tumor, lung tumor, colon tumor, lymphoid tumor, breast tumor, or melanoma.

[0307] Blastoma types include hepatoblastoma, glioblastoma, neuroblastoma, or retinoblastoma. Carcinoma types include breast cancer, endometrial cancer, colorectal cancer or hepatocellular carcinoma, pancreatic cancer, prostate cancer, gastric cancer, urothelial cancer, renal cancer, Merkel cell carcinoma, esophageal cancer, cervical cancer, and head and neck cancer, and adenocarcinoma. Sarcoma types include Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, or any other soft tissue sarcoma. Melanoma types include lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentigo melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, neurotropic melanoma, amelanotic melanoma, soft tissue melanoma, melanoma with small nevus-like cells, melanoma with features of Spitz nevus, and uveal melanoma. Lymphoma types include precursor T-cell leukemia / lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma, Burkitt lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosis type of Hodgkin lymphoma, mixed cellularity subtype of Hodgkin lymphoma. Lung tumor types include non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) and small cell lung cancer tumors.

[0308] In one embodiment of the invention, the cancer may be a microsatellite instability-high (MSI-H) cancer, and / or a deficient mismatch repair (dMMR) cancer, and / or a cancer associated with high tumor mutational burden (i.e., TMB-high).

[0309] In one embodiment of the invention, the cancer may be mesothelioma.

[0310] The method of the invention comprises: (a) administering to a subject a therapeutically effective amount of an antibody that specifically binds to CD137; and (b) systemically administering to the subject a therapeutically effective amount of an additional immunotherapeutic agent, where the additional immunotherapeutic agent is a PD-1 inhibitor, and optionally, the PD-1 inhibitor is administered systemically. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed with step (a) being provided sequentially prior to step (b). In step (a), the anti-CD137 antibody is preferably administered systemically to the tumor, and most preferably, the anti-CD137 antibody is administered intravenously.

[0311] A "therapeutically effective amount" of a substance means that a given substance is administered to a subject suffering from a condition in an amount sufficient to cure, alleviate, or partially suppress the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods. The effective amount for a given purpose and a given agent depends on the severity of the disease or injury, as well as the weight and general condition of the subject. As used herein, the term "subject" includes any mammal, preferably a human.

[0312] The present invention also provides the following: - an antibody that specifically binds to CD137 for use in a method for treating a solid tumor in a subject, the method comprising: (a) administering to the subject a therapeutically effective amount of an antibody that specifically binds to CD137 as described above; and (b) systemically administering to the subject a therapeutically effective amount of a PD-1 inhibitor. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, with step (a) being provided prior to step (b). In step (a), the anti-CD137 antibody as described above is preferably administered locally to the tumor. - Use of an antibody that specifically binds to CD137 in the manufacture of a medicament for treating a solid tumor in a subject, the treating comprising: (a) administering to the tumor a therapeutically effective amount of said antibody that specifically binds to CD137; and (b) systemically administering to the subject a therapeutically effective amount of a PD-1 inhibitor. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed with step (a) being provided sequentially prior to step (b). In step (a), said anti-CD137 antibody is preferably administered systemically, most preferably said anti-CD137 antibody is administered intravenously. - A product containing (1) an antibody that specifically binds to CD137 and (2) a PD-1 inhibitor for simultaneous, separate or sequential use in a method for treating a solid tumor in a subject, the method comprising (a) systemically administering to the tumor a therapeutically effective amount of said antibody that specifically binds to CD137, and optionally (b) systemically administering to the subject a therapeutically effective amount of a PD-1 inhibitor. Steps (a) and (b) may be performed simultaneously. Alternatively, steps (a) and (b) may be performed sequentially, with step (a) being provided prior to step (b). In step (a), the anti-CD137 antibody described above is preferably administered locally to the tumor.

[0313] Timing and sequence of steps (a) and (b) In one embodiment, steps (a) and (b) may be performed sequentially (i.e., at different times), with step (a) being performed before step (b).

[0314] Steps (a) and (b) may be separated by an interval such that the combined antitumor effect is optimized. Step (b) may be performed at a sufficiently long interval after step (a) that at least one physiological effect of step (a) is at or near its peak level. For example, anti-CD137 antibodies typically stimulate CD137 and activate T cells and / or other immune cells (e.g., to induce the release of interferon gamma from CD8+ cells). Activated T cells may begin to express higher levels of immune system checkpoint molecules (such as PD-1) within about 24 hours of treatment with anti-CD137. These immune system checkpoint molecules may negatively regulate antitumor responses. The additional immunotherapeutic agent administered in step (b) is a PD-1 inhibitor, and thus may preferably be an anti-PD-1 or anti-PDL1 antibody that blocks or inhibits such activity of PD-1. If the further immunotherapeutic agent administered in step (b) is such an agent, step (b) may be performed at a sufficiently long interval after step (a) such that the expression level of an immune system checkpoint molecule (such as PD-1) in the subject's cells, or the number of cells in the subject expressing said immune system checkpoint molecule, is elevated relative to the level or number mentioned above in the subject before step (a) or relative to the level or number mentioned above in a healthy subject. In this context, step (b) is performed within 24 hours after step (a), between 24 hours and 2 weeks after step (a), between 24 hours and 1 week after step (a), between 24 hours and 72 hours after step (a), or between 24 hours and 48 hours after step (a). Preferably, step (b) is performed within 24 hours after step (a).

[0315] Alternatively, step (b) may be performed at a point during step (a) if it is determined that the level of expression of an immune system checkpoint molecule (such as PD-1) in the subject's cells, or the number of cells in the subject expressing said immune system checkpoint molecule, is elevated relative to said level or number in the subject prior to step (a), or relative to said level or number in a healthy subject.

[0316] The expression level of an immune system checkpoint molecule (such as PD-1) in cells of a subject, or the number of cells in a subject that express such a molecule, may be determined by any suitable means, for example, by flow cytometric analysis of a sample taken from the subject.

[0317] Alternatively, in the most preferred embodiment, steps (a) and (b) are performed on the same day. It may be preferable to perform steps (a) and (b) simultaneously (i.e., at the same time) or within 24 hours of each other, so that both steps can be performed on the same day or during the same visit to the treatment center. This may be particularly advantageous when access to the treatment center is limited. In this context, steps (a) and (b) may be performed simultaneously, or may be performed less than 24 hours apart, less than 12 hours apart, less than 10 hours apart, less than 6 hours apart, less than 4 hours apart, less than 3 hours apart, or less than 2 hours apart.

[0318] In further embodiments, steps (a) and (b) are performed simultaneously or step (b) is performed between 24 hours and 2 weeks after step (a), between 24 hours and 1 week after step (a), between 24 hours and 72 hours after step (a), or between 24 hours and 48 hours after step (a).

[0319] In any of the above-mentioned embodiments, step (a) may be performed on multiple additional occasions after the first occasion. That is, the subject may receive a series of doses of anti-CD137 antibody. These doses are administered so that the subject has only intermittent exposure to the anti-CD137 antibody, preferably so that the subject's immune cells are not depleted and / or the subject does not suffer from tachyphylaxis to the anti-CD137 antibody. Upon detection of any of these symptoms, the next administration of anti-CD137 antibody may be delayed or canceled. When multiple doses of anti-CD137 are administered, step (b) is preferably performed in a manner that allows continuous exposure of the subject to the additional immunotherapeutic agent (PD-1 inhibitor) during the method, including any second and further occasions of step (a), after the initiation of step (b). This may be particularly appropriate when the additional agent is an anti-PD-1 or anti-PDL1 antibody that blocks or inhibits such activity of the immune system checkpoint molecule PD-1. Continuous receptor blockade may be particularly important for the therapeutic effect of such agents.

[0320] Thus, in one embodiment, step (a) is performed on multiple separate occasions and step (b) is performed such that exposure of the subject to the additional immunotherapeutic agent is continuous over the duration of the method.

[0321] Step (a) Step (a) of the method involves local or systemic administration of an anti-CD137 antibody to a subject with a solid tumor. Preferably, step (a) involves systemic administration of the anti-CD137 antibody, for example by intravenous or subcutaneous administration. In a most preferred embodiment, step (a) involves intravenous administration of the anti-CD137 antibody.

[0322] In alternative embodiments, the anti-CD137 antibody, or antigen-binding fragment thereof, is administered locally to the subject at the site of the tumor. Local administration to the tumor site includes peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracranial and intravesical administration by any suitable means, such as injection. Local administration can also include intracavity injection and inhalation, depending on the site of the tumor.

[0323] A high percentage of anti-CD137 antibodies can be retained at the tumor site in vivo, i.e., within the tumor microenvironment, for extended periods following administration of the antibodies described above. That is, the antibodies exhibit reduced leakage from the tumor site into the vascular or lymphatic circulation, especially when administered locally to the tumor site. Preferably, at least 30% of the antibody dose administered to a tumor according to the present methods is retained at the tumor site 4 hours after administration, more preferably, at least 40% of the dose is retained 4 hours after administration, and most preferably, at least 50% of the dose is retained 4 hours after administration.

[0324] "Retained at the site of a solid tumor" includes that the anti-CD137 antibody is only slowly released from the tumor region. Antibody retention in the tumor microenvironment can be studied by injecting the antibody into the tumor in a mouse model and measuring the serum levels of the antibody over time after administration. Alternatively, the distribution of the antibody can be measured using a radiolabeled antibody injected into the tumor in a mouse model. Suitable techniques are known to those skilled in the art. For example, the retention of the antibody at the tumor site can be assessed by monitoring the serum levels of the antibody after administration (see Mangsbo et al., 2014, Clin. Cancer Res. 21(5):1115-1126, the disclosure of which is incorporated herein by reference). For example, in one embodiment, the serum levels of anti-CD137 4 hours after intratumoral injection of 30 μg (in 60 μL) of the antibody are less than 1 μL / ml.

[0325] Step (b) Step (b) of the method involves systemic administration of a PD-1 inhibitor to a subject. Systemic administration of any agent described herein (including the anti-CD137 antibody of step (a)) refers to administration to the subject's circulatory system, including the vascular and / or lymphatic systems. Such administration may be by any suitable route, but is typically parenteral.

[0326] Thus, in one embodiment, the PD-1 inhibitor is administered locally to the subject at the site of the tumor. In one embodiment, the PD-1 inhibitor is administered systemically to the subject, e.g., intravenously or subcutaneously. In a preferred aspect, the systemic administration of the PD-1 inhibitor is intravenous.

[0327] As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, which are typically accomplished by injection, infusion, or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, intracerebral, intrathecal, intraosseous, or other parenteral routes of administration.

[0328] Kits and Pharmaceutical Compositions The present invention also provides a kit for treating cancer, preferably solid tumors, in a subject, comprising a combination therapy as defined above. For example, the kit may comprise (a) a therapeutically effective amount of an antibody that specifically binds to CD137 and optionally is retained at the tumor site after administration, and (b) a therapeutically effective amount of a PD-1 inhibitor. The antibody that specifically binds to CD137 is preferably provided in a form suitable for local administration to the tumor site.

[0329] The kits of the invention may further comprise one or more other reagents or equipment that enable any of the above-described embodiments to be carried out, including one or more of a suitable buffer(s) (aqueous solutions) and a means for administering the anti-CD137 antibody and / or PD-1 inhibitor (e.g., a container or equipment including a needle).

[0330] The anti-CD137 antibody and PD-1 inhibitor used in the methods of the invention or provided in the kits of the invention can each be provided as separate pharmaceutical compositions formulated with a pharma- ceutically acceptable carrier. As used herein, a "pharma- ceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and compatible with the desired route of administration.

[0331] Thus, the carrier of the anti-CD137 antibody and PD-1 inhibitor may be suitable for systemic administration, which as defined above means administration to the circulatory system of a subject, including the vascular and / or lymphatic systems. Such administration may be by any suitable route, but is typically parenteral. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, which are typically achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration.

[0332] However, the anti-CD137 antibody carrier is preferably suitable for local administration, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracranial and intravesical administration, as defined above, by any suitable means, such as injection. Local administration may also include intracavity injection and inhalation, depending on the site of the tumor.

[0333] Depending on the route of administration, the antibody and / or drug may be coated with a material to protect the antibody from the action of acids and other natural conditions that may inactivate or denature the antibody and / or drug. Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, buffered water, and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include an isotonic agent, for example, a sugar, a polyalcohol such as mannitol, sorbitol, or sodium chloride in the composition.

[0334] Those skilled in the art will understand that the antibody components of the combination therapies of the invention will typically be provided in the form of one or more pharmaceutical compositions, each containing a therapeutically effective amount of the antibody component(s) together with a pharma- ceutically acceptable buffer, excipient, diluent or carrier.

[0335] It will be appreciated by those skilled in the art that the pharmaceutical compositions may also include additional compounds, including chelating agents such as EDTA, citrate, EGTA, or glutathione.

[0336] "Pharmaceutically acceptable" refers to a non-toxic substance that does not reduce the effectiveness of the CD137 binding activity of the antibody polypeptide of the present invention. Such pharma-ceutically acceptable buffers, carriers or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company (1990), and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

[0337] As used herein, "therapeutically effective amount" or "effective amount" or "therapeutically effective" refers to an amount that provides a therapeutic effect for a given condition and administration regimen. It is a predetermined amount of active antibody calculated to produce a desired therapeutic effect in association with necessary additives and diluents, i.e., carriers or administration vehicles. It is further intended to mean an amount sufficient to reduce or prevent clinically significant deficits in host activity, function, and response. Alternatively, a therapeutically effective amount is an amount sufficient to produce an improvement in a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. A suitable dosage may contain a predetermined amount of active composition calculated to produce a desired therapeutic effect in association with necessary diluents.

[0338] A therapeutically effective amount can be determined by an ordinarily skilled medical or veterinary practitioner based on patient characteristics such as age, weight, sex, condition, comorbidities, other diseases, etc., as is well known in the art.

[0339] Pharmaceutical compositions may contain pharma- ceutically acceptable antioxidants. These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures (see above) and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents in the compositions, such as sugars, sodium chloride, and the like. In addition, prolonged absorption of the injectable formulation can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0340] Pharmaceutical compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. Sterile injectable solutions can be prepared by incorporating the required amount of active agent (e.g., antibody) in a suitable solvent, which may contain one or a combination of the above-listed ingredients as needed, followed by sterile microfiltration. In general, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is high vacuum drying and freeze-drying (lyophilization), which obtains a powder of the active agent and any additional desired ingredients from its previously sterile-filtered solution. Pharmaceutical compositions can contain additional active ingredients, as well as those listed above.

[0341] Suitable pharma- ceutically acceptable buffers, diluents, carriers and excipients are well known in the art (Remington's Pharmaceutical Sciences, 18 thedition, AR Gennaro, Ed., Mack Publishing Company (1990), and handbook of Pharmaceutical Excipients, 3 rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference.

[0342] The term "buffer" is intended to include an aqueous solution containing an acid-base mixture for the purpose of stabilizing pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartaric acid, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0343] The term "diluent" is intended to include aqueous or non-aqueous solutions for the purpose of diluting a drug in a pharmaceutical preparation. The diluent may be one or more selected from saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil).

[0344] The term "adjuvant" is intended to include any compound added to the formulation to increase the biological effect of the agent of the present invention. The adjuvant may be one or more zinc salts, copper salts or silver salts with different anions, such as, but not limited to, fluorides, chlorides, bromides, iodides, thiocyanates, sulfites, hydroxides, phosphates, carbonates, lactates, glycolates, citrates, borates, tartrates and acetates with different acyl compositions. The adjuvant may also be a cationic polypeptide, such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinylimidazole), and cationic polypeptides such as polyhistines, polylysines, polyarginines and peptides containing these amino acids.

[0345] The excipient may be one or more of a carbohydrate, a polymer, a lipid, and a mineral. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, cyclodextrin, etc., which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginate, carrageenan, hyaluronic acid and their derivatives, polyacrylic acid, polysulfonic acid, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, and polyvinylpyrrolidone, all of different molecular weights, which are added to the composition, for example, to control viscosity, achieve bioadhesion, or protect lipids from chemical and proteolytic degradation. Examples of lipids include fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids, and glycolipids, all with different acyl chain lengths and degrees of saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, etc., are added to the composition for reasons similar to those of polymers. Examples of minerals include talc, magnesium oxide, zinc oxide, and titanium dioxide, which are added to the composition for benefits such as reduced pooling and favorable pigment properties.

[0346] The active antibody-based agents of the present invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for their delivery.

[0347] In one embodiment, the pharmaceutical composition of the present invention may be in the form of liposomes, in which the drug is combined with amphiphilic agents such as lipids that exist in aggregated form as micelles, insoluble monolayers, and liquid crystals, in addition to other pharma- ceutically acceptable carriers. Suitable lipids for liposomal formulations include, but are not limited to, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, and the like. Suitable lipids also include the above lipids modified with poly(ethylene glycol) in the polar head group to extend bloodstream circulation time. Preparation of such liposomal formulations can be found, for example, in US 4,235,871 and EP 0 213 303, the disclosures of which are incorporated herein by reference.

[0348] The pharmaceutical composition of the present invention may also be in the form of biodegradable microparticles. Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL) and polyanhydrides are widely used as biodegradable polymers in the production of microparticles. Preparations of such microparticles can be found in US 5,851,451 and EP 0 213 303, the disclosures of which are incorporated herein by reference.

[0349] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of nanoparticles, for example based on poly-gamma glutamic acid.Details of the preparation and use of such nanoparticles can be found in WO2011 / 128642, the disclosure of which is incorporated herein by reference.Those skilled in the art will understand that one or more of the active ingredients of the combination therapy of the present invention may be formulated in separate nanoparticles, or both active ingredients may be formulated in the same nanoparticle.

[0350] In a further embodiment, the pharmaceutical composition of the present invention is provided in the form of a polymer gel, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulfonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate with different degrees of hydrolysis, polyvinylpyrrolidone are used to thicken the solution containing the drug. The polymer may also include gelatin and collagen.

[0351] Alternatively, the drugs may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil), tragacanth gum, and / or various buffers.

[0352] It is understood that the pharmaceutical compositions of the present invention may contain ions and a defined pH to enhance the action of the active agent. In addition, the compositions may be subjected to conventional pharmaceutical operations such as sterilization, and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0353] The pharmaceutical compositions according to the present invention can be administered by any suitable route known to those skilled in the art. Thus, routes of administration include parenteral (intravenous, subcutaneous, intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, vaginal, and rectal. Administration via implants is also possible.

[0354] Advantageously, the pharmaceutical composition is suitable for administration at or near the site of a tumor, for example, intratumoral or peritumoral.

[0355] The pharmaceutical composition is preferably suitable for parenteral administration, for example the pharmaceutical composition is preferably suitable for administration intravenously, intraventricularly, intraarticularly, intra-articularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or by infusion techniques. Methods for formulating antibodies into pharmaceutical compositions, such as pharmaceutical compositions suitable for parenteral administration, will be well known to those skilled in the art of medicine and pharmacy. Preferred compositions are described in the accompanying examples.

[0356] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may also contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials, and may be stored in a lyophilized (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0357] Thus, the pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, for example intravenous administration.

[0358] The combination therapy of the present invention can be delivered using an injectable sustained release drug delivery system. These are specifically designed to reduce injection frequency. One example of such a system is Nutropin Depot, which encapsulates recombinant human growth hormone (rhGH) in biodegradable microparticles that, once injected, slowly release rhGH over a sustained period of time. Preferably, delivery is intramuscular (im) and / or subcutaneous (sc) and / or intravenous (iv).

[0359] The combination therapy of the present invention can be administered by a surgically implanted device that releases the drug directly to the required area. For example, Vitrasert releases ganciclovir directly to the eye to treat CMV retinitis. By applying this toxic agent directly to the diseased area, effective therapy is achieved without the significant systemic side effects of the drug.

[0360] Electroporation therapy (EPT) systems can also be used for administration of the combination therapy of the present invention. Devices that deliver pulsed electric fields to cells increase the permeability of cell membranes to drugs, resulting in significantly enhanced intracellular drug delivery.

[0361] The combination therapy of the present invention can also be delivered by electroincorporation (EI). EI occurs when small particles, up to 30 microns in diameter, on the surface of the skin are subjected to an electrical pulse identical or similar to that used in electroporation. In EI, these particles are driven through the stratum corneum into deeper layers of the skin. The particles can carry or be coated with drugs or genes, or simply act as "bullets" that generate holes in the skin through which drugs can enter.

[0362] An alternative combination therapy of the present invention is the ReGel injection system, which is temperature sensitive. Below body temperature, ReGel is an injectable liquid, but at body temperature it quickly forms a gel reservoir that slowly erodes and dissolves into a known, safe, biodegradable polymer. The active agent is delivered over time as the biopolymer dissolves.

[0363] The combination therapy of the present invention may also be delivered orally. This process involves co-delivering proteins and peptides by ingesting vitamin B 2 in the body. 12 and / or employing natural processes for oral uptake of vitamin D. 12 and / or by engaging the vitamin D uptake system, the agents, medicaments and pharmaceutical compositions of the present invention are capable of transporting through the intestinal wall. 12 Vitamin B analogues and / or vitamin D analogues in complexes 12 The compound is a compound between a drug moiety / vitamin D moiety that retains both significant affinity for intrinsic factor (IF) and significant biological activity of the active substance of the complex.

[0364] The combination therapy of the present invention can be introduced into cells by "Trojan peptides". These are a type of polypeptide called penetratins that have translocation properties and can carry hydrophilic compounds across the plasma membrane. This system allows for direct targeting of oligopeptides to the cytoplasm and nucleus, is not cell type specific, and can be very efficient. See Derossi et al. (1998), Trends Cell Biol. 8, 84-87.

[0365] Preferably, the combination therapy of the present invention is a unit dosage containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of the active ingredient.

[0366] The combination therapy of the present invention is usually administered in the form of a pharmaceutical composition containing the active ingredients, optionally in the form of non-toxic organic or inorganic acid or base addition salts in a pharma- ceutically acceptable dosage form, by oral administration or any parenteral route. Depending on the disorder and patient to be treated, and the route of administration, the compositions may be administered in various doses.

[0367] In human therapy, the combination therapies of the present invention can be administered alone, but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

[0368] For example, the combination therapy of the present invention may be administered orally, buccally or sublingually in the form of a tablet, capsule, ovule, elixir, solution or suspension for immediate, delayed or controlled release applications, and may contain flavoring or coloring agents. The agents, drugs and pharmaceutical compositions of the present invention may also be administered via intracavernous injection.

[0369] Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0370] Solid compositions of a similar type may also be used as fillers for gelatin capsules.Preferred excipients in this respect include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.For aqueous suspensions and / or elixirs, the medicaments, medicaments and pharmaceutical compositions of the present invention may be combined with various sweeteners or flavorings, colorants or pigments, emulsifying and / or suspending agents, and diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

[0371] The combination therapies of the invention may be administered parenterally, e.g., intravenously, intraarterially, intraperitoneally, intramedullary, intraventricular, intrasternal, intracranial, intramuscular, or subcutaneously, or by infusion techniques. For example, they are best used in the form of a sterile aqueous solution, which may contain sufficient salts and other substances, such as glucose, to make the solution isotonic with blood. The aqueous solution should be suitably buffered (preferably to a pH of 3-9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0372] Medicines and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions (which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient) and aqueous and non-aqueous sterile suspensions (which may contain suspending agents and thickening agents). Medicines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0373] Thus, the pharmaceutical compositions of the present invention are particularly suitable for parenteral administration, for example intravenous administration.

[0374] The combination therapy of the invention may also be administered intranasally or by inhalation, conveniently in the form of a dry powder inhalant or in the form of a fluorous liquid, such as, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA The active agent is delivered in the form of an aerosol spray presentation from a pressurized container, pump, spray, or nebulizer using a suitable propellant, such as a hydrofluoroalkane, such as 227EA3, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebulizer may contain a solution or suspension of the active agent, for example, using a mixture of ethanol and a propellant as a solvent, which may further contain a lubricant, for example, sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mix of the agent of the invention and a suitable powder base, such as lactose or starch.

[0375] Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff" contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the total daily dosage administered by aerosol will vary from patient to patient and can be administered in a single administration or, more usually, in divided doses throughout the day.

[0376] Alternatively, the combination therapy of the present invention can be administered in the form of a suppository or pessary, or may be applied topically in the form of a lotion, solution, cream, gel, ointment or dusting powder. The agents, medicaments and pharmaceutical compositions of the present invention may also be administered transdermally, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

[0377] For ophthalmic use, the combination therapy of the invention can be formulated as a micronized suspension in isotonic, pH-adjusted, sterile saline, optionally combined with a preservative such as benzylalkonium chloride, or, preferably, as a solution in isotonic, pH-adjusted, sterile saline, or can be incorporated into an ointment such as petrolatum.

[0378] For topical application to the skin, the combination therapy of the invention can be formulated, for example, as a suitable ointment containing the active agent suspended or dissolved in a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax, and water. Alternatively, it can be formulated as a suitable lotion or cream, for example, suspended or dissolved in a mixture of one or more of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0379] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0380] Generally, in humans, local administration of the combination therapy of the invention at or near the site of a tumor, particularly intratumoral or peritumoral administration, is the preferred route.

[0381] The pharmaceutical composition will be administered to the patient in a pharma- ceutical effective dose / therapeutically effective amount, as described above. In the method of making and using the composition of the present invention, a therapeutically effective amount of the active ingredient is provided. The therapeutically effective amount can be determined by an ordinary skilled medical or veterinary practitioner based on patient characteristics such as age, weight, sex, condition, comorbidities, other diseases, etc., as is well known in the art.

[0382] The administration of a pharma- ceutically effective dose can be carried out both by single administration in the form of individual dose units or several smaller dose units, and by multiple administration of subdivided doses at specific intervals. Alternatively, the dose can be provided as a continuous infusion over time. The antibody polypeptide can be formulated in various concentrations depending on the efficacy / toxicity of the polypeptide used. For example, the formulation can contain the active antibody polypeptide in a concentration of 0.1 μM to 1 mM, more preferably 1 μM to 500 μM, 500 μM to 1 mM, 300 μM to 700 μM, 1 μM to 100 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 800 μM to 900 μM, or 900 μM to 1 mM. Typically, the formulation contains the active antibody polypeptide at a concentration of from 300 μM to 700 μM.

[0383] Typically, a therapeutic dose of an antibody polypeptide in a human patient (with or without a therapeutic moiety) ranges from 100 μg to 1 g per administration (e.g., 300 μg to 700 mg per administration, based on a body weight of 70 kg). For example, a maximum therapeutic dose may range from 0.1 to 10 mg / kg per administration, e.g., 1 to 10 mg / kg, or 0.1 to 5 mg / kg, or 1 to 5 mg / kg, or 0.1 to 2 mg / kg. Most preferably, the therapeutic dose is 1 to 10 mg / kg, optionally 2.5 to 7.5 mg / kg. It will be understood that such doses may be administered at different intervals as determined by the oncologist / physician, e.g., doses may be administered daily, twice weekly, weekly, biweekly, or monthly.

[0384] Those skilled in the art will further appreciate that the polypeptides and pharmaceutical formulations of the present invention have utility in both the medical and veterinary fields. Thus, the methods of the present invention can be used in the treatment of both humans and non-human animals, such as horses, dogs, and cats. Preferably, however, the patient is a human.

[0385] For veterinary use, the combination therapies of the present invention will be administered in a suitably acceptable formulation in accordance with normal veterinary practice, with the veterinarian determining the administration regime and route of administration that will be most suitable for a particular animal.

[0386] The invention also provides a kit for treating a solid tumor in a subject, the kit comprising: (a) a therapeutically effective amount of an antibody that specifically binds to CD137; and (b) a therapeutically effective amount of a further immunotherapeutic agent suitable for systemic administration to the subject. The further immunotherapeutic agent is optionally a PD-1 inhibitor as defined in the first aspect. The antibody that specifically binds to CD137 is preferably provided in a form suitable for local administration to the tumor.

[0387] Nucleic Acids, Vectors, and Hosts The present invention also relates to a method for the preparation of a method for the preparation of a medicament for the treatment of a pulmonary artery disease comprising administering to a patient a therapeutically effective amount ... (i) a PD-1 inhibitor, and / or (ii) a second isolated nucleic acid molecule (or a component peptide chain thereof) encoding an antibody to PD-1 or an antigen-binding fragment thereof that specifically binds to PD-1.

[0388] "Nucleic acid molecule" includes DNA, which may be single-stranded or double-stranded (e.g., genomic or complementary DNA), and mRNA molecules. "Isolated" means that the nucleic acid molecule is not located or otherwise provided within a cell.

[0389] In one embodiment, the first and / or second nucleic acid molecule(s) is / are a cDNA molecule(s).

[0390] In one embodiment, the first and / or second isolated nucleic acid molecule encodes an antibody heavy chain or a variable region thereof, and / or encodes an antibody light chain or a variable region thereof.

[0391] Preferably, the first nucleic acid molecule comprises one or more nucleotide sequences selected from either SEQ ID NO: 9 and SEQ ID NO: 10, reproduced below. Nucleotide sequence encoding the VH region of "1630" GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTGGTTACTCTTACATGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTGGTTCTGGTTCTTCTTACACATAC TATGCAGACTCCGTGAAGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGTTTACTCTTCTCCGGGTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA [SEQ ID NO: 9] Nucleotide sequence encoding the VL region of "1631" GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACACTTGGGTTCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA [SEQ ID NO: 10]

[0392] In an alternative preferred embodiment, the first nucleic acid molecule comprises one or more nucleotide sequences selected from either SEQ ID NO: 27 and / or SEQ ID NO: 28, reproduced below. Nucleotide sequence encoding the VH region of "2674" [SEQ ID NO:27] Nucleotide sequence encoding the VL region of "2675" gacatccagatgacccagtctccatcctctctgtctgcctctgtgggcgacagagtgaccatcacctgtcgggcttctcagtccatcggcagcaccctgaactggtatcagcagaagcctggcaaggcccctaagctgctgatctatggcgctagctctct gcagtctggcgtgccctctagattttccggctctggctctggcaccgacttcaccctgacaatcagttccctgcagcctgaggacttcgccacctactactgccagcagtactacacctgggtgccctttacctttggccagggcaccaagctggaaatca agagaaccgtggccgctccttccgtgttcatcttcccaccatctgacgagcagctgaagtccggcacagcttctgtcgtgtgcctgctgaacaacttctaccctcgggaagccaaggtgcagtggaaggtggacaatgccctgcagtccggcaactcccaa gagtctgtgaccgagcaggactccaaggactctacctacagcctgtcctccacactgaccctgtctaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccatcagggactgtctagccccgtgaccaagtccttcaacagaggcgagtgt [SEQ ID NO:28]

[0393] It will be understood by those of skill in the art that the first nucleic acid molecule can be codon optimized for expression of the antibody polypeptide in a particular host cell, e.g., expression in a human cell (see, e.g., Angov, 2011, Biotechnol. J. 6(6):650-659, the disclosure of which is incorporated herein by reference).

[0394] The present invention also relates to a first isolated nucleic acid molecule encoding an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, or a component peptide chain thereof; (i) a PD-1 inhibitor, and / or (ii) a second isolated nucleic acid molecule encoding an antibody, or an antigen-binding fragment thereof, that specifically binds to PD-1, or a component peptide chain thereof, and a vector comprising the second isolated nucleic acid molecule.

[0395] In one embodiment, the vector is an expression vector.The first and / or second isolated nucleic acid may be as described above.

[0396] The present invention also relates to a first isolated nucleic acid molecule encoding an antibody that specifically binds to CD137, or an antigen-binding fragment thereof, or a component peptide chain thereof; (i) a PD-1 inhibitor, and / or (ii) a second isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, or a component peptide chain thereof.

[0397] Preferably, the host cell may be a mammalian cell (e.g., a human cell, or a Chinese hamster ovary cell, e.g., a CHOK1SV cell), a bacterial cell, or a yeast cell. The first and / or second isolated nucleic acid may be as described above. The first and / or second isolated nucleic acid may be comprised in a vector, such as an expression vector.

[0398] Brief Description of the Sequence Listing Sequence number 1 is the amino acid sequence of the VH region of "1630".

[0399] Sequence number 2 is the amino acid sequence of the VL region of "1631".

[0400] SEQ ID NO:3 is the amino acid sequence of HCDR1 of "1630".

[0401] SEQ ID NO:4 is the amino acid sequence of HCDR2 of "1630".

[0402] SEQ ID NO:5 is the amino acid sequence of HCDR3 of "1630".

[0403] SEQ ID NO:6 is the amino acid sequence of LCDR1 of "1631".

[0404] SEQ ID NO:7 is the amino acid sequence of LCDR2 of "1631".

[0405] SEQ ID NO:8 is the amino acid sequence of LCDR3 of "1631".

[0406] SEQ ID NO:9 is the nucleotide sequence encoding the VH region of "1630".

[0407] SEQ ID NO:10 is the nucleotide sequence encoding the VL region of "1631".

[0408] SEQ ID NO: 11 is the amino acid sequence of human CD137 (amino acids 66 to 107 correspond to domain 2 of human CD137).

[0409] SEQ ID NO: 12 is the amino acid sequence of the IgG1 heavy chain constant region.

[0410] SEQ ID NO: 13 is the amino acid sequence of the modified IgG4 constant region.

[0411] SEQ ID NO: 14 is the amino acid sequence of the modified IgG4 constant region.

[0412] SEQ ID NO: 15 is the amino acid sequence of the wild-type IgG4 constant region.

[0413] SEQ ID NO:16 is the amino acid sequence of the kappa chain constant region.

[0414] SEQ ID NO:17 is the complete amino acid sequence of the heavy chain of "1630".

[0415] SEQ ID NO:18 is the complete amino acid sequence of the light chain of "1631".

[0416] Sequence number 19 is the amino acid sequence of the VH region of "2674".

[0417] SEQ ID NO: 20 is the amino acid sequence of the VL region of "2675".

[0418] SEQ ID NO: 21 is the amino acid sequence of HCDR1 of "2674".

[0419] SEQ ID NO: 22 is the amino acid sequence of HCDR2 of "2674".

[0420] SEQ ID NO: 23 is the amino acid sequence of HCDR3 of "2674".

[0421] SEQ ID NO: 24 is the amino acid sequence of LCDR1 of "2675".

[0422] SEQ ID NO: 25 is the amino acid sequence of LCDR2 of "2675".

[0423] SEQ ID NO: 26 is the amino acid sequence of LCDR3 of "2675".

[0424] SEQ ID NO:27 is the nucleotide sequence encoding the VH region of "2674".

[0425] SEQ ID NO:28 is the nucleotide sequence encoding the VL region of "2675".

[0426] SEQ ID NO:29 is the complete amino acid sequence of the heavy chain "2674".

[0427] SEQ ID NO:30 is the complete amino acid sequence of the light chain of "2675".

[0428] SEQ ID NO:31 is the heavy chain amino acid sequence of nivolumab.

[0429] SEQ ID NO:32 is the light chain amino acid sequence of nivolumab.

[0430] SEQ ID NO: 33 is the heavy chain amino acid sequence of pembrolizumab.

[0431] SEQ ID NO: 34 is the light chain amino acid sequence of pembrolizumab.

[0432] SEQ ID NO:35 is the amino acid sequence of the human PD-1 sequence.

[0433] SEQ ID NO: 36 is the amino acid sequence of human PD-L1 sequence.

[0434] SEQ ID NO: 37 is the heavy chain sequence of pidilizumab.

[0435] SEQ ID NO: 38 is the light chain sequence of pidilizumab.

[0436] SEQ ID NO: 39 is the heavy chain sequence of cemiplimab.

[0437] SEQ ID NO: 40 is the light chain sequence of cemiplimab.

[0438] SEQ ID NO: 41 is the heavy chain sequence of spartalizumab.

[0439] SEQ ID NO: 42 is the light chain sequence of spartalizumab.

[0440] SEQ ID NO: 43 is the heavy chain sequence of camrelizumab.

[0441] SEQ ID NO: 44 is the light chain sequence of camrelizumab.

[0442] SEQ ID NO: 45 is the heavy chain sequence of tislelizumab.

[0443] SEQ ID NO: 46 is the light chain sequence of tislelizumab.

[0444] SEQ ID NO: 47 is the heavy chain sequence of toripalimab.

[0445] SEQ ID NO: 48 is the light chain sequence of toripalimab.

[0446] SEQ ID NO: 49 is the heavy chain sequence of dostarlimab.

[0447] SEQ ID NO:50 is the light chain sequence of dostarlimab.

[0448] SEQ ID NO:51 is the heavy chain sequence of INCMGA00012.

[0449] SEQ ID NO:52 is the light chain sequence of INCMGA00012.

[0450] SEQ ID NO:53 is the heavy chain sequence of atezolizumab.

[0451] SEQ ID NO:54 is the light chain sequence of atezolizumab.

[0452] SEQ ID NO:55 is the heavy chain sequence of durvalumab.

[0453] SEQ ID NO:56 is the light chain sequence of durvalumab.

[0454] SEQ ID NO:57 is the heavy chain sequence of avelumab.

[0455] SEQ ID NO:58 is the light chain sequence of avelumab.

[0456] SEQ ID NO:59 is the heavy chain sequence of CK-301.

[0457] SEQ ID NO:60 is the light chain sequence of CK-301.

[0458] SEQ ID NO:61 is the heavy chain sequence of JTX-4014.

[0459] SEQ ID NO: 62 is the light chain sequence of JTX-4014.

[0460] Embodiments of the present invention include, but are not limited to, the following.

[0461] A. A method for treating a solid tumor in a subject, the method comprising: (a) administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, that specifically binds to CD137 (preferably which is retained at the tumor site following administration); and (b) systemically administering to the subject a therapeutically effective amount of an additional immunotherapeutic agent, where the additional immunotherapeutic agent is a PD-1 inhibitor.

[0462] B. The method of embodiment A, wherein the PD-1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody.

[0463] C. The method of any one of the preceding embodiments, wherein the solid tumor is an adenoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms tumor, lung tumor, colon tumor, lymphoid tumor, breast tumor, or melanoma.

[0464] D. The method of any one of the preceding embodiments, wherein the solid tumor is a lung tumor (e.g., non-small cell lung cancer or small cell lung cancer); a head and / or neck tumor, a gastric tumor, an esophageal tumor, a renal tumor, a urothelial tumor, an MSI high tumor, a dMMR tumor, a TMB high tumor, a breast tumor, a cervical tumor, a prostate tumor, or a melanoma, preferably, the solid tumor is metastatic.

[0465] E. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises at least one CDR selected from SEQ ID NOs: 3, 4, 5, 6, 7 and 8.

[0466] F. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises the CDR sequences of SEQ ID NOs: 3, 4, and 5 and / or SEQ ID NOs: 6, 7, and 8.

[0467] G. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises a light chain variable region of SEQ ID NO:2 and / or a heavy chain variable region of SEQ ID NO:1.

[0468] H. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises a light chain constant region of SEQ ID NO: 16 and / or a heavy chain constant region of SEQ ID NO: 13.

[0469] I. The method of any one of the preceding embodiments, wherein the antibody of step (a) competes for binding to human CD137 with an antibody comprising a light chain variable region of SEQ ID NO:2 and a heavy chain variable region of SEQ ID NO:1.

[0470] J. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises at least one CDR selected from 21, 22, 23, 24, 25 and 26, and optionally wherein the antibody of step (a) comprises the CDR sequences of SEQ ID NOs: 21, 22 and 23 and / or SEQ ID NOs: 24, 25 and 26.

[0471] K. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises a light chain variable region of SEQ ID NO:20 and / or a heavy chain variable region of SEQ ID NO:19.

[0472] L. The method of any one of the preceding embodiments, wherein the antibody of step (a) comprises a light chain constant region of SEQ ID NO: 16 and / or a heavy chain constant region of SEQ ID NO: 13.

[0473] M. The method of any one of the preceding embodiments, wherein the antibody of step (a) competes for binding to human CD137 with an antibody comprising a light chain variable region of SEQ ID NO: 20 and a heavy chain variable region of SEQ ID NO: 19.

[0474] N. The method of any one of the preceding embodiments, wherein steps (a) and (b) are performed simultaneously or step (b) is performed within 24 hours after step (a), between 24 hours and 2 weeks after step (a), between 24 hours and 1 week after step (a), between 24 hours and 72 hours after step (a), or between 24 hours and 48 hours after step (a), preferably, step (b) is performed within 24 hours after step (a).

[0475] O. The method of any one of the preceding embodiments, wherein step (a) comprises systemic administration of an antibody to a subject, and optionally, the antibody is formulated as a composition suitable for systemic administration with at least one pharma- ceutically acceptable diluent or carrier.

[0476] P. The method of any one of the preceding embodiments, wherein at least 30% of the amount of antibody administered in step (a) is retained at the tumor site 4 hours after administration, and preferably at least 40% of that amount is retained at the tumor site 4 hours after administration.

[0477] Q. The method of any one of the preceding embodiments, wherein the additional immunotherapeutic agent of step (b) is formulated as a composition suitable for systemic administration with at least one pharma- ceutically acceptable diluent or carrier.

[0478] R. The method of any one of the preceding embodiments, wherein step (a) is performed on multiple separate occasions and step (b) is performed such that exposure of the subject to the additional immunotherapeutic agent is continuous over the duration of the method.

[0479] S. The method of any one of the preceding embodiments, wherein the subject is a human.

[0480] T. A kit for treating a solid tumor in a subject, the kit comprising: (a) a therapeutically effective amount of an antibody that specifically binds to CD137, and preferably retained at the tumor site following administration; and, optionally, (b) a therapeutically effective amount of an additional immunotherapeutic agent suitable for systemic administration to the subject, wherein the additional immunotherapeutic agent is a PD-1 inhibitor.

[0481] U. An antibody, or antigen-binding portion thereof, capable of specifically binding to CD137 and preferably being retained at the tumor site following administration, for use in treating cancer, such as a solid tumor, in a subject in combination with one or more further immunotherapeutic agents, wherein the one or more further immunotherapeutic agent(s) is a PD-1 inhibitor.

[0482] V. The antibody, or antigen-binding portion thereof, of embodiment U, wherein the PD-1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody.

[0483] W. The antibody, or antigen-binding portion thereof, of any of embodiments U or V, wherein the solid tumor is an adenoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms tumor, lung tumor, colon tumor, lymphoid tumor, breast tumor or melanoma.

[0484] X. The antibody, or antigen-binding portion thereof, of any one of embodiments U-W, wherein the solid tumor is a lung tumor (e.g., non-small cell lung cancer or small cell lung cancer); a head and / or neck tumor, a gastric tumor, an esophageal tumor, a renal tumor, a urothelial tumor, a MSI high tumor, a dMMR tumor, a TMB high tumor, a breast tumor, a cervical tumor, a prostate tumor, or a melanoma, preferably wherein the solid tumor is metastatic.

[0485] Y. An antibody of any one of embodiments U to X, or an antigen-binding portion thereof, comprising at least one CDR selected from SEQ ID NOs: 3, 4, 5, 6, 7 and 8.

[0486] Z. An antibody of any one of embodiments U to Y, or an antigen-binding portion thereof, comprising at least one CDR selected from SEQ ID NOs: 21, 22, 23, 24, 25 and 26.

[0487] AA. The antibody of any one of embodiments U to Z, or an antigen-binding portion thereof, comprising the CDR sequences of SEQ ID NOs: 3, 4, 5, 6, 7 and 8 or SEQ ID NOs: 21, 22, 23, 24, 25 and 26.

[0488] BB. An antibody or antigen-binding portion thereof described in any one of embodiments U to AA, comprising a light chain variable region of SEQ ID NO:2 and / or a heavy chain variable region of SEQ ID NO:1, or comprising a light chain variable region of SEQ ID NO:20 and / or a heavy chain variable region of SEQ ID NO:19.

[0489] CC. The antibody of any one of embodiments U to BB, or an antigen-binding portion thereof, comprising a light chain constant region of SEQ ID NO: 16 and / or a heavy chain constant region of SEQ ID NO: 13.

[0490] DD. An antibody or antigen-binding portion thereof described in any one of embodiments U to CC, wherein the antibody, or antigen-binding portion thereof, competes for binding to human CD137 with an antibody comprising a light chain variable region of SEQ ID NO: 1 and a heavy chain variable region of SEQ ID NO: 2, and / or competes for binding to human CD137 with an antibody comprising a light chain variable region of SEQ ID NO: 19 and a heavy chain variable region of SEQ ID NO: 20.

[0491] EE. The antibody, or antigen-binding portion thereof, of any one of embodiments U-DD, wherein the antibody is formulated as a composition suitable for topical administration with at least one pharma- ceutically acceptable diluent or carrier, and the composition comprises a PD-1 inhibitor.

[0492] FF. The combination therapy of embodiment EE, wherein the PD-1 inhibitor is optionally an anti-PD-1 or anti-PD-L1 antibody, as defined according to any aspect herein.

[0493] GG. Use of an antibody, or antigen-binding portion thereof, of any one of embodiments U to DD in the preparation of a medicament for treating a solid tumor in a subject.

[0494] It should be understood that the different applications of the disclosed combination therapy and methods can be tailored to the specific needs of the art. It should also be understood that the terms used herein are only intended to describe certain embodiments of the present invention and are not intended to be limiting. When a feature is described with reference to a particular aspect, those skilled in the art will understand that the feature can also be applied to other related aspects.

[0495] Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an antibody" includes "antibodies," reference to "an antigen" includes two or more such antigens, reference to "a subject" includes two or more such subjects, etc.

[0496] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0497] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0498] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0499] Preferred, non-limiting examples embodying certain aspects of the present invention will now be described with reference to the following drawings. [Brief description of the drawings]

[0500] [Figure 1] Shows the effect of administration of anti-CD137 antibody (ATOR-1017) and / or PD-1 inhibitor (RMP1-14 clone) on tumor volume in female h4-1BBtg mice infected with MC38 colon adenocarcinoma cells. [Figure 2A] Shows the effect of administration of anti-CD137 antibody (ATOR-1017) and / or PD-1 inhibitor (RMP1-14 clone) on survival in female h4-1BBtg mice infected with MC38 colon adenocarcinoma cells. [Figure 2B] Shows the effect of administration of anti-CD137 antibody (ATOR-1017) and / or PD-1 inhibitor (RMP1-14 clone) on survival in female h4-1BBtg mice infected with MC38 colon adenocarcinoma cells. [Diagram 3] FIG. 1 shows the effect of administration of anti-CD137 antibody (ATOR-1017) and / or PD-1 inhibitor (RMP1-14 clone) on tumor growth inhibition in female h4-1BBtg mice infected with MC38 colon adenocarcinoma cells. [Figure 4A] 1 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 4B] 1 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 5A] 1 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (pembrolizumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 5B] 1 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (pembrolizumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 6A] 1 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 6B] 1 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 7A] 1 shows T cell activation by anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (atezolizumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 7B] 1 shows T cell activation by anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (atezolizumab) in a mixed lymphocyte reaction (MLR) assay. [Figure 8A] Figure 2 shows T cell activation by anti-CD137 antibody (ATOR-1017) in combination with PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay with exhausted CD4+ T cells. [Figure 8B] Figure 2 shows T cell activation by anti-CD137 antibody (ATOR-1017) in combination with PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay with exhausted CD4+ T cells. [Figure 8C] Figure 2 shows T cell activation by anti-CD137 antibody (ATOR-1017) in combination with PD-1 inhibitor (nivolumab) in a mixed lymphocyte reaction (MLR) assay with exhausted CD4+ T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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[0502] Example 1 Antitumor efficacy of anti-CD137 antibodies in combination with PD-1 inhibitors in h4-1BB transgenic mice bearing MC38 colon cancer tumors ATOR-1017, a human anti-4-1BB (anti-CD137) agonistic IgG4 antibody, induces potent dose-dependent antitumor efficacy in mice bearing MC38 colon cancer tumors transgenic for human 4-1BB (h4-1BBtg), as demonstrated by reduced tumor volume, improved tumor growth inhibition, and induction of complete responders, as well as prolonged mouse survival.

[0503] The inventors have surprisingly found that combination therapy of an anti-CD137 antibody (such as ATOR-1017) with a PD-1 inhibitor (surrogate anti-PD-1 antibody clone RPM1-14) results in further improved anti-tumor efficacy.

[0504] Materials and Methods 5 × 10 5 MC38 colon adenocarcinoma cells were inoculated subcutaneously (sc) in the right front flank in a volume of 100 μl. Seven days after inoculation, mice were randomized into six test groups and treated intraperitoneally (ip) with the anti-CD137 antibody ATOR-1017 given at two doses of 0.5 mg / kg or 5 mg / kg with or without additional treatment with a surrogate anti-PD-1 antibody (clone RMP1-14, catalog number BE0146, supplied by BioXCell, 39 Labombard Rd, Lebanon, NH 03766, USA) at 5 mg / kg. Controls received a human IgG4 isotype control antibody at 5 mg / kg. Treatments were given twice weekly for three weeks, for a total of up to six dosing opportunities. Tumor growth was measured twice weekly, and tumor volumes were determined to be >3000 mm 3 Mice were euthanized when they approached the ethical limit of

[0505] Results and Conclusions Figure 1 shows that treatment with the anti-CD137 antibody ATOR-1017 given at 0.5 mg / kg results in significantly reduced tumor volume (p=0.0015 at day 18) compared to isotype control, an effect that is more pronounced when ATOR-1017 is given at 5 mg / kg. In mice receiving either of the two doses of ATOR-1017 in combination with anti-PD-1, tumor volume is further reduced when compared to the corresponding ATOR-1017 monotherapy. This effect of combination therapy is statistically significant in a one-tailed Mann-Whitney test for ATOR-1017 given at a dose of 5 mg / kg (p=0.0482 at day 35).

[0506] Treatment with the anti-CD137 antibody ATOR-1017 at doses of 10 μg / mouse and / or 100 μg / mouse, or with the surrogate anti-PD-1 antibody clone RPM1-14, increased survival in female mice compared to huIgG4 isotype controls (Figure 2A). Mice receiving the combination of ATOR-1017 and anti-PD-1 showed a further increase in survival compared to the corresponding monotherapies (Figures 2A and 2B).

[0507] On day 46, the survival rate of mice receiving combination therapy containing 10 μg / mouse ATOR-1017 and 100 μg / mouse anti-PD-1 antibody was significantly increased compared to the individual monotherapies (Figure 2B).

[0508] Figure 3 demonstrates the effect of treatment with a combination of 100 μg / mouse of anti-CD137 antibody ATOR-1017 and 100 μg / mouse of surrogate anti-PD-1 antibody clone RPM1-14 on tumor growth inhibition (TGI, %) compared to the corresponding monotherapy. The inventors surprisingly identified that the effect on TGI was further increased in mice receiving the combination therapy compared to the corresponding ATOR-1017 monotherapy. The effect of the combination therapy was statistically significant by T-test (p=0.0366), demonstrating a synergistic effect between the two agents.

[0509] Furthermore, the number of complete responders was monitored (Table 3) and increased in mice receiving the combination of ATOR-1017 and an anti-PD-1 antibody. [Table 6]

[0510] Overall, the anti-cancer efficacy of the combination of CD137 and PD-1 inhibitor targeted therapy is increased compared to monotherapy alone. In conclusion, these data demonstrate the unexpected efficacy of the combination of ATOR-1017 and anti-PD-1 antibody in the treatment of cancer.

[0511] Example 2A T cell activation of ATOR-1017 in combination with PD-1 inhibitors in a mixed lymphocyte reaction (MLR) assay Background and Objectives To demonstrate the synergistic effect of the combination of ATOR-1017 with a PD-1 inhibitor on T cell activation, T cell activation was assessed in a mixed lymphocyte reaction (MLR) using human primary CD4+ T cells and mature monocyte-derived dendritic cells (mMo-DCs), in which both targets (4-1BB and PD-1) are endogenously expressed.

[0512] Materials and Methods Generation of expanded CD4+ T cells: Human primary CD4+ T cells were isolated from the leukocyte concentrate using the CD4+ T Cell Isolation Kit (130-096-533, Miltenyi Biotec Ltd) according to the manufacturer's instructions. Cells were expanded for 7 days using Dynabeads™ Human T-Activator CD3 / CD28 (11131D, Gibco) in the presence of 50 IU / mL recombinant human IL-2 (202-IL, R&D) at a 1:1 bead-to-cell ratio. Dynabeads were removed and CD4+ T cells were rested overnight with reduced 10 IU / mL recombinant human IL-2.

[0513] Differentiation of mMo-DCs: Monocytes were isolated from human PBMCs using the Human CD14 Isolation Kit (130-050-201, Miltenyi Biotec Ltd, UK) according to the manufacturer's instructions. Monocytes were differentiated into monocyte-derived DCs (Mo-DCs) using Mo-DC differentiation medium containing IL-4 and GM-CSF (130-094-812, Miltenyi). Mo-DCs were further matured into mature Mo-DCs (mMo-DCs) using a cocktail of Il-1β (130-093-563, Miltenyi), IL-6 (130-095-352, Miltenyi), TNFα (130-094-023, Miltenyi) and PGE2 (P0409-1MG, Merck Millipore).

[0514] A 1:10 mixture of mMo-DC cells and expanded CD4+ T cells was treated for 7 days with ATOR-1017 in the presence of a fixed concentration of 45 nM F(ab)2 anti-Ig crosslinker (109-006-008, Jackson) and a titration of PD-1 inhibitors. Supernatants were analyzed for interferon gamma (IFN-γ) using a Monkey IFN gamma Elisa development Kit (3421M-1H-20, Mabtech). PD-1 inhibitors tested by the inventors include the anti-PD-1 antibody nivolumab (Opdivo®, Bristol Myers Squibb) (shown in Figure 4) and the anti-PD-1 antibody pembrolizumab (Keytruda®, Merck) (shown in Figure 5). Heavy chain sequence of Nivolumab (SEQ ID NO:31): [ka] Nivolumab light chain sequence (SEQ ID NO:32) [ka] Pembrolizumab heavy chain sequence (SEQ ID NO:33): [ka] Pembrolizumab light chain sequence (SEQ ID NO:34) [ka]

[0515] Results and Conclusions Although both PD-1 inhibitors were able to activate CD4+ T cells alone, ATOR-1017 alone induced poor CD4+ T cell activation in the MLR assay (Figure 4A, nivolumab; Figure 5A, pembrolizumab). The dotted lines in Figures 4A and 5A indicate the baseline (background) levels of allogeneic stimulation of CD4+ T cells in the MLR assay. However, combination treatment of ATOR-1017 and PD-1 inhibitors synergistically improved the efficacy of T cell activation compared to the individual monotherapies alone (Figures 4B and 5B). The dotted lines shown in Figures 4B and 5B indicate the additive effect of ATOR-1017 and PD-1 inhibitor monotherapy.

[0516] In conclusion, these data demonstrate the unexpected efficacy of combination therapy with ATOR-1017 and a PD-1 inhibitor (e.g., an anti-PD-1 antibody) for the treatment of T cell activation and cancer.

[0517] Example FIG. 2B T cell activation of ATOR-1017 in combination with PD-1 inhibitors in a mixed lymphocyte reaction (MLR) assay Example 2A was repeated with the same materials and methods as described in Example 2A, but using the anti-PD-1 antibody nivolumab (Figure 6) or the anti-PD-L1 antibody atezolizumab (Tecentriq®, Roche) (Figure 7) as the PD-1 inhibitor, and using various concentrations of anti-F(ab)2 used for cross-linking. SEQ ID NO: 53 is the heavy chain sequence of atezolizumab, and SEQ ID NO: 54 is the light chain sequence of atezolizumab. Anti-F(ab)2 was administered at a ratio of ATOR-1017 doses (anti-F(ab)2 / ATOR-1017 ratio 5:1) instead of a fixed concentration of anti-F(ab)2. Combination treatment of ATOR-1017 and PD-1 inhibitors synergistically improved the efficacy of T cell activation compared to the individual monotherapies alone (Figure 6A, nivolumab; Figure 7A, atezolizumab). The dotted lines in Figures 6A and 7A indicate baseline levels (background levels) of allogeneic stimulation of CD4+ T cells in the MLR assay.

[0518] The dosages investigated for monotherapy and combination studies were 0.005, 0.02, 0.09, 0.35, 1.4, 5.62, and 22.5 nM. For the 5.62 nM dosage for monotherapy, the monotherapy concentration is 5.62 nM. For the 5.62 nM dosage for combination therapy, each component (ATOR-1017 and PD-1 inhibitor) is present at 5.62 nM.

[0519] A dose of 22.5 nM in the MLR assay corresponds to approximately a dose of 0.1 mg / kg when administered to a human patient.

[0520] As shown in Figure 6B (nivolumab) and Figure 7B (atezolizumab), synergistic improvement of the combination therapy was observed at high concentrations (22.5 nM). The dotted lines shown in Figures 6B and 7B indicate the additive effect of ATOR-1017 and PD-1 inhibitor monotherapy, indicating the synergistic effect of the combination.

[0521] Example 3 ATOR-1017 in combination with anti-PD-1 antibodies activates exhausted T cells in a mixed lymphocyte reaction (MLR) assay Background and Objectives To demonstrate the synergistic activity of combining ATOR-1017 with anti-PD-1 on exhausted T cell activation. T cell activation was assessed in a mixed lymphocyte reaction (MLR) using human primary CD4+ T cells with an exhausted phenotype and mature monocyte-derived dendritic cells (mMo-DCs), where both targets (4-1BB and PD-1) are endogenously expressed.

[0522] Materials and Methods Generation of exhausted CD4+ T cells: CD4+ T cells were expanded for 8 days as previously described for generation of expanded CD4+ T cells, except that fresh CD3 / CD28 Dynabeads were added to the CD4+ T cells every 2 days for a total of three times during the 8-day expansion period. After 8 days, exhausted CD4+ T cells were characterized by flow cytometry as having increased expression of PD-1, TIM-3, and LAG-3.

[0523] Differentiation of mMo-DCs: Mature mMo-DCs were generated as previously described for differentiation of mMo-DCs.

[0524] A 1:10 mixture of mMo-DC cells and expanded CD4+ T cells was treated for 7 days with ATOR-1017 and a titration of PD-1 inhibitor (nivolumab) in the presence of F(ab)2 anti-Ig crosslinker (109-006-008, Jackson). Supernatants were analyzed for interferon gamma (IFN-γ) using the Monkey IFN gamma Elisa development Kit (3421M-1H-20, Mabtech).

[0525] The dosages investigated for monotherapy and combination studies were 0.005, 0.02, 0.09, 0.35, 1.4, 5.62, and 22.5 nM. For the 5.62 nM dosage for monotherapy, the monotherapy concentration is 5.62 nM. For the 5.62 nM dosage for combination therapy, each component (ATOR-1017 and PD-1 inhibitor) is present at 5.62 nM.

[0526] A dose of 22.5 nM in the MLR assay corresponds to approximately a dose of 0.1 mg / kg when administered to a human patient.

[0527] Results and Conclusions Exhausted CD4+ T cells are characterized by increased expression of PD-1, TIM-3, and LAG-3, and a reduced ability to respond to allogeneic stimuli. Anti-PD-1 alone was able to activate exhausted CD4+ T cells, whereas ATOR-1017 alone induced poor CD4+ T cell activation in the MLR assay (Figure 8A). However, combined treatment with ATOR-1017 and a PD-1 inhibitor significantly improved the activation of exhausted CD4+ T cells compared to each monotherapy alone. + synergistically improved the efficacy of T cell activation (Figures 8B and 8C).

[0528] In conclusion, these data support the rationale for combination therapy with ATOR-1017 and PD-1 inhibitors, particularly PD-1 / PD-L1 blocking antibodies, to activate exhausted T cells in cancer patients and result in enhanced antitumor activity compared with monotherapy with either agent alone.

Claims

1. A combination therapy for use in the treatment or prevention of cancer in a subject, the combination therapy comprising: (a) an antibody that specifically binds to CD137, or an antigen-binding fragment thereof; and (b) a PD-1 inhibitor.

2. wherein the cancer is a solid tumor and / or (i) the cancer and / or solid tumor is selected from the group consisting of prostate cancer; breast cancer; colorectal cancer; kidney cancer; pancreatic cancer; ovarian cancer; lung cancer; cervical cancer; rhabdomyosarcoma; neuroblastoma; bone cancer; multiple myeloma; leukemia (e.g., acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g., melanoma), bladder cancer and glioblastoma, adenoma, blastoma, cancer tumor, desmoid tumor, fibromatosis, small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, sarcoma, Wilms tumor, lung tumor, colon tumor, lymphoma tumor, breast tumor and melanoma, and / or (ii) the cancer and / or solid tumor is lung cancer (e.g., non-small cell lung cancer [NSCLC] or small cell lung cancer [SCLC]), head and / or neck cancer, gastric cancer, esophageal cancer, kidney cancer, urothelial cancer, melanoma, mesothelioma, breast cancer, cervical cancer, prostate cancer, high-frequency microsatellite instability (MSI) cancer, cancer associated with DNA mismatch repair (dMMR), and / or high tumor mutation burden (TMB) cancer, preferably, the cancer and / or solid tumor is metastatic, and / or (ii) the antibody that specifically binds to CD137, or an antigen-binding fragment thereof, a) has binding specificity for domain 2 of human CD137 and / or b) is a CD137 agonist and / or c) can inhibit the binding of the reference antibody "1630 / 1631" to human CD137, optionally, the antibody, or antigen-binding fragment, has binding specificity for domain 2 of human CD137, is a CD137 agonist, and can inhibit the binding of the reference antibody "1630 / 1631" to human CD137, and / or d) can inhibit the binding of the reference antibody "2674 / 2675" to human CD137, optionally, the antibody, or antigen-binding fragment, has binding specificity for domain 2 of human CD137, is a CD137 agonist, and can inhibit the binding of the reference antibody "2674 / 2675" to human CD137, and / or e) The following characteristics: (I) The ability to stimulate CD137 via a crosslinking-dependent mechanism and activate T cells and other immune cells, optionally, the ability to activate T cells and other immune cells depends on binding to both CD137 and Fc receptor, and / or (II) Cross-reactivity with cynomolgus CD137 antibody, showing one or more of the following, and / or f) An antibody or antigen-binding fragment thereof that can bind to the Fc receptor and optionally specifically bind to the CD137, and can bind to CD137 and Fc receptor simultaneously, and / or g) When binding to cells expressing CD137, (I) Antibody-dependent cell-mediated cytotoxicity (ADCC), (II) Antibody-dependent cell phagocytosis (ADCP), and / or (III) Complement-dependent cytotoxicity (CDC) cannot be substantially induced, and / or h) Can induce tumor immunity, and / or i) Comprising or consisting of intact antibodies, such as IgG1, IgG2, IgG3 or IgG4 antibodies, preferably comprising or consisting of IgG4 antibodies, and / or j) An antigen-binding fragment selected from the group consisting of an Fv fragment (e.g., single-chain Fv and disulfide-bonded Fv), Fab-like fragments (e.g., Fab fragment, Fab' fragment, and F(ab) ２ fragment), and domain antibodies (e.g., a single V Ｈ variable domain or V Ｌ variable domain), preferably comprising or consisting of an scFv, and / or k) Is a recombinant polypeptide, and / or l) Is monoclonal, and / or m) The combination therapy according to claim 1, which is human or humanized.

3. The antibody or antigen-binding fragment thereof that specifically binds to the CD137, (i) a) A heavy chain CDR1 sequence having a consensus sequence G, F, T / N, F, G, Y, S, Y, and b) A heavy chain CDR2 sequence having a consensus sequence I, G, S, G / T, S, S, Y / H, T, and c) A heavy chain CDR3 sequence having the sequence ARVYSSPGIDY, including, and / or (ii) a) A light chain CDR1 sequence having a consensus sequence Q, S, I, S / G, S, Y / T, and b) A light chain CDR2 sequence having a consensus sequence A / G, A, S, and c) A light chain CDR3 sequence having the sequence QQYYTWVPFT, The combination therapy according to claim 1.

4. The antibody or antigen-binding fragment thereof that specifically binds to the CD137, (i) The following CDRs: a) GFTFGYSY [SEQ ID NO: 3], or a CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 3, such as 1, 2 or 3 mutations, b) the CDR of an amino acid sequence containing IGSSSYT [SEQ ID NO: 4], or up to 3 amino acid mutations compared to SEQ ID NO: 4, for example, 1, 2, or 3 mutations, c) the CDR of an amino acid sequence containing ARVYSSPGIDY [SEQ ID NO: 5], or up to 3 amino acid mutations compared to SEQ ID NO: 5, for example, 1, 2, or 3 mutations, comprising a heavy chain variable region, preferably, the heavy chain variable region comprises the CDRs of SEQ ID NOs: 3, 4, and 5, and / or (ii) the following CDRs: a) QSISSY [SEQ ID NO: 6], or the CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 6, for example, 1, 2, or 3 mutations, b) AAS [SEQ ID NO: 7], or the CDR of an amino acid sequence containing up to 2 amino acid mutations compared to SEQ ID NO: 7, for example, 1 or 2 mutations, c) QQYYTWVPPT [SEQ ID NO: 8], or the CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 8, for example, 1, 2, or 3 mutations, comprising a light chain variable region, preferably, the light chain variable region comprises the CDRs of SEQ ID NOs: 6, 7, and 8, the combination therapy according to claim 1.

5. The antibody specifically binding to CD137, or an antigen-binding fragment thereof, comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 60% sequence identity thereto, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, and / or The antibody specifically binding to CD137, or an antigen-binding fragment thereof, comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 60% sequence identity thereto, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, the combination therapy according to claim 4.

6. The antibody specifically binding to CD137, or an antigen-binding fragment thereof, comprises the CDRs of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, the combination therapy according to claim 1.

7. The antibody specifically binding to CD137, or an antigen-binding fragment thereof, comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 2, the combination therapy according to claim 1. Claim 8 The antibody that specifically binds to CD137, or an antigen-binding fragment thereof, is (i) the following CDRs: a) GFNFGYSY [SEQ ID NO: 21], or a CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 21, for example, 1, 2, or 3 mutations, b) IGSTSSTH [SEQ ID NO: 22], or a CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 22, for example, 1, 2, or 3 mutations, c) ARVYSSPGIDY [SEQ ID NO: 23], or a CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 23, for example, 1, 2, or 3 mutations, and includes a heavy chain variable region, preferably the heavy chain variable region includes the CDRs of SEQ ID NOs: 21, 22, and 23, and / or (ii) the following CDRs: a) QSIGST [SEQ ID NO: 24], or a CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 24, for example, 1, 2, or 3 mutations, b) GAS [SEQ ID NO: 25], or a CDR of an amino acid sequence containing up to 2 amino acid mutations compared to SEQ ID NO: 25, for example, 1 or 2 mutations, c) QQYYTWVPFT [SEQ ID NO: 26], or a CDR of an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 26, for example, 1, 2, or 3 mutations, and includes a light chain variable region, preferably the light chain variable region includes the CDRs of SEQ ID NOs: 24, 25, and 26. The combined therapy according to claim 1. Claim 9 The antibody that specifically binds to CD137, or an antigen-binding fragment thereof, is the amino acid sequence of SEQ ID NO: 19, or has a heavy chain variable region having an amino acid sequence with at least 60% sequence identity thereto, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, and / or the amino acid sequence of SEQ ID NO: 20, or has a light chain variable region having an amino acid sequence with at least 60% sequence identity thereto, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. The combined therapy according to claim 8. Claim 10 The antibody that specifically binds to CD137, or an antigen-binding fragment thereof, includes the CDRs of SEQ ID NOs: 21, 22, 23, 24, 25, and 26. The combined therapy according to claim 8.

11. The combined therapy according to claim 8, wherein the antibody specifically binding to CD137, or an antigen-binding fragment thereof, comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

20.

12. The antibody specifically binding to CD137, or an antigen-binding fragment thereof, (a) comprises a heavy chain constant region, or a part thereof, and optionally, the heavy chain constant region of the antibody specifically binding to CD137, or an antigen-binding fragment thereof, is of an immunoglobulin subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, preferably, the heavy chain constant region of the antibody specifically binding to CD137, or an antigen-binding fragment thereof, is of immunoglobulin subtype IgG4, or comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 13, 14 and 15, and / or (b) comprises a light chain constant region, or a part thereof, and optionally, the light chain constant region of the antibody specifically binding to CD137, or an antigen-binding fragment thereof, is of kappa or lambda light chain, preferably, the light chain constant region of the antibody specifically binding to CD137, or an antigen-binding fragment thereof, comprises or consists of the amino acid sequence of SEQ ID NO: 16, and / or (c) comprises an Fc region, preferably, the Fc region of the antibody specifically binding to CD137, or an antigen-binding fragment thereof, is naturally occurring, or the Fc region of the antibody specifically binding to CD137, or an antigen-binding fragment thereof, is not naturally occurring, preferably, the Fc region is not naturally occurring and comprises a mutation for shortening the half-life of the antibody or antigen-binding fragment. The combined therapy according to claim 1.

13. The antibody specifically binding to CD137, or an antigen-binding fragment thereof, (a) a heavy chain comprising a variable region of SEQ ID NO: 1 together with the constant region of SEQ ID NO: 13, or an amino acid sequence having at least 60% sequence identity thereto, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and / or 13, The variable region of SEQ ID NO: 2 in combination with the constant region of SEQ ID NO: 16, or an amino acid sequence having at least 60% sequence identity therewith, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2 and / or 16, a light chain, and comprising, and / or (b) the variable region of SEQ ID NO: 19 in combination with the constant region of SEQ ID NO: 13, or an amino acid sequence having at least 60% sequence identity therewith, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19 or 13, a heavy chain, and The variable region of SEQ ID NO: 20 in combination with the constant region of SEQ ID NO: 16, or an amino acid sequence having at least 60% sequence identity therewith, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 20 or 16, a light chain, and comprising, and / or (c) two heavy chains having the amino acid sequence of SEQ ID NO: 17 and two light chains having the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 60% sequence identity therewith, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17 and / or 18, an intact IgG4 molecule comprising or consisting of the same, and / or (d) two heavy chains having the amino acid sequence of SEQ ID NO: 29 and two light chains having the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 60% sequence identity therewith, for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29 and / or 30, an intact IgG4 molecule comprising or consisting of the same, the combination therapy according to claim 1.

14. The antibody specifically binding to CD137, or an antigen-binding fragment thereof, further comprises a cytotoxic moiety, and / or further comprises a detectable moiety, optionally, (a) the cytotoxic moiety comprises or consists of a radioisotope or a cytotoxic drug, and / or (b) the detectable moiety comprises or consists of a radioisotope, and / or, (c) the cytotoxic moiety and / or the detectable moiety is connected to an antibody or antigen-binding fragment thereof that specifically binds indirectly to CD137 via a linking moiety, preferably the linking moiety is a chelating agent, and most preferably the chelating agent is selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), the combination therapy according to claim 1. **Claim 15** The PD-1 inhibitor is (a) an anti-PD-1 antibody or an antigen-binding fragment thereof capable of inhibiting PD-1 function, optionally selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, semiprimab, AMP-224, PDR-001, MEDI-0680, JTX-4014 (pimavizumab), spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, and INCMGA00012 (retifanlimab), preferably the anti-PD-1 antibody is pembrolizumab or nivolumab, the anti-PD-1 antibody or antigen-binding fragment thereof, and / or, (b) an anti-PD-L1 antibody or an antigen-binding fragment thereof capable of inhibiting PD-1 function, selected from the group consisting of atezolizumab (Tecentriq™, MPDL3280A), durvalumab (MEDI-4736), avelumab, MDX-1105, KN035 (embafolimab), and CK-301 (cosibelimab), preferably the anti-PD-L1 antibody is atezolizumab (Tecentriq™, MPDL3280A), the anti-PD-L1 antibody or antigen-binding fragment thereof, or (c) A small molecule or peptide-based inhibitor of PD-1 or PD-L1, optionally a small molecule or peptide-based inhibitor of D-1 or PD-L1 selected from CA-170, AUNP12, and BMS-986189, and / or (d) The PD-1 inhibitor blocks the PD-1 / PD-L1 interaction, and optionally, the PD-1 inhibitor binds to PD-1 or PD-L1 in a manner that inhibits the ability of PD-L1 to bind to PD-1, and / or (e) The PD-1 inhibitor preferably reactivates T cells expressing PD-1 by blocking inhibitory signaling mediated by the tyrosine phosphatase SHP-2, which dephosphorylates signaling molecules downstream of the T cell receptor signaling molecule. The combination therapy according to claim 1.

16. A composition comprising an antibody that specifically binds to CD137, or an antigen-binding portion thereof, for use in a method of treating solid tumors, wherein the antibody that specifically binds to CD137, or an antigen-binding portion thereof, is for use in combination with a further immunotherapeutic agent, the further immunotherapeutic agent is a PD-1 inhibitor, and optionally, the antibody, or an antigen-binding portion thereof, is as defined in claim 2, and / or the PD-1 inhibitor is as defined in claim 15. The composition.

17. Use of an antibody that specifically binds to CD137, or an antigen-binding portion thereof, in the preparation of a medicament for treating solid tumors, wherein the antibody that specifically binds to CD137, or an antigen-binding portion thereof, is for use in combination with a further immunotherapeutic agent, the further immunotherapeutic agent is a PD-1 inhibitor, and optionally, the antibody, or an antigen-binding portion thereof, is as defined in claim 2, and / or the PD-1 inhibitor is as defined in claim 15. The use.

18. (a) An antibody that specifically binds to CD137, or an antigen-binding portion thereof, and (b) a further immunotherapeutic agent, the further immunotherapeutic agent being a PD-1 inhibitor, optionally, the antibody, or an antigen-binding portion thereof, is as defined in claim 2, and / or the PD-1 inhibitor is as defined in claim 15. The pharmaceutical composition.

19. The pharmaceutical composition according to claim 18 for use in medicine.

20. The pharmaceutical composition according to claim 18 for use in the treatment of cancer.

21. A kit comprising (a) an antibody that specifically binds to CD137, or an antigen-binding portion thereof, and (b) a further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor, optionally the antibody, or an antigen-binding portion thereof, is as defined in claim 2 and / or the PD-1 inhibitor is as defined in claim 15, the kit.

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