Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention relates to combination therapies for treating cancer in a subject, as well as methods for use thereof.
• the combination therapies comprise (a) an antibody, or antigen-binding fragment thereof, that specifically binds to CD137 and (b) a further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor.
• the invention also relates to pharmaceutical compositions comprising, uses of, methods of using, and kits comprising the combination therapies of the invention.
• the cancer may be a solid tumour.
• Cancer is a leading cause of premature deaths in the developed world.
• the aim of immunotherapy in cancer is to mount an effective immune response by the body against a tumour, particularly a solid tumour. This may be achieved by, for example, breaking tolerance against tumour antigen, augmenting anti-tumor immune responses, and stimulating local cytokine responses at the tumor site.
• the key effector cell of a long lasting anti-tumor immune response is the activated tumor specific effector T cell. Potent expansion of activated effector T cells can red i rect the immune response towards the tumour.
• regulatory T cells (Treg) play a role in inhibiting the anti-tumour immunity.
• NK cells play an important role in tumour immunology by attacking tumour cells with down-regulated human leukocyte antigen (HLA) expression and by inducing antibody dependent cellular cytotoxicity (ADCC). Stimulation of NK cells may thus also reduce tumour growth.
• HLA human leukocyte antigen
• CD137 (4-1BB, TNFRSF9) is a TNF receptor (TNFR) superfamily member and is expressed on activated CD4 + and CD8 + T cells, Treg, DC, monocytes, mast cells and eosinophils.
• CD137 activation plays an important role in CD8 + T cell activation and survival (Lee et al., 2002; Pul le et al., 2006). It sustains and augments, rather than initiates, effector functions and preferentially supports Thl cytokine production (Shuford et al. , 1997).
• CD137 stimulation initially results in activation and later in activation-induced cell death, explaining why CD137 agonistic antibodies have shown therapeutic effect in tumour immunity as well as in autoimmunity (Zhang, JCI, 2007, Sun, Trends Mol Med, 2003). CD137 also suppresses Treg function (So, Cytokine Growth Factor Rev, 2008). Activation of CD137 is dependent on receptor oligomerization (Rabu et at., 2005; Wyzgol et al., 2009).
• CD137 agonistic antibody has been shown to activate endothelial cells in the tumour environment, leading to upregulation of ICAM-1 and VCAM-1 and improved T cell recruitment (Palazon, Cancer Res, 2011).
• CD137 is upregulated on NK cells activated by cytokines or CD16, in mice or humans, respectively (see Melero, CCR 19 (5)1044-53, 2013 and references cited therein). CD137 has been shown to activate NK cells in mice as well as humans, potentiating ADCC (Kohrt et al., 2014), though there are reports suggesting opposite effects on NK cells in mice and humans, leading to NK cell activation in mice and inhibition in humans (Baessler, Blood, 2010).
• Urelumab is a strong 4-1BB agonist that has demonstrated limited clinical efficacy (Chester et al. 2017; Chin et al. 2018). Development of urelumab was however hampered by hepatotoxicity at doses 30.3 mg/kg (including 2 fatal events at doses 3lmg/kg) (Segal et al. 2017). The maximum tolerated dose was therefore set to 0.1 mg/kg (or a flat dose of 8 mg). In the subsequent studies, no clear objective responses was observed for urelumab as monotherapy (Chester et al. 2017). The mechanism behind the hepatotoxicity is not fully understood.
• Utomilumab is regarded as 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 a tolerable clinical safety profile up to 10 mg/ kg with no dose limiting toxicity (DLT),
• Utomilumab is dependent on FcyR-crosslinking to execute its agonistic effect.
• FcyRs in the blood are saturated by endogenous circulating human IgG, at approximately 10 g/L, FcyR-crosslinking dependent antibodies such as utomilumab need to compete with IgG to bind to FcyRs (Jolliff 1982).
• Endogenous IgG of 10 g/L is more than 60-fold higher than the maximum serum concentration (Cmax) reached with the highest clinical dose of utomilumab (155 pg/mL at 10 mg/ kg) (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, it can be expected that FcyR- crosslinking dependent 4-1BB activation is also reduced in the liver, due to competition with endogenous IgG. This reduced possibility for FcyR-crosslinking of utomilumab in the liver may explain the absence of liver toxicity with utomilumab that was detected with urelumab. Since 4-1BB activation with ALG.APV-527 is 5T4-crosslinking dependent and 5T4 is not expressed in liver, liver toxicity is not expected with ALG.APV-527.
• ADG106 administered at doses of 0.03 to 10 mg/kg, currently in phase I/II (Liu et al. 2017), and CTX-471, AGEN2373, LVGN6051 ATOR-1017, EU101 (IND/CTA), PE0116, STA551 and HOT1030 have entered clinical development during 2018-2021 and are currently being evaluated for safety in phase I studies.
• CD137 The agonistic effect of CD137 antibodies is affected by the isotype of the Fc region.
• the antibodies tested in the clinic are either IgG2 or IgG4.
• CD137 depends on cross linking for activation (Wilson 2011, Cancer Cell).
• the CD137L expressed on the membrane of an APC may induce significant multiple cross linking of the receptor.
• An antibody can by itself only cross link two CD137 receptors, and to induce a strong signal, further cross linking via FcyRs expressed on other cells (in trans) may be necessary for induction of a strong CD137 mediated signal.
• An exception to this may be IgG2 antibodies, which induce a cross linking independent signaling by an unknown mechanism (White et al, 2015 Cancer Cell).
• T cells do not express FcyRs, and the FcyR mediated cross linking in vivo is thought to be mediated by monocytes, macrophages, DCs and potentially B cells and other cell types.
• FcyR receptors may also induce ADCC, antibody-dependent cellular phagocytosis (ADCP) and complement- dependent cytotoxicity (CDC) on cells coated with antibodies (for simplicity ADCC below includes ADCP and CDC).
• ADCC antibody-dependent cellular phagocytosis
• CDC complement- dependent cytotoxicity
• human IgGl is a strong inducer of NK/Macrophage dependent ADCC, depending on the nature of the target, the cell type and the receptor density.
• IgG4 antibodies may also induce ADCC but to a lower extent than IgGl (Wang 2015, Front Imm; Vidarson 2014 Front Imm).
• the effect of a CD137 agonistic antibody with different isotypes may thus be affected by the balance between 1) inducing cross linking, which results in a stronger immune activation, and 2) inducing ADCC, which may lead to killing of both effector T cells (predominantly CD8 T cells) and Tregs.
• the net effect of 1) and 2) will likely depend on the distribution of CD137 expressing cells, the possibility of the target cells to engage with FcyR expressing immune cells, the receptor density and affinity and the sensitivity of Teff vs Treg to ADCC.
• the CD137 expression is high both on CD8 and Tregs in melanoma tumours (Quezada, presentation SITC 2015).
• the IgG4 format would allow for FcyRI mediated cross linking by macrophages and monocytes, yet minimizing NK mediated ADCC of effector CD8 T cells.
• CD137 agonists that blocks the CD137L, and thus not allow for simultaneous activation via C137L and the CD137 agonistic antibody, have a reduced risk of inducing exaggerated activation and systemic toxicity.
• the toxicity seen in mouse models has been detected following repeated dosing in a time dependent but not dose dependent manner (Ascierto 2010 Semin One, Dubrot 2010 Can Imm, Niu 2007 JI).
• the toxicity includes skin toxicity and liver toxicity: aspartate amino transferase/alanine amino transferase ratio (ASAT/ALAT) and cytokine release.
• ASAT/ALAT aspartate amino transferase/alanine amino transferase ratio
• cytokine release This suggests that either the toxicity requires CD137 mediated pre activation of immune cell populations (likely T cells) or it depends on secondary effects caused by antidrug-antibodies (ADA) response, potentially forming aggregations of CD137 antibodies that may lead to enhanced cross-linking.
• mice The toxicities seen in mice are reversible and seems to depend on TNFa/CD8 cell dependent manner (Ascierto 2010 Sem One). Toxicology studies in monkeys showed that both single and repeated dosing of up to lOOmg/kg once weekly for four weeks was tolerable with no skin or liver toxicity detected (Ascierto 2010, Semin One).
• TNF receptor family members may lead to immune exhaustion. Therefore, it may be of advantage to administer such antibodies in a manner allowing resting periods for the cells expressing the receptors.
• One approach to increase the resting period in a specific dosing protocol is to reduce the half-life of an antibody by for example decreasing the binding to the neonatal Fc receptor (FcRn). This could, depending on the administration route, also reduce the toxicity associated with the treatment.
• FcRn neonatal Fc receptor
• the programmed death-1 (PD-1) receptor is a negative regulator of anti-tumor T cell effector function when engaged by its ligand PD-L1, expressed on the surface of cells within a tumor (Ribas and Wolchok 2018).
• the PD-1 is an immune checkpoint, with its inhibitory function mediated by the tyrosine phosphatase SHP-2 that de- phosphorylates signaling molecules downstream of the T cell receptor (TCR) signaling molecules.
• PD-1 has two ligands, programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1), which is broadly expressed by many somatic cells mainly upon exposure to pro-inflammatory cytokines, and programmed death-ligand 2 (PD-L2, also known as CD273 or B7-DC), which has more restricted expression in antigen- presenting cells.
• PD-L1 programmed death-ligand 1
• PD-L2 also known as CD273 or B7-DC
• Inflammation-induced PD-L1 expression in the tumor microenvironment results in PD-l-mediated T cell exhaustion, inhibiting the antitumor cytotoxic T cell response.
• PD-L1 is expressed on both tumor cells and myeloid cells.
• PD-1 resistance can broadly be subdivided into primary resistance or secondary (acquired) resistance. (Kluger et al. 2020). It is imperative to understand the nature of PD-1 resistance in order to select the right type of combination treatment (
• cancer therapies in particular anti- CD137 antibodies suitable for use in treating solid tumours and combination therapies thereof.
• a combination therapy comprising an anti- CD137 antibody or antigen-binding fragments thereof and a PD-1 inhibitor (such as an anti-PD-1 antibody, an anti-PD-Ll antibody or antigen binding fragments thereof) is surprisingly efficacious in the treatment of cancer.
• a PD-1 inhibitor such as an anti-PD-1 antibody, an anti-PD-Ll antibody or antigen binding fragments thereof
• a first aspect of the invention provides a combination therapy for use in the treatment or prevention of cancer in a subject comprising (a) an antibody, or 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.
• a second aspect of the invention provides an antibody, or antigen-binding portion thereof, that specifically binds to CD137 for use in a method of treating cancer, wherein the antibody or antigen-binding portion thereof that specifically binds to CD137 is for use in combination with a PD-1 inhibitor.
• the cancer is a solid tumour.
• a related, third aspect of the invention provides the use of an antibody, or antigen binding portion thereof, that specifically binds to CD137 in the preparation of a medicament for treating a solid tumour, wherein the antibody or antigen-binding portion thereof that specifically binds to CD137 is for use in combination with a PD-1 inhibitor.
• a fourth aspect of the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising (a) an antibody, or 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.
• a fifth aspect of the invention provides a kit for treating a solid tumour comprising (a) an antibody, or antigen-binding portion thereof, that specifically binds to CD137, and (b) a PD-1 inhibitor.
• a sixth aspect of the invention provides a method for treating or preventing cancer, for example a solid tumour, in a subject, the method comprising administering to the subject a therapeutically effective amount of (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.
• the method comprises administering the (a) an antibody, or antigen-binding portion thereof, that specifically binds to CD137, and (b) a PD-1 inhibitor simultaneously. In other embodiments the method comprises administering (a) an antibody, or antigen-binding portion thereof, that specifically binds to CD137 prior to administration of (b) a PD-1 inhibitor. In other embodiments the method comprises administering the PD-1 inhibitor prior to administration of the antibody, or antigen-binding portion thereof, that specifically binds to CD137.
• 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 to CD137.
• the cancer is a solid tumour.
• 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 tumour, wherein the PD-1 inhibitor is for use in combination with an antibody, or antigen-binding portion thereof that specifically binds to CD137.
• a further aspect of the invention provides a kit comprising a first isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 (or a component polypeptide chain thereof) and:
• a further aspect of the invention provides a kit comprising a vector comprising a first isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 or a component polypeptide chain thereof, and: (i) a PD-1 inhibitor; and/or (ii) a second isolated nucleic acid molecule encoding an antibody of antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 or a component polypeptide chain thereof.
• a further aspect of the invention provides a kit comprising a host cell comprising a first isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 or a component polypeptide chain thereof, and:
• a further aspect provides a kit comprising any two or more of: a) an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 (or a component polypeptide chain thereof); b) a PD-1 inhibitor, and/or c) an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 (or a component polypeptide chain thereof); d) an antibody, or antigen-binding portion thereof, that specifically binds to CD137; e) a PD-1 inhibitor; f) a vector comprising an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 or a component polypeptide chain thereof; g) a vector comprising an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 or a component polypeptide chain
• a first aspect of the invention provides a combination therapy for use in the treatment or prevention of cancer in a subject comprising (a) an antibody, or 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
• the cancer is a solid tumour.
• the cancer and/or solid tumour is selected from the groups consisting of lung cancer (such as a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC)), a head and/or neck cancer, a gastric cancer, an oesophageal cancer, a renal cancer, a urothelial cancer, a melanoma, a breast cancer, a cervical cancer, a prostate cancer, an microsatellite instability (MSI)-high cancer, a cancer associated with DNA mismatch repair (dMMR) and/or a tumour mutational burden (TMB)-high cancer colorectal cancer; kidney cancer; pancreatic cancer; ovarian cancer; rhabdomyosarcoma; neuroblastoma; bone cancer; multiple myeloma; leukemia (such as acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g.
• lung cancer such as a
• melanoma bladder cancer, glioblastoma, adenoma, a blastoma, a carcinoma, a desmoid tumour, a desmopolastic small round cell tumour, an endocrine tumour, a germ cell tumour, a lymphoma, a sarcoma, a Wilms tumour, a lung tumour, a colon tumour, a lymph tumour, a breast tumour and a melanoma.
• the cancer and/or solid tumour is a lung cancer (such as a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC)), a head and/or neck cancer, a gastric cancer, an oesophageal cancer, a renal cancer, a urothelial cancer, a melanoma, a breast cancer, a cervical cancer, a prostate cancer, an microsatellite instability (MSI)-high cancer, a cancer associated with DNA mismatch repair (dMMR) and/or a tumour mutational burden (TMB)-high cancer, preferably wherein the cancer and/or solid tumour is metastatic.
• a lung cancer such as a non-small cell lung cancer (NSCLC) or a small cell lung cancer (SCLC)
• a head and/or neck cancer a gastric cancer, an oesophageal cancer, a renal cancer, a urothelial cancer, a melanoma, a breast cancer, a cervical cancer,
• the cancer and/or solid tumour is metastatic.
• the antibody or an antigen-binding fragment thereof that specifically binds to CD137: a) has binding specificity for domain 2 of human CD137; b) is a CD137 agonist; and/or c) is capable of inhibiting the binding of reference antibody ⁇ 630/163 to human CD137.
• 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 is capable of inhibiting the binding of reference antibody ⁇ 630/163 to human CD137.
• the antibody or an antigen-binding fragment thereof ('antibody polypeptides') that specifically binds to CD137 has: a) binding specificity for domain 2 of CD137; b) is a CD137 agonist; and/or c) is capable of inhibiting the binding of reference antibody '2674/2675' to human CD137.
• 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 the binding of reference antibody '2674/2675' to human CD137.
• the antibody or antigen binding fragment that specifically binds to CD137 is capable of inhibiting the binding of reference antibody ⁇ 630/1631' and/or '2674/2675' to human CD137.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 is capable of inhibiting the binding of one or more reference antibodies to human CD137, for example is capable of inhibiting the binding of reference antibody ⁇ 630/1631' and/or '2674/2675' to human CD137.
• Exemplary anti-CD137 antibodies are disclosed in WO 2018/091740 to Alligator Bioscience AB (the disclosures of which are incorporated herein by reference).
• anti-CD-137 antibodies are explicitly disclosed on pages 7-8, 11-12, 16-17 and 19 of WO 2018/091740, the disclosures of which are incorporated herein by reference.
• CD137 we specifically include the human CD137 protein, for example as described in GenBank Accession No. AAH06196.1 (the sequence of which is set out in SEQ ID NO: 11, below). CD137 is also known in the scientific literature as 4-1BB and TNFRSF9.
• domain 2 corresponds to amino acids 66 to 107 of human CD137 (see bold, underlined region in SEQ ID NO: 11 above).
• the combination therapy of the invention comprises an antibody or antigenbinding fragment that specifically binds to CD137 i.e. has specificity for CD137.
• specificity we mean that the antibody polypeptide is capable of binding to CD137 in vivo, i.e. under the physiological conditions in which CD137 exists within the human body.
• the antibody polypeptide does not bind to any other protein in vivo.
• binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blots and flow cytometry using transfected cells expressing CD137.
• the antibody or antigen-binding fragments that specifically binds to CD137 preferably binds to human CD137 with a Kd value which is less than 10xl0 ⁇ 9 M or less than 7x10 9 M, more preferably less than 4, or 2xlO ⁇ 9 M, most preferably less than 1.2xlO ⁇ 9 M,
• the antibody polypeptide is capable of binding selectively to CD137, i.e. it bind at least 10-fold more strongly to CD137 than to any other proteins.
• the anti-CD137 antibody preferably specifically binds to CD137, i.e. it binds to CD137 but does not bind, or binds at a lower affinity ⁇ e.g.
• the Kd for the antibody with respect to human CD137 will be 2-fold, preferably 5-fold, more preferably 10-fold less than Kd with respect to the other, non-target molecule, such as murine CD137, other TNFR superfamily members, or any other unrelated material or accompanying material in the environment. More preferably, the Kd will be 50-fold less, even more preferably 100-fold less, and yet more preferably 200-fold less.
• CD137 typically refers to human CD137.
• the antibody may have some binding affinity for CD137 from other mammals, such as CD137 from a non-human primate, for example Macaca fascicularis (cynomolgus monkey).
• the antibody preferably does not bind to murine CD137 and/or does not bind to other human TNFR superfamily members, for example human 0X40 or CD40.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may have affinity for CD137 in its native state, and in particular for CD137 localised on the surface of a cell.
• CD137 is associated with the cell such that one or more region of CD137 is present on the outer face of the cell surface.
• CD137 may be inserted into the cell plasma membrane (i.e. orientated as a transmembrane protein) with one or more regions presented on the extracellular surface. This may occur in the course of expression of CD137 by the cell.
• “localised on the surface of a cell” may mean “expressed on the surface of a cell.”
• CD137 may be outside the cell with covalent and/or ionic interactions localising it to a specific region or regions of the cell surface.
• the antibodies and antigen-binding fragments thereof that specifically bind to CD137 as defined herein are CD137 agonists.
• they may be capable of inducing the release of interferon-gamma from CD8+ T cells.
• Agonistic activity of anti-CD137 antibodies may be evaluated in a T cell assay based on primary CD8+ T cells (see Examples).
• the antibody or antigen-binding fragment that specifically binds to CD137 may modulate the activity of a cell expressing CD137, wherein said modulation is an increase or decrease in the activity of said cell.
• the cell is typically a T cell.
• the antibody may increase the activity of a CD4+ or CD8+ effector cell, or may decrease the activity of, or deplete, a regulatory T cell (T reg). In either case, the net effect of the antibody will be an increase in the activity of effector T cells, particularly CD4+, CD8+ or NK effector T cells. Methods for determining a change in the activity of effector T cells are well known and are as described earlier.
• the antibody or antigen-binding fragment that specifically binds to CD137 preferably causes an increase in activity in a CD8+ T cell in vitro, optionally wherein said increase in activity is an increase in proliferation, IFN-y production and/or IL-2 production by the T cell.
• the increase is preferably at least 2-fold, more preferably at least 10-fold and even more preferably at least 25-fold higher than the change in activity caused by an isotype control antibody measured in the same assay.
• antibodies or antigen-binding fragments thereof which are capable of inhibiting the binding of one or more reference antibodies to human CD137 are provided.
• the reference antibodies described herein are reference antibody 1630/1631 and reference antibody 2674/2675.
• anti-CD137 antibodies are disclosed in WO 2018/091740 to Alligator Bioscience AB (the disclosures of which are incorporated herein by reference) .
• the reference antibody ⁇ 630/163 binds to domain 2 of CD137.
• Reference antibody 2674/2675 also binds to domain 2 of CD137.
• the antibody or an antigen-binding fragment thereof that specifically binds CD137 in the combination therapy of the invention also binds to domain 2 of CD137. Accordingly in some embodiments the antibody or antigen-binding fragment thereof that specifically binds to CD137 binds to domain 2 of CD137.
• Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using BIAcore chips with immobilised CD137 and incubating in the presence the reference antibody ⁇ 630/1631' or '2674/2675' with and without an antibody polypeptide to be tested.
• a pair-wise mapping approach can be used, in which the reference antibody ⁇ 630/163 or '2674/2675' is immobilised to the surface of the BIAcore chip, CD137 antigen is bound to the immobilised antibody, and then a second antibody is tested for simultaneous CD137-binding ability (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; the disclosures of which are incorporated herein by reference).
• competitive binding inhibition can be determined using flow cytometry.
• cells expressing the antigen can be p re-incubated with the test antibody for 20 min before cells are washed and incubated with the reference 1630/1631 or 2674/2675 antibody conjugated to a fluorophore, which can be detected by flow cytometry. If the pre incubation with the test antibody reduces the detection of the reference 1630/1631 or 2674/2675 antibody in flow cytometry, the test antibody inhibits the binding of the reference antibody to the cell surface antigen. If the antibody to be tested exhibits high affinity for CD137, then a reduced pre-incubation period may be used (or even no pre-incubation at all).
• competitive binding inhibition can be determined using an ELISA .
• the antibodies and antigen binding fragments that specifically bind CD137 of the combination therapy of the invention are defined by reference to the variable regions of reference antibodies 1630/1631 and 2674/2675.
• the reference antibody designated ⁇ 630/163 comprises:
• the reference antibody designated '2674/2675' comprises:
• amino acid as used herein includes the standard twenty genetically- encoded amino acids and their corresponding stereoisomers in the ⁇ ' form (as compared to the natural V form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g. a,a-disubstituted amino acids, N -a Iky I amino acids, etc.) and chemically derivatised amino acids (see below).
• amino acid When an amino acid is being specifically enumerated, such as “alanine” or “Ala” or “A”, the term refers to both L-alanine and D-alanine unless explicitly stated otherwise.
• Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide.
• each encoded amino acid residue where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
• the antibody polypeptides as defined herein comprise or consist of L-amino acids.
• 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.
• polypeptide thus includes short peptide sequences and also longer polypeptides and proteins.
• amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
• CDRs complementarity determining regions
• any intact IgG antibody comprising the above variable regions may be used as the reference antibody to identify antibody polypeptides of the combination therapy of the invention that competitively inhibit 1630/1631 or 2674/2675 binding to CD137.
• reference antibody 1630/1631 consists of heavy and light chains as defined in SEQ ID NOs: 17 and 18, respectively
• reference antibody 2674/2675 consists of heavy and light chains as defined in SEQ ID NOs:29 and 30, respectively
• test antibody binds at, or at least very close to, the epitope on the antigen to which binds the reference antibody (in this case, 1630/1631 or 2674/2675).
• reference antibody in this case, 1630/1631 or 2674/2675.
• competitive binding may also arise by virtue of steric interference; thus, the test antibody may bind at an epitope different from that to which the reference antibody binds but may still be of sufficient size or configuration to hinder the binding of the reference antibody to the antigen.
• 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, on the basis of exhibiting properties that make them particularly suitable as diagnostic and therapeutic agents for cancer.
• the 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 release of interferon-gamma from CD8+ T cells; see Examples); and/or b) cross-reactivity with cynomolgus CD137 (see Examples).
• the 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 a fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137, and in some embodiments retains at least one of functional characteristics (a) to (b) above.
• cross linking dependent mechanism we include an Fc cross linking dependent mechanism wherein the antibody has to bind both CD137 and an Fc receptor in order to stimulate CD137. As such, in some embodiments the antibody has to be capable of binding both CD137 and an Fc receptor.
• the antibody or antigen binding domain that specifically binds to CD137 is capable of binding an Fc receptor. In one embodiment, the antibody or antigen binding domain is capable of simultaneous binding to CD137 and a Fc receptor. In a preferred embodiment, the ability of the antibody and/or antigen binding domain thereof that specifically binds to CD137 to activate T cells is dependent upon binding to both CD137 and Fc receptors.
• the Fc receptor that is targeted is an FCYR. Examples of FCYRS include, FcyRI, FCYRIIA and FCYRIIB
• the FCYR may be FCYRIIA, By FCYRIIA, we include both the R131 and H131 allotypes of FCYRIIA.
• the FCYR to be targeted is the R131 allotype of FCYRIIA.
• the antibody that specifically binds to CD137 could be Fc crosslinking independent, such that it can stimulate CD137 in the absence of binding to an Fc receptor.
• exemplary antibodies 2674/2675 and 1630/1631 are FcYR-crosslinking dependent agonistic antibodies targeting the co-stimulatory CD137 receptor. They are therefore only active in tissues or tumours containing cells expressing CD137 and FcyR.
• tumours or tumour draining lymph nodes comprising tumour cells and/or tumour infiltrating immune cells (such as monocytes, macrophages, dendritic cells, NK cells, T cells, B cells and granulocytes) expressing CD137 and FcyR.
• CD137 and FcyR may be expressed on separate cells within the tumour and/or co-expressed in the same cells.
• Reference antibodies 2674/2675 and 1630/1631 will thus provide a tumour directed immune activation in indications associated with cells that express both CD137 and FcyR in the tumour microenvironment; this contrasts with FcyR independent CD137 agonists ( e.g . Urelumab), which capable of inducing systemic immune activation.
• FcyR independent CD137 agonists e.g . Urelumab
• the tumour localizing effect of antibodies 2674/2675 and 1630/1631 will primarily depend on the number of tumour infiltrating macrophages/myeloid cells expressing different FcyRs.
• IgG4 binds with high affinity to FcyRI and with moderate/low affinity to FcyRIIa and FcyRIIb.
• FcyRI and FcyRIIa are expressed on monocytes and FcyRIIb is expressed with a high density on B cells.
• Crosslinking of antibodies 2674/2675 and 1630/1631 will preferentially occur intratumorally as well as in adjacent draining lymph nodes.
• serum IgG levels are high, the availability of free non-blocked FcyRs are believed to be too low for an effective crosslinking to occur. Therefore, the risk for a systemic immune activation of is believed to be low which improves the risk-benefit profile compared to other CD137 mAbs.
• Patient selection and a biomarker rationale for treatment with antibodies that specifically bind CD137 and form part of the combination therapy of the invention, such as 2674/2675 and 1630/1631, may be guided by tumour types that have infiltrating cells expressing CD137 and FcyRs.
• the combination therapy of the invention may be for use in patients selected on the basis of having a tumour containing cells expressing CD137 and FcyRs ( i.e , a as companion diagnostic test).
• tumour infiltrating immune cells such as monocytes, macrophages, dendritic cells, NK cells, T cells, B cells and granulocytes
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 is capable of inducing tumour immunity.
• Tumour immunity can be demonstrated using methods well known in the art, for example by re-challenging mice that have been cured from a given tumour by CD317 antibody treatment with the same tumour and/or by re-challenging mice that have been cured from a given tumour by the combination therapy of the present invention with the same tumour. If tumour immunity has been induced by the antibody therapy and/or combination therapy, then the tumour is rejected upon re-challenge.
• the antibody or antigen binding fragment thereof that specifically binds to CD137 is substantially incapable of inducing the following upon binding to cells expressing CD137: a) antibody-dependent cellular cytotoxicity (ADCC); b) antibody-dependent cellular phagocytosis (ADCP); and/or c) complement-dependent cytotoxicity (CDC).
• ADCC antibody-dependent cellular cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement-dependent cytotoxicity
• the antibody may be or may comprise a variant or a fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment reta i ns specificity for CD137 and is incapable of inducing one or more of (a) to (c) upon binding to cells expressing CD137.
• a chromium-51 release assay for determining the level of ADCC-mediated lysis or apoptosis in a sample of cells.
• a chromium-51 release assay for determining the level of ADCC-mediated lysis or apoptosis in a sample of cells.
• a chromium-51 release assay for determining the level of ADCC-mediated lysis or apoptosis in a sample of cells.
• sulphur-35 release assay may be used.
• a previously labelled target cell line expressing the antigen is incubated with an antibody to be tested .
• effector cells typically expressing Fc receptor CD16
• Target cell lysis is subsequently measured by release of intracellular label by a scintillation counter or spectrophotometry.
• lysis is detected by measuring the release of enzymes naturally present in the target cells. This may be achieved by detection (for example bioluminescent detection) of the products of an enzyme-catalysed reaction. No previous labelling of the cells is required in such an assay.
• a typical cellular enzyme detected with such an assay is GAPDH.
• the tumor antigen-expressing cancer cells may be incubated in the presence of a titration of mAb and the human leukemia monocytic cell line THP-1. Both effector and target cells may be fluorescently labelled and cell engulfment may be measured by flow cytometry. Phagocytosis may also be confirmed using microscopy or imaging cytometry.
• CDC assays may also be used, which may determine the release of abundant cell components, such as GAPDH, with fluorescent or luminescent determination.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 is capable of binding to an epitope on the extracellular domain of CD137 which overlaps, at least in part, with the epitope on CD137 to which reference antibody 1630/1631 and/or 2674/2675 is capable of binding.
• the antibody or antigenbinding fragment may be capable of binding to an epitope located at/within domain 2 of CD137 (i.e. amino acids 66 to 107 of human CD137).
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises or consists of an intact antibody ((for example an IgGl, IgG2, IgG3 or IgG4 antibody).
• the antibody is an IgG4 antibody.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises or consists of an antigen-binding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)2 fragments) and domain antibodies (e.g. single VH variable domains or VL variable domains).
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be a scFv,
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises or consists of an antibody mimic selected from the group comprising or consisting of a ffi bodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.
• CTLDs tetranectins
• monobodies monobodies
• anticalins DARPins (ankyrins)
• DARPins avimers
• iMabs iMabs
• microbodies peptide aptamers
• Kunitz domains and affilins Kunitz domains and affilins.
• the antibody or antigen binding fragment thereof that specifically binds to CD137 comprises: a) a heavy chain CDR1 sequence with the consensus sequence G, F, T/N, F, G,
• Y, S, Y a heavy chain CDR2 sequence with the consensus sequence I, G, S, G/T, S, S, Y/H, T; and c) a heavy chain CDR3 sequence with the sequence ARVYSSPGIDY.
• the antibody or antigen binding fragment thereof that specifically binds to CD137 comprises: a) a light chain CDR1 sequence with the consensus sequence Q, S, I, S/G, S,
• Y/T a light chain CDR2 sequence with the consensus sequence A/G, A, S; and c) a light chain CDR3 sequence with the sequence QQYYTWVPFT.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a heavy chain variable region comprising the following CDRs: a) GFTFGYSY [SEQ ID NO: 3] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 3, for example 1, 2 or 3 mutations; b) IGSGSSYT [SEQ ID NO: 4] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 4, for example 1, 2 or 3 mutations; and c) 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.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising one, two or all three of the CDRs of SEQ ID NOs 3, 4 and 5.
• 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.
• the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the following CDRs: a) GFNFGYSY [SEQ ID NO: 21] or 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) IGSTSSHT [SEQ ID NO: 22] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 22, for example 1, 2 or 3 mutations; and c) ARVYSSPGIDY [SEQ ID NO: 23] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 23, for example 1, 2 or 3 mutations.
• the antibody or antigen-binding fragment thereof that specifically binds CD137 may comprise a heavy chain variable region comprising one, two or all three of the CDRs of SEQ ID NOs 21, 22 and 23.
• the 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.
• SEQ ID NO: 19 the amino acid sequence of the corresponding region of the 2674/2675 reference antibody.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising the CDRs as defined above, wherein the HI and H2 CDRs are mutated versions of SEQ ID NO: 3 and 4, respectively, and wherein the H3 CDR is SEQ ID NO: 5.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising the CDRs as defined above, wherein the HI and H2 CDRs are mutated versions of SEQ ID NO: 21 and 22, respectively, and wherein the H3 CDR is SEQ ID NO: 23.
• the 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 therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.
• Percent sequence identity can be determined by, for example, the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357, the disclosures of which are incorporated herein by reference) at the Expasy facility site (http://www.ch.embnet.org/software/LALIGN___form.html) using as parameters the global alignment option, scoring matrix BLOSUM62, opening gap penalty -14, extending gap penalty -4.
• the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent sequence identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
• the alignment may alternatively be carried out using the Clustal W program (as described in Thompson et at., 1994, Nucl. Acid Res. 22:4673-4680, which is incorporated herein by reference).
• the parameters used may be as follows: Fast pair-wise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.
• the BESTFIT program may be used to determine local sequence alignments.
• 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) QSISSY [SEQ ID NO: 6] or 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 an amino acid sequence containing up to 2 amino acid mutations compared to SEQ ID NO: 7; for example 1 or 2 mutations and c) QQYYTWVPFT [SEQ ID NO: 8] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 8, for example 1, 2 or 3 mutations.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs of SEQ ID NOs 6, 7 and 8.
• the 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 1630/1631 reference antibody, i.e. SEQ ID NO: 2 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.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs as defined above, wherein the LI and L2 CDRs are mutated versions of SEQ ID NO: 6 and 7, respectively, and wherein the L3 CDR is SEQ ID NO:8.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 and 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) QSIGST [SEQ ID NO: 24] or 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 an amino acid sequence containing up to 2 amino acid mutations compared to SEQ ID NO: 25; for example 1 or 2 mutations and c) QQYYTWVPFT [SEQ ID NO: 26] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 26, for example 1, 2 or 3 mutations.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs of SEQ ID NOs 24, 25 and 26.
• the 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.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs as defined above, wherein the LI and L2 CDRs are mutated versions of SEQ ID NO: 24 and 25, respectively, and wherein the L3 CDR is SEQ ID NO: 26.
• An anti-CD137 antibody used in the combination therapies and methods of the invention may be an antibody comprising one, two or all three of the CDR sequences of SEQ ID NOs: 3 to 5 and/or one, two, or all three of the CDR sequences of SEQ ID NOs: 6 to 8.
• the antibody may comprise all six CDR sequences of SEQ ID NOs: 3 to 8
• 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 therewith, 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.
• the antibody may be, or may bind to the same epitope as, an antibody comprising the light chain variable region sequence of SEQ ID NO: 2 and the heavy chain variable region sequence of SEQ ID NO: 1.
• 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 therewith, 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.
• An anti-CD137 antibody used in the combination therapies and methods of the invention may be an antibody comprising one, two or all three of the CDR sequences of SEQ ID NOs: 21 to 23 and/or one, two, or all three of the CDR sequences of SEQ ID NOs: 24 to 26.
• the antibody may comprise all six CDR sequences of SEQ ID NOs: 21 to 26.
• the antibody may comprise the light chain variable region sequence of SEQ ID NO: 20 and/or the heavy chain variable region sequence of SEQ ID NO: 19 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 and/or 19.
• the antibody may be, or may bind to the same epitope as, 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.
• 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 therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 and/or 13.
• human or humanised antibodies are preferably used.
• Humanised forms of non-human (e.g . murine) antibodies are genetically engineered chimaeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies.
• Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat of rabbit having the desired functionality.
• donor antibody such as mouse, rat of rabbit having the desired functionality.
• Fv framework residues of the human antibody are replaced by corresponding non-human residues.
• Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences.
• the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence.
• Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321 :522-525; Riechmann et a!., 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596, the disclosures of which are incorporated herein by reference).
• the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et a!., 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) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions.
• humanised antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. Chimeric antibodies are discussed by Neuberger et al (1998, 8 th International Biotechnology Symposium Part 2, 792-799).
• Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, 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).
• phage display libraries see, for example, 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
• humanised antibodies or antigen-binding fragments that specifically bind to CD137 may further comprise a heavy chain constant region, or part thereof (see below).
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a CHI, CH2 and/or CH3 region of an IgG heavy chain (such as an IgGl, IgG2, IgG3 or IgG4 heavy chain).
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise part or all of the constant regions from an IgG4 heavy chain.
• the antibody or antigen binding fragment thereof that specifically binds to CD137 may be a Fab fragment comprising CHI and CL constant regions, combined with any of the above-defined heavy and light variable regions respectively.
• the above-defined antibodies or antigen-binding fragments that specifically bind to CD137 may further comprise a light chain constant region, or part thereof (see below).
• the antibody polypeptide may comprise a CL region from a kappa or lambda light chain.
• the antibodies or antigen-binding fragments that specifically bind to CD137 and are comprised in the combination therapy of the invention comprise an antibody Fc-region.
• the Fc portion may be from an IgG antibody, or from a different class of antibody (such as IgM, IgA, IgD or IgE).
• the Fc region is from an IgGl, IgG2, IgG3 or IgG4 antibody.
• the Fc region is from an IgG4 antibody.
• the Fc region may be naturally-occurring (e.g. part of an endogenously produced antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring Fc region).
• a variant of an Fc region typically binds to Fc receptors, such as FcyR and/or neonatal Fc receptor (FcRn) with altered affinity providing for improved function and/or half-life of the polypeptide.
• Fc receptors such as FcyR and/or neonatal Fc receptor (FcRn) with altered affinity providing for improved function and/or half-life of the polypeptide.
• the biological function and/ or the half-life may be either increased or a decreased relative to the half-life of a polypeptide comprising a native Fc region.
• ADCC antibody dependent cell cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement-dependent cytotoxicity
• the Fc region may be naturally-occurring (e.g. part of an endogenously produced human antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring human Fc region).
• 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 cell phagocytosis (ADCP).
• CDC complement-dependent cytotoxicity
• ADCC antibody-dependent cellular cytotoxicity
• ADCP antibody-dependent cell phagocytosis
• One approach to improve the efficacy of a therapeutic antibody is to increase its serum persistence, thereby allowing higher circulating levels, less frequent administration and reduced doses.
• FcRn which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation.
• ADCC effector function
• abrogating effector functions may be required for certain clinical indications.
• the four human IgG isotypes bind the activating Fey receptors (FcyRI, FcyRIIa, FcyRIIIa), the inhibitory FcyRIIb receptor, and the first component of complement (Clq) with different affinities, yielding very different effector functions (Bruhns et al., 2009, Blood. 113(16) :3716-25, the disclosures of which are incorporated herein by reference).
• FcyRs Human FcyRs are primarily expressed by cells of the myeloid lineage, which has been demonstrated in numerous studies for circulating myeloid cell subsets.
• Classical monocytes generally identified as CD14 + CD16 ⁇ display high levels of FcyRII (CD32), intermediate levels of FcyRI and low levels of FcyRIII (CD16) (Almeida et al. 2001, 100(3) :325-38, Cheeseman et al. 2016, PLoS One ll(5) :e0154656, the disclosures of which are incorporated herein by reference).
• CD14 ⁇ CD16 + non-classical monocytes display high levels of FcyRIII, intermediate levels of FcyRII and low levels of FcyRI (Almeida et al. 2001).
• FcyRIII intermediate levels of FcyRII
• low levels of FcyRI Almeida et al. 2001.
• a summary and compilation of several published microarray data sets showing the expression of human FcyR genes on different myeloid cell subsets confirms these observations (Guilliams et al. 2014, Nat Rev Immunol. !4(2) :94-108, the disclosures of which are incorporated herein by reference).
• monocytes Once within tissues, monocytes differentiate towards macrophages and, depending on environmental cues, these macrophages obtain specific phenotypes.
• peripheral blood monocytes were polarized towards different macrophage lineages by using various inflammatory stimuli and the expression profile of these cells evaluated.
• IFN-y stimulated monocytes resulted in a highly elevated expression specifically of CD64.
• SLE patients where increased CD64 expression was detected on circulating CD14 + monocytes, which correlated with expression of interferon-stimulated genes (Li et al. 2010, Arthritis Res Ther 12(3) : R90, the disclosures of which are incorporated herein by reference).
• myeloid cell subsets such as inflammatory monocytes, monocytic myeloid- derived suppressor cells (MDSC) and macrophages have, in numerous studies, been shown to accumulate in cancer patients (Solito etal. 2014, Ann N Y Acad Sci 1319:47- 65., Hu et al. 2016, Clin Transl Oncol.18(3):251-8, the disclosures of which are incorporated herein by reference). Although recent attempts have aimed at proposing strategies to standardize the characterization of these cells (Bronte et al. 2016, Nat Commun.
• tumor-associated macrophages are commonly identified by the expression of CD64 and CD68 (Ml-polarized, anti-tumorigenic), or CD163 and CD206 (M2-polarized, pro-tumorigenic) (Elliott et al. 2017).
• CDllb + myeloid cells were also 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 disclosures of which are incorporated herein by reference). An even higher frequency of CDllb + cells was observed in pancreatic cancer where over 60% of the CD45 + cells were CDllb + CD15 + CD33 + (Porembka et al. 2012, Cancer Immunol Immunother 61(9) : 1373-85, the disclosures of which are incorporated herein by reference). Also, one study concluded that the macajor myeloid cell population within non-small cell lung carcinoma is a CDllb + CD15 + CD66b + neutrophil-like population.
• FcyRI expression has also been shown for other types of tumors.
• Grugan et al (Grugan et al. 2012, J Immunol. 189(ll) : 5457-66, the disclosures of which are incorporated herein by reference) demonstrated the presence of CDllb + CD14 + cells within human breast tumor tissue. These cells were shown to express high levels of FcyRI and FcyRIIa, as well as FcyRIIb and FcyRIII.
• CD45 + CDllb + CD14 + CD68 + TAM were identified in gastrointestinal stromal tumors displaying expression of FcyRI (Cavnar et al. 2013, J Exp Med.
• CD45 + CDllb + FcyRI + cells were also identified in colorectal cancer patients and these cells displayed a higher expression of FcyRI in tumor tissue, compared to healthy control tissue (Norton et al. 2016, Clin Transl Immunology. 5(4) :e76, the disclosures of which are incorporated herein by reference). FcyRI expression has also been demonstrated for melanoma meta stases (Hansen etal. 2006, Acta Oncol 45(4) :400-5, the disclosures of which are incorporated herein by reference).
• Binding of IgG to the FcyRs or Clq depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and Clq binding, and have unique sequences in IgG2 and IgG4. Substitutions into human IgGl of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 were shown to greatly reduce ADCC and CDC (Armour et a!., 1999, Eur J Immunol. 29(8) :2613-24; Shields et at., 2001, J Biol Chem. 276(9) :6591-604, the disclosures of which are incorporated herein by reference).
• IgG4 antibodies Due to their lack of effector functions, IgG4 antibodies represent a preferred IgG subclass for receptor modulation without cell depletion. IgG4 molecules can exchange half-molecules in a dynamic process termed Fab-arm exchange. This phenomenon can also occur in vivo between therapeutic antibodies and endogenous IgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of less unpredictable therapeutic IgG4 antibodies (Labrijn et al., 2009, Nat Biotechnol. 27(8) :767-71, the disclosures of which are incorporated herein by reference).
• the effector function of the Fc region may be altered through modification of the carbohydrate moieties within the CH2 domain therein, for example by modifying 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)
• Low fucose antibody polypeptides may be produced by expression in cells cultured in a medium containing an inhibitor of mannosidase, such as kinfunensine (see Example I below).
• Another method to create low fucose antibodies is by inhibition or depletion of alpha- ( 1 , 6 ) -f u cosy 11 ra n sf e ra se in the antibody-producing cells (e.g. using the Potelligent® CHOK1SV technology of Lonza Ltd, Basel, Switzerland).
• An exemplary heavy chain constant region amino acid sequence which may be combined with any VH region sequence disclosed herein (to form a complete heavy chain) is the IgGl heavy chain constant region sequence reproduced here:
• a preferred constant region is a modified IgG4 constant region such as that reproduced here:
• This modified IgG4 sequence results in stabilization of the core hinge of IgG4 making the IgG4 more stable, preventing Fab arm exchange.
• Another preferred constant region is a modified IgG4 constant region such as that reproduced here:
• This modified IgG4 sequence exhibits reduced FcRn binding and hence results in a reduced serum half-life relative to wild type IgG4. In addition, it exhibits stabilization of the core hinge of IgG4 making the IgG4 more stable, preventing Fab arm exchange.
• polypeptides of the invention are also suitable for use in the polypeptides of the invention.
• a wild type IgG4 constant region such as that reproduced here:
• the antibody or antigen binding fragment that specifically binds to CD137 may comprise the IgG4 constant regions of SEQ ID NOs: 13 and 16, respectively.
• exemplary antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise:
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be an intact IgG4 molecule comprising or consisting of two heavy chains having an amino acid sequence of SEQ ID NO: 17 and two light chains having an amino acid sequence of SEQ ID NO: 18.
• Alternative exemplary antibodies or antigen-binding fragments that specifically bind to CD137 may comprise:
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be an intact IgG4 molecule comprising or consisting of two heavy chains having an amino acid sequence of SEQ ID NO: 29 and two light chains having an amino acid sequence of SEQ ID NO: 30.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 is or comprises a "fusion" polypeptide.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 that forms part of the combination therapy of the invention may also be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be fused to an oligo-histidine tag, such as His6, or to an epitope recognised by an antibody such as the well-known Myc tag epitope.
• Fusions to any variant or derivative of said antibody or antigen binding fragment thereof are also included in the scope of the invention. It will be appreciated that fusions (or variants, derivatives or fusions thereof) which retain or improve desirable properties, such as IL-1R binding properties or in vivo half-life are preferred.
• the fusion may comprise an amino acid sequence as detailed above together with a further portion which confers a desirable feature on the said polypeptide comprised in the combination therapy of the invention; for example, the portion may useful in detecting or isolating the polypeptide, or promoting cellular uptake of the polypeptide.
• the portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art.
• GFP green fluorescent protein
• the moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprised in the combination therapy of the invention may comprise or consist of one or more amino acids which have been modified or derivatised.
• Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group.
• derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, t- butyloxy carbonyl groups, chloroacetyl groups or formyl groups.
• Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides.
• Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives.
• Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids.
• 4-hydroxyproline may be substituted for proline
• 5-hydroxylysine may be substituted for lysine
• 3-methylhistidine may be substituted for histidine
• homoserine may be substituted for serine and ornithine for lysine.
• Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained.
• Other included modifications are amidation, amino terminal acylation ⁇ e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.
• peptidomimetic compounds may also be useful.
• the term 'peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent.
• the 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 the peptide bond is reversed.
• retro- inverso peptidomimetics may be made using methods known in the art, for example 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 containing changes involving the backbone, and not the orientation of side chains. Retro- inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
• the said polypeptide may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -yCCI-teNH)- bond in place of the conventional amide linkage.
• the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.
• the said antibody or antigen-binding fragment thereof may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exo-proteolytic digestion.
• a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber et al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et a!., 1983, Biochem. Biophys. Res. Comm. 111 : 166, which are incorporated herein by reference.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 will be a 'naked' antibody polypeptide, i.e. without any additional functional moieties such as cytotoxic or detectable moieties.
• the therapeutic effect is mediated by a direct effect of the antibody comprised 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.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be augmented with a functional moiety to facilitate their intended use, for example as a diagnostic (e.g. in vivo imaging) agent or therapeutic agent.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 is linked, directly or indirectly, to a therapeutic moiety.
• a suitable therapeutic moiety is one that is capable of reducing or inhibiting the growth, or in particular killing, a cancer cell (or associated stem cells or progenitor cells).
• the therapeutic agent may be a cytotoxic moiety, such as a radioisotope ( e.g . 90 Y, 177 Lu, "Tc m , etc) or cytotoxic drug ⁇ e.g. anti metabolites, toxins, cytostatic drugs, etc).
• 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 irradiation therapy or low energy X-ray photon activation therapy.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may further comprise a detectable moiety.
• a detectable moiety may comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of 99m Tc, m In, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I and 201 TI
• the agent may comprise a pair of detectable and cytotoxic radionuclides, such as 86 Y/ 90 Y or 124 I/ 211 At.
• the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a radioisotope that is capable of simultaneously acting in a multi-modal manner as a detectable moiety and also as a cytotoxic moiety to provide so-called "Multimodality theragnostics".
• the binding moieties may thus be coupled to nanoparticles that have the capability of multi-imaging (for example, SPECT, PET, MRI, Optical, or Ultrasound) together with therapeutic capability using cytotoxic drugs, such as radionuclides or chemotherapy agents.
• Therapeutic and/or detectable moieties may be linked directly, or indirectly, to the antibody or fragment thereof.
• Suitable linkers include, for example, prosthetic groups, non- phenolic linkers (derivatives of N-succimidyl- benzoates; dodeca borate), chelating moieties of both macrocyclics and acyclic chelators, such as derivatives of 1,4,7,10- tetraazacyclododecane- 1,4,7, 10, tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid (DTPA), derivatives of S-2-(4- Isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA) and derivatives of 1,4,8, 11-tetraazacyclodocedan- 1,4,8, 11-t
• DOTA deferoxamine
• DTPA diethylenetriaminepentaacetic
• linker is DTPA, for example as used in 177 Lu-DTPA- [anti body polypeptide].
• a further preferred linker is deferoxamine, DFO, for example as used in
• combination therapy of the invention comprising an antibody or antigen-binding fragment thereof that specifically binds to CD137 will not require the presence of a cytotoxic or diagnostic moiety.
• 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 hosts cells (for example, see Green & Sam brook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference).
• the antibody that specifically binds to CD137 may be a polyclonal antibody, it is preferred if it is a monoclonal antibody, or that the antigen-binding fragment, variant, fusion or derivative thereof, is derived from a monoclonal antibody.
• Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Hurrell (CRC Press, 1982). Polyclonal antibodies may be produced which are poly specific or mono-specific. It is preferred that they are mono-specific.
• Antibody polypeptides comprised in the combination therapy of the invention can also be produced using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate (available from Promega).
• the translation system is rabbit reticulocyte lysate.
• the translation system may be coupled to a transcription system, such as the TNT transcription-translation system (Promega). This system has the advantage of producing suitable mRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation.
• antibody or antigen-binding fragment thereof that specifically binds to CD137 may alternatively be synthesised artificially, for example using well known liquid-phase or solid phase synthesis techniques (such as f-Boc or Fmoc solid-phase peptide synthesis).
• the combination therapies of the invention comprise 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.
• the PD-1 inhibitor is an immunotherapeutic agent with efficacy in the treatment of cancer.
• immunotherapeutic agent is intended to include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumour or cancer in the subject.
• Various immunotherapeutic agents are useful in the compositions and methods described herein.
• the immunotherapeutic agent is an antibody or antigen-binding fragment thereof, such as an anti-PD-1 antibody that is capable of specifically binding PD-1 or an anti-PD-Ll antibody which is capable of specifically binding PD-L1 .
• 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 cellular cytotoxicity.
• immune response includes immune responses that arc indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
• Immune checkpoint molecules include a group of proteins on the cell surface of immune cells, such as CD4+ and/or CD8+ T cells, dendritic cells, NK cells and macrophages but also on certain tumor cells, that modulate immune responses. It will be appreciated by persons skilled in the art that PD-1 is an inhibitory immune check point molecule.
• Blocking or neutralisation of inhibitory immune checkpoint molecules can block or otherwise neutralise inhibitory signalling to thereby upregulate an immune response in order to more efficaciously treat cancer.
• agents useful for blocking inhibitory immune checkpoint include antibodies, small molecules, peptides, peptidomimctics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit inhibitory immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of inhibitory immune checkpoint nucleic acids, or fragments thereof.
• Exemplary agents for upregulating an immune response include antibodies against one or more inhibitory immune checkpoint proteins that blocks the interaction between the proteins and its natural receptor(s); a non activating form of one or more immune checkpoint inhibitor proteins ⁇ e.g., a dominant negative polypeptide): small molecules or peptides that block the interaction between one or more inhibitory immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block inhibitory immune checkpoint nucleic acid transcription or translation; and the like.
• a non activating form of one or more immune checkpoint inhibitor proteins ⁇ e.g., a dominant negative polypeptide
• small molecules or peptides that block the interaction between one or more inhibitory immune checkpoint proteins and its natural receptor(s)
• fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an
• agents can directly block the interaction between the one or more inhibitory immune checkpoint and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signalling and upregulate an immune response.
• agents can indirectly block the interaction between one or more inhibitory immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signalling and upregulate an immune response.
• a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
• anti-PD-1 antibodies and/or anti-PD-Ll antibodies either alone or in combination, are used to inhibit immune checkpoint inhibitors.
• the further immunotherapeutic agent is a PD-1 inhibitorthat binds to and inhibits the function of an inhibitory immune checkpoint molecule.
• PD-1 inhibitor (or “PD-1 pathway inhibitor”) we include an entity which is capable of inhibiting the PD-1 pathway.
• PD-1 serves as a negative regulator of T cell activation when engaged with its ligands PD-L1 or PD-L2.
• PD-L1 in particular is expressed by many solid tumors, including melanoma. These tumours may therefore down regulate immune mediated anti-tumor effects through activation of the inhibitory PD-1 receptors on T cells.
• a check point of the immune response may be removed, leading to augmented anti-tumour T cell responses. This interaction may be blocked by an antibody specific for PD-1 or PD-Llor any other suitable agent.
• Such antibodies and agents may be generally referred to as PD-1 inhibitors.
• the further immunotherapeutic agent in step (b) of the method of the invention is a PD-1 inhibitor.
• PD-1 inhibitors block the interaction of PD-1 (programmed cell death protein 1) with its ligand PD-L1 (programmed death-ligand 1). Such PD-1 inhibitors can therefore act on either, or both, PD-L1 and PD-1.
• the term PD-1 inhibitors includes both PD-1 and PD-L1 inhibitors.
• PD-1 inhibitors block the activity of PD-1 and PD-L1 immune checkpoint proteins.
• PD-1 we specifically include the human PD-1 protein, for example as described in GenBank Accession No. NP_005009.2 (the sequence of which is set out in SEQ ID NO: 35, below). PD-1 is also know in the scientific literature as PD1, CD279, PDCD1 and SLEB2.
• PD-L1 we specifically include the human PD-L1 protein, for example as described in GenBank Accession No. AAI13735.1 (the sequence of which is set out in SEQ ID NO: 36, below). PD-L1 is also know in the scientific literature as CD274, B7-H1, B7-H, PDCD1L1 and PDCD1LG1
• the combination therapy of the invention comprises a PD-1 inhibitor that specifically binds to PD-1 or PD-L1 i.e. has specificity for PD-1 or PD-L1.
• specificity we mean that the inhibitor e is capable of binding to PD-1 or PD-L1 in vivo, i.e. under the physiological conditions in which PD-1 or PD-L1 exists within the human body.
• the PD-1 inhibitor does not bind to any other protein (other than PD-1 or PD-L1) in vivo.
• binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blots and flow cytometry using transfected cells expressing PD-1 or PD-L1.
• the PD-1 inhibitor that specifically binds to PD-1 or PD-L1 preferably binds to human PD-1 or PD-L1 with a Kd value which is less than 10xl0 ⁇ 9 M or less than 7xlO ⁇ 9 M, more preferably less than 4, or 2xl0 ⁇ 9 M, most preferably less than 1.2xlO ⁇ 9 M.
• the PD-1 inhibitor is capable of binding selectively to PD-1 or PD-L1, i.e. it bind at least 10-fold more strongly to PD-1 or PD-L1 than to any other proteins.
• the PD-1 inhibitor preferably specifically binds to PD-1 or PD-L1, i.e.
• the Kd for the antibody with respect to human PD-1 or PD-L1 will be 2-fold, preferably 5-fold, more preferably 10-fold less than Kd with respect to the other, non- target molecule, such as murine PD-1 or PD-L1, other immune checkpoint molecules, or any other unrelated material or accompanying material in the environment. More preferably, the Kd will be 50-fold less, even more preferably 100-fold less, and yet more preferably 200-fold less.
• PD-1 and PD-L1 typically refers to human PD-1 and PD-L1.
• the inhibitor may have some binding affinity for PD-1 or PD-L1 from other mammals, such as PD-1 or PD-L1 from a non-human primate, for example Macaca fascicularis (cynomolgus monkey).
• the antibody preferably does not bind to murine PD-1 or PD- Ll and/or does not bind to other immune checkpoint molecules.
• the PD-1 inhibitor thereof that specifically binds to PD-1 or PD-L1 may have affinity for PD-1 or PD-L1 in its native state, for example for PD-1 or PD-L1 localised on the surface of a cell.
• the PD-1 inhibitor blocks the PD-1 PD-L1 interaction.
• 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-Ll interaction.
• PD-1 may be inserted into the cell plasma membrane (i.e. orientated as a transmembrane protein) with one or more regions presented on the extracellular surface. This may occur in the course of expression of PD-1 by the cell.
• “localised on the surface of a cell” may mean “expressed on the surface of a cell.”
• PD-1 may be outside the cell with covalent and/or ionic interactions localising it to a specific region or regions of the cell surface.
• the PD-1 inhibitors described here are capable of inducing antitumour immunity, via immune checkpoint blockade.
• the PD-1 inhibitor binds to PD-1 or PD-L1 in a manner that inhibits PD-L1 to bind to PD-1, i.e. blocks the PD-l/PD- Ll interaction.
• the PD-1 inhibitor may be capable of enhancing T cell responses, for example it may be capable of enhancing or restoring T cell effector function.
• the PD-1 inhibitor may promote infiltration of tumour reactive CD8+ T cells into established tumours.
• PD-1 inhibitor may modulate the activity of a cell expressing PD-1 or PD-L1, wherein said modulation is an increase or decrease in the activity of said cell.
• the cell is typically a T cell.
• the inhibitor may increase the activity of a CD4+ or CD8+ effector cell, or may decrease the activity of, or deplete, a regulatory T cell (T reg).
• T reg regulatory T cell
• the net effect of the antibody will be an increase in the activity of effector T cells, particularly CD4+, CD8+ or NK effector T cells.
• the PD-1 inhibitor preferably causes an increase in activity in a T cell in vitro, preferable a CD8+ T cell, optionally wherein said increase in activity is an increase in proliferation, IFN-y production and/or IL-2 production by the T cell.
• the increase is preferably at least 2-fold, more preferably at least 10-fold and even more preferably at least 25-fold higher than the change in activity caused by an isotype control antibody measured in the same assay.
• the PD-1 inhibitors are capable of improving efficacy of another immunotherapy.
• the PD-1 inhibitor blocks the programmed death-1 (PD-1) receptor to its ligand PD-L1, expressed on the surface of cells within a tumor (Ribas and Wolchok 2018).
• PD-1 is an immune checkpoint, with its inhibitory function mediated by the tyrosine phosphatase SHP-2 that de-phosphorylates signaling molecules downstream of the T cell receptor (TCR) signaling molecules.
• the PD-1 inhibitor reactivates PD-1 expressing T cells, preferably by blocking the inhibitory signaling mediated by the tyrosine phosphatase SHP-2 (that de- phosphorylates signaling molecules downstream of the T cell receptor (TCR) signaling molecules.
• the PD-1 inhibitor may be an anti-PD-1 antibody, or antigen-binding fragment thereof capable of inhibiting PD-1 function (for example, 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, Dostarlimab and INCMGA00012 (Retifanlimab).
• the PD-1 inhibitor may comprise or consist of an anti-PD-Ll antibody, or antigen-binding fragment thereof capable of inhibiting PD-1 function (for example, Atezolizumab (TecentriqTM, MPDL3280A), Durvalumab (MEDI-4736), Avelumab, MDX- 1105, KN035 (Envafolimab) and CK-301 (Cosibelimab)).
• an anti-PD-Ll antibody for example, Atezolizumab (TecentriqTM, MPDL3280A), Durvalumab (MEDI-4736), Avelumab, MDX- 1105, KN035 (Envafolimab) and CK-301 (Cosibelimab)).
• the PD-1 inhibitor may be a small molecule or peptide based inhibitor of PD-1 or PD-L1.
• the PD-1 inhibitor may be a small molecule inhibitor of PD-L1 such as CA-170.
• the PD-1 inhibitor may be a peptide inhibitor of PD-L1 such as AUNP12 or BMS-986189.
• the PD-1 inhibitor binds to an epitope that blocks the PD-1 PD-L1 interaction.
• the PD-1 inhibitor is capable of inhibiting binding of reference antibodies Pembrolizumab or Nivolumab to human PD-1.
• the PD-1 inhibitor is capable of inhibiting binding of reference antibody Atezolizumab to human PD-L1.
• Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using BIAcore chips with immobilised PD-1 and incubating in the presence the reference antibody Pembrolizumab and Nivolumab with and without an antibody polypeptide to be tested.
• a pair-wise mapping approach can be used, in which the reference antibody Pembrolizumab or Nivolumab is immobilised to the surface of the BIAcore chip, PD-1 antigen is bound to the immobilised antibody, and then a second antibody is tested for simultaneous PD-l-binding ability (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; the disclosures of which are incorporated herein by reference).
• PD-L1 By “capable of inhibiting the binding of reference antibody Atezolizumab to human PD-L1” we mean that the presence of the antibody polypeptides of the combination therapy of invention inhibits, in whole or in part, the binding of Atezolizumab to human PD-L1,
• the PD-1 inhibitors used in the combination therapies of the invention may therefore compete for binding to PD-L1 with 'reference antibody' Atezolizumab.
• Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using BIAcore chips with immobilised PD-L1 and incubating in the presence the reference antibody Atezolizumab with and without an antibody polypeptide to be tested.
• a pair-wise mapping approach can be used, in which the reference antibody Atezolizumab is immobilised to the surface of the BIAcore chip, PD-L1 antigen is bound to the immobilised antibody, and then a second antibody is tested for simultaneous PD-Ll-binding ability (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; the disclosures of which are incorporated herein by reference).
• competitive binding inhibition can be determined using flow cytometry.
• test antibody to test whether a test antibody is able to inhibit the binding of the Pembrolizumab or Nivolumab (or Atezolizumab) reference antibody to a cell surface antigen, cells expressing the antigen can be p re-incubated with the test antibody for 20 min before cells are washed and incubated with the reference Pembrolizumab or Nivolumab (or Atezolizumab) antibody conjugated to a fluorophore, which can be detected by flow cytometry.
• the test antibody inhibits the binding of the reference antibody to the cell surface antigen. If the antibody to be tested exhibits high affinity for PD-1 (or PD-L1), then a reduced pre-incubation period may be used (or even no pre-incubation at all).
• Proteinab we mean an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOS: 33 and 34, respectively.
• Nevolumab we mean an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOS: 31 and 32, respectively.
• Nivolumab (SEQ ID NO: 31): QVQLVESGGG VVQPGRSLRL DCKASGITFS NSG HWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFY
• Camrelizumab DIQMTQSPSS LSASVGDRVT ITCLASQTIG TWLTWYQQKP GKAPKLLIYT ATSLADGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ VYSIPWTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC Heavy chain sequence of Tislelizumab (SEQ ID NO: 45)
• DIVMTQSPDS LAVS LG E RAT INCKSSESVS NDVAWYQQKP GQPPKLLINY AFHRFTGVPD RFSGSGYGTD FTLTISSLQA EDVAVYYCHQ AYSSPYTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
• Antezolizumab we mean an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOS: 53 and 54, respectively.
• Avelumab (SEQ ID NO: 58)
• Heavy chain sequence of CK-301 (Cosibelimab) (SEQ ID NO: 59)
• NFMLTQPHSV SESPGKTVTI SCTRSSGSID SNYVQWYQQR PGSAPTTVIY EDNQRPSGVP DRFSGSIDSS SNSASLTISG LKTEDEADYY CQSYDSNNRH VIFGGGTKLT VLGQPKAAPS VTLFPPSSEE LQANKATLVC LISDFYPGAV TVAWKADSSP VKAGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTECS
• PD-1 inhibitors are also described in US8354509 B2 and US8779105 B2, and the PD-1 inhibitors (in particular anti-PD-1 antibodies) of US8354509 B2 and US8779105 B2 are incorporated herein by reference.
• the antibodies or antigen-binding fragments that specifically bind to PD-1 or PD-L1 and are comprised in the combination therapy of the invention comprise an antibody Fc-region.
• the Fc portion may be from an IgG antibody, or from a different class of antibody (such as IgM, IgA, IgD or IgE).
• the Fc region is from an IgGl, IgG2, IgG3 or IgG4 antibody.
• the Fc region is from an IgG4 antibody.
• the Fc region may be naturally-occurring (e.g. part of an endogenously produced antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring Fc region).
• a variant of an Fc region typically binds to Fc receptors, such as FcyR and/or neonatal Fc receptor (FcRn) with altered affinity providing for improved function and/or half-life of the polypeptide.
• Fc receptors such as FcyR and/or neonatal Fc receptor (FcRn) with altered affinity providing for improved function and/or half-life of the polypeptide.
• the biological function and/ or the half-life may be either increased or a decreased relative to the half-life of a polypeptide comprising a native Fc region.
• ADCC antibody dependent cell cytotoxicity
• ADCP antibody-dependent cellular phagocytosis
• CDC complement-dependent cytotoxicity
• the Fc region may be naturally-occurring (e.g. part of an endogenously produced human antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring human Fc region).
• 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 cell phagocytosis (ADCP).
• CDC complement-dependent cytotoxicity
• ADCC antibody-dependent cellular cytotoxicity
• ADCP antibody-dependent cell phagocytosis
• Fc regions may be engineered as described above in relation to the CD137 antibodies of the combination therapy of the invention.
• the following definitions apply to either or both of the CD137 and PD-1 inhibitors of the invention, wherein the PD-1 inhibitor is an antibody (either an antibody specific for PD-1 or an antibody specific for PD-L1).
• antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e ., "antigen-binding portion") or single chains thereof.
• An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
• Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
• Each light chain is comprised 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 a binding domain that interacts with an antigen.
• VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• FR framework regions
• the constant regions of the antibodies may 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 (Ciq) of the classical complement system.
• an antibody or an antigen-binding fragment thereof we include substantially intact antibody molecules, as well as chimeric antibodies, humanised 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 of the same.
• Suitable antigen-binding fragments and derivatives include, but are not necessarily limited to, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab) ⁇ fragments), single variable domains (e.g.
• VH and VL domains VH and VL domains
• domain antibodies dAbs, including single and dual formats [i.e. dAb-linker-dAb]
• the potential 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 better penetration of solid tissue.
• antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
• the antigen-binding fragment may comprise an scFv molecule, i.e. wherein the VH and VL partner domains are linked via a flexible oligopeptide.
• Heavy chains can be of any isotype, including IgG (IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM and IgE.
• Light chains include kappa chains and lambda chains.
• Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multi-specific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intra bodies, scFvs (e.g. including mono- specific and bi-specific, etc.), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-id) antibodies, and epitope-binding fragments of any of the above.
• synthetic antibodies monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multi-specific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intra bodies, scFvs (e.g. including mono- specific and bi-specific, etc.), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (
• antibodies and their antigen-binding fragments that have been "isolated” so as to exist in a physical milieu distinct from that in which it may occur in nature or that have been modified so as to differ from a naturally occurring antibody in amino acid sequence
• an antibody or an antigen-binding fragment thereof is also intended to encompass antibody mimics (for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions).
• antibody mimics for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions.
• Exemplary antibody mimics include: a ffi bodies (also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins; Innovations Pharma c. Technol. (2006), 27-30); adnectins (also called monobodies; Meth.
• combination therapies comprising modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, e.g. modified by the covalent attachment of polyethylene glycol or another suitable polymer (see below).
• An antibody may be a polyclonal antibody or a monoclonal antibody.
• the antibody may be produced by any suitable method.
• antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833- 3837; Winter et al., 1991, Nature 349:293-299, the disclosures of which are incorporated herein by reference) or generation of monoclonal antibody molecules by cell lines in culture.
• antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York, the disclosures of which are incorporated herein by reference).
• antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells ( e.g . Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
• antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
• 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, such as 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 within the term “antigen-binding portion” of an antibody include a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment and an isolated complementarity determining region (CDR).
• CDR complementarity determining region
• Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
• 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.
• binding activity and "binding affinity” are intended to refer to the tendency of an molecule (e.g. an antibody molecule) to bind or not to bind to a target. Binding affinity may be quantified by determining the dissociation constant (Kd) for an antibody and its target. Similarly, the specificity of binding of an antibody to its target may be defined in terms of the comparative dissociation constants (Kd) of the antibody for its target as compared to the dissociation constant with respect to the antibody and another, non-target molecule.
• the Kd for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than Kd with respect to the other, non-target molecule such as unrelated material or accompanying material in the environment. More preferably, the Kd will be 50- fold less, even more preferably 100-fold less, and yet more preferably 200-fold less.
• this dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984).
• the Kd may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993).
• Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, E LISAs, Western blots, RIAs, and flow cytometry analysis.
• the binding kinetics (e.g., binding affinity) of the antibody also can be assessed by standard assays known in the art, such as by BiacoreTM system analysis.
• a competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another, known ligand of that target, such as another antibody.
• the concentration at which 50% inhibition occurs is known as the Ki.
• the Ki is equivalent to Kd.
• the Ki value will never be less than the Kd, so measurement of Ki can conveniently be substituted to provide an upper limit for Kd.
• 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 that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than its affinity for binding to another non-target molecule.
• a PD-1 inhibitor (such as anti-PD-1 antibody or anti-PD-Ll antibody) used in the combination therapies and methods of the invention is preferably capable of binding to its target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than its affinity for binding to another non-target molecule.
• An 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 also is derived from human germline immunoglobulin sequences.
• 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).
• human antibody is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences - such antibodies are typically referred to as chimeric or humanised.
• a human antibody for use the methods of the invention is typically a human monoclonal antibody.
• a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
• Human antibodies may also be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.
• the term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.
• An antibody for use in the methods of the invention may alternatively be a humanised antibody.
• humanised refers to an antibody molecule, generally prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin from a non-human species and a remaining immunoglobulin structure based upon the structure and /or sequence of a human immunoglobulin.
• the antigen binding site may comprise either complete non-human antibody variable domains fused to human constant domains, or only the complementarity determining regions (CDRs) of such variable domains grafted to appropriate human framework regions of human variable domains.
• CDRs complementarity determining regions
• the framework residues of such humanised 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.
• variable regions of both heavy and light chains contain three complementarity- determining regions (CDRs) which vary in response to the antigens in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
• CDRs complementarity- determining regions
• FRs framework regions
• the variable regions can be "reshaped” or “humanised” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.
• Riechmann, L. et /. (1988) "Reshaping
• humanised antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies).
• humanised antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody.
• CDRs one, two, three, four, five, six
• the ability to humanise an antigen is well known (see, e.g., US Patents No. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).
• the antibody may be or may comprise a variant or a fragment of one of the specific antibodies disclosed herein, provided that said variant or fragment retains specificity for its target.
• the antibody may be or may comprise a variant or a fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137.
• the antibody may be or may comprise a variant or a 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.
• a fragment is preferably an antigen binding portion of a said antibody.
• a fragment may be made by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and/or C terminal in this way. Fragments may also be generated by one or more internal deletions.
• a variant may comprise one or more substitutions, deletions or additions with respect to the sequences of a specific anti-CD137 antibody or other antibody (e.g. anti-PD-1 antibody or anti-PD-Ll antibody) disclosed herein.
• a variant may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions from the specific sequences disclosed herein.
• “Deletion” variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features.
• substitution preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions.
• an amino acid may be substituted with an alternative amino acid having similar properties, for example, 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.
• an alternative amino acid having similar properties, for example, 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.
• variants include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected.
• Variants may be prepared during synthesis of the antibody or by post- production modification, or when the antibody is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
• variant antibodies have an amino acid sequence which has more than 60%, or more than 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more 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 seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.
• sequence identity refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:
• Pairwise alignment parameters -Method accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;
• An anti-CD137 antibody or PD-1 inhibitor for use in the combination therapies and methods of the invention may bind to the same epitope as a specific antibody as disclosed herein (e.g. an anti-CD137 antibody may bind domain 2 of CD137), since such an antibody is likely to mimic the action of the disclosed antibody.
• 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 be using a competitive binding assay. Methods for carrying out competitive binding assays are well known in the art. For example they may involve contacting together an antibody and a target molecule under conditions under which the antibody can 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 is able to displace the first antibody from antibody/target complexes may be assessed.
• a second (test) antibody may be assessed.
• Such assessment may use any suitable technique, including, for example, Surface Plasmon Resonance, ELISA, or flow cytometry.
• the ability of a test antibody to inhibit the binding of a first antibody to the target demonstrates that the test antibody can compete with said first antibody for binding to the target and thus that the test antibody binds to the same epitope or region on the target as the first antibody, and may therefore mimic the action of the first antibody.
• Any antibody referred to herein may be provided in isolated form or may optionally be provided linked (directly or indirectly) to another moiety.
• the other moiety may be a therapeutic molecule such as a cytotoxic moiety or a drug.
• the therapeutic molecule may be directly attached, for example by chemical conjugation, to an antibody of the invention.
• Methods for conjugating molecules to an antibody are known in the art.
• carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) may be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides.
• the water-soluble carbodiimide, l-ethyl-3-(3-dimethyla mi nopropyl) carbodiimide (EDC) is particularly useful for conjugating a functional moiety to a binding moiety.
• a cytotoxic moiety may be directly and/or indirectly cytotoxic.
• directly cytotoxic it is meant that the moiety is one which on its own is cytotoxic.
• indirectly cytotoxic it is meant that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it.
• the cytotoxic moiety may be cytotoxic only when intracellular and is preferably not cytotoxic when extracellular.
• the antibody or antigen-binding fragment is linked to a cytotoxic moiety which is a directly cytotoxic chemotherapeutic agent.
• cytotoxic moiety is a directly cytotoxic polypeptide.
• Cytotoxic chemotherapeutic agents are well known in the art.
• Cytotoxic chemotherapeutic agents include: alkylating agents including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexa methyl melamine, thiotepa; alkyl sulphonates such as busulfane; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoi midazole-carboxamide); Anti metabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil;
• 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 alphenomes.
• VLB vinblastine
• 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-asparaginas
• Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methyl hydrazine, MIH); and adrenocortical suppressant such as mitotane (o,r'-DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.
• platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin
• anthracenedione such as mitoxantrone and anthracycline
• substituted urea such as hydroxyurea
• methyl hydrazine derivative such as procarbazine (N-methyl hydrazine, MI
• the cytotoxic moiety may be a cytotoxic peptide or polypeptide moiety which leads to cell death.
• Cytotoxic peptide and polypeptide moieties are well known in the art and include, 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 WO 96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide. Certain cytokines, such as TNFo and IL-2, may also be useful as cytotoxic agents.
• radioactive atoms may also be cytotoxic if delivered in sufficient doses.
• the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as 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 which emits enough energy to destroy neighbouring cells, organelles or nucleic acid.
• the isotopes and density of radioactive atoms in the agents of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus.
• the radioactive atom may be attached to the antibody, antigen-binding fragment, variant, fusion or derivative thereof in known ways.
• EDTA or another chelating agent may be attached to the binding moiety and used to attach lllln or 90Y.
• Tyrosine residues may be directly labelled with 1251 or 1311.
• the cytotoxic moiety may be a suitable indirectly-cytotoxic polypeptide.
• the indirectly cytotoxic polypeptide may be a polypeptide which has enzymatic activity and can convert a non-toxic and/or relatively non-toxic prodrug into a cytotoxic drug.
• ADEPT Antibody-Directed Enzyme Prodrug Therapy
• the system requires that the antibody locates the enzymatic portion to the desired site in the body of the patient and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound.
• the object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues.
• the cytotoxic moiety may be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.
• the enzyme and prodrug of the system using a targeted enzyme as described herein may be any of those previously proposed.
• the cytotoxic substance may be any existing anti-cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinases or topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent.
• Etoposide is an example of a topoisomerase inhibitor.
• Suitable enzymes for forming part of an enzymatic portion include: exopeptidases, such as carboxypeptidases G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2-Q- aminocyl MTC prodrugs); endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (e.g. alkaline phosphatase) or sulphatases (e.g.
• aryl sulphatases (for phosphylated or sulphated prodrugs); amidases, such as penicillin amidases and arylacyl amidase; lactamases, such as b- lactamases; glycosidases, such as b-glucuronidase (for b-glucuronomide anthracyclines), a-galactosidase (for amygdalin) and b-galactosidase (for b-galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for gancyclovir); reductases, such as nitroreductase (for CB1954 and analogues), azoreductase (for azobenzene mustards) and DT- diaphorase (for CB195
• 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.
• each moiety is a polypeptide
• the two portions may be linked together by any of the conventional ways of cross-linking polypeptides.
• the antibody or antigen-binding fragment may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl- 3-(2-pyridyldithio) propionate (SPDP).
• NHS iodoacetic acid
• SPDP N-succinimidyl- 3-(2-pyridyldithio) propionate
• Amide and thioether bonds for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.
• the cytotoxic moiety may be a radiosensitizer.
• Radiosensitizers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-ami nobenzodiamide, etanixadole, pimonidazole and misonidazole.
• delivery of genes into cells can radiosensitise them, for example delivery of the p53 gene or cyclin D.
• the further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation.
• the boron-10 isotope when appropriately irradiated, releases a particles which are cytotoxic.
• the cytotoxic moiety may be one which is useful in photodynamic therapy such as photofrin.
• the invention provides a combination therapy for use in treating cancer, such as a solid tumour, in a subject comprising (a) an antibody, or 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.
• the antibody, or antigen-binding portion thereof, that specifically binds to CD137 and the PD-1 inhibitor are as described above.
• synergistic we include that the therapeutic effect of the two agents in combination (e.g. as determined by reference to the rate of growth or the size of the tumour) is greater than the additive therapeutic effect of the two agents administered on their own.
• Such synergism can be identified by testing the active agents, alone and in combination, in a relevant cell line model of the solid tumour.
• combination therapy or “combined treatment” or “in combination” as used herein denotes any form of simultaneous or sequential treatment with at least two different therapeutic agents.
• 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.
• 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 prior to or after the administration of the PD- 1 inhibitor.
• the administration of the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor are concurrent, i.e., the administration period of the anti-CD137 antibody, or antigen-binding fragment thereof, and that of the PD-1 inhibitor overlap with each other.
• the administration of the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor are non-concurrent, or sequential.
• the administration of the anti-CD137 antibody, or antigen-binding fragment thereof is terminated before the PD-1 inhibitor is administered.
• the administration of the PD-1 inhibitor is terminated before the anti- CD137 antibody, or antigen-binding fragment thereof, is administered.
• the anti-CD137 antibody, or antigen-binding fragment thereof, and the PD-1 inhibitor are administered as a single therapeutic composition.
• the therapeutic composition further comprises therapeutically acceptable diluents or carrier.
• CD137 stimulation results in activation of tumour infiltrating T cells enabling them to kill the tumour cells.
• CD137 mediated activation of T cells also results in an upregulation of PD-1 on T cells (and indirectly to upregulation of PD-L1 on tumour cells).
• PD-1 inhibition results in re-activation (or removal of inhibition of) tumour specific T cells.
• T cells that are re-activated by a PD-1 inhibitor express CD137.
• CD137 stimulation of CD137 expressing T cells increase their ability to kill tumor cells and prevent them from being exhausted (or rescue them from exhaustion)
• the further immunotherapeutic agent is a PD-1 inhibitor and may preferably be an antibody or other agent which specifically binds to at least one of PD-1 or PD-L1 (as already described above).
• the further immunotherapeutic agent is an antibody or bi-specific molecule comprising an antibody
• the definitions of target specificity/affinity and methods for determining specificity/affinity set out above for anti-CD137 antibodies will apply equally to an antibody that is the further immunotherapeutic agent, except the specific target of the agent will be read in place of CD137.
• Variants and fragments of an antibody which is the further immunotherapeutic agent may also be defined in the same way as the variants and fragments of anti-CD137 antibodies.
• the invention provides a method for treating a cancer, preferably a solid tumour, in a subject.
• the tumour is typically malignant and may be metastatic.
• the combination therapy of the invention may be used to treat patients or subjects who suffer from or are at risk of suffering from a cancer.
• 'treatment' we include both therapeutic and prophylactic treatment of the patient.
• the term 'prophylactic' is used to encompass the use of an agent, or formulation thereof, as described herein which either prevents or reduces the likelihood of a 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, as described herein to prevent recurrence of a cancer in a patient who has previously been treated for the neoplastic disorder.
• the cancer may be associated with formation of solid tumours or may be a haematologic cancer.
• Cancer types that may be treated include carcinomas, sarcomas, lymphomas, leukemias, blastomas and germ cell tumours.
• the cancer may be 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 (such as acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g. melanoma), bladder cancer and glioblastoma.
• prostate cancer breast cancer; colorectal cancer; kidney cancer; pancreatic cancer; ovarian cancer; lung cancer; cervical cancer; rhabdomyosarcoma; neuroblastoma; bone cancer; multiple myeloma; leukemia (such as acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g. melanoma), bladder cancer and glioblastoma.
• ALL acute lymphoblastic leukemia
• AML acute
• the cancer may be selected from the list of cancers in Table 4 or Table 5 below (taken from WO 2018/091740).
• Table 4 Mean expression values of solid human tumors with an above average expression (mean expression level >10) of both Fey receptor and CD137 (TNFRSF9). The ten tumors with the highest expression of the six Fey receptors are shown.
• Table 5 Mean expression values of hematological malignancies with an above average expression (mean expression level 310) of both FCY receptor and CD137. The ten malignancies with the highest expression of the six FCY receptors are shown.
• 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/near the site of a tumour.
• the therapeutic agents in the combination therapy of the invention are administered intravenously.
• the combination therapy and or methods of the invention can be used for treating a patient who has been pre-screened and identified as having a tumour with cells expressing CD137 and FcyR, such as FcyRI, FcyRIIA, FcyRIIB or combinations thereof.
• combination therapy of the invention may be used as a sole treatment for cancer in a patient or as part of an additional combination treatment (which additional treatment may be a pharmaceutical agent, radiotherapy and/or surgery).
• the cancer may be a solid tumour.
• Solid tumours are classically defined by the tissue from which they originate, e.g. breast, colon etc.
• immunotherapy acts on the immune system, and not the tumour itself, the immune status of the tumour may be more predictive of the response than the origin of the tumour.
• the MC38 colon cancer model is evaluated in more detail, which is generally immunogenic and responds to PD-1 therapy alone.
• the tumour is immunogenic.
• Such tumours are characterised 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 tumour profile, correlates with a good prognosis following therapy, for example in colon cancer, (Galon ef a/., 2014, J. Pathol. 232(2) : 199-209).
• the tumour is non-immunogenic or poorly-immunogenic. Poorly immunogenic tumours often have low or absent MHO expression and are characterized by lower number of infiltrating immune cells, such as T cells and cells of myeloid origin (Lechner ef a/., 2013, J Immunotherapy 36(9) :477- 89).
• the tumour may be an adenoma, an adenocarcinoma, a blastoma, a ca rcinoma, a desmoid tumour, a desmopolastic small round cell tumour, an endocrine tumour, a germ cell tumour, a lymphoma, a sarcoma, a Wilms tumour, a lung tumour, a colon tumour, a lymph tumour, a breast tumour or a melanoma.
• Types of blastoma include hepatblastoma, glioblastoma, neuroblastoma or retinoblastoma.
• Types of carcinoma include breast, endometrial, colorectal carcinoma or hepatocellular carcinoma, pancreatic, prostate, gastric, urothelial, renal, Merkel cell, oesophageal, cervical, and head and neck carcinomas, and adenocarcinoma.
• Types of sarcoma include Ewing sarcoma, osteosarcoma, rhabdomyosarcoma, or any other soft tissue sarcoma.
• Types of melanoma include Lentigo maligna, Lentigo maligna melanoma, Superficial spreading melanoma, Acral lentiginous melanoma, Mucosal melanoma, Nodular melanoma, Polypoid melanoma, Desmoplastic melanoma, Amelanotic melanoma, Soft-tissue melanoma, Melanoma with small nevus-like cells, Melanoma with features of a Spitz nevus and Uveal melanoma.
• Types of lymphoma 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's lymphoma, Mycosis fungoides, Peripheral T-cell lymphoma, Nodular sclerosis form of Hodgkin lymphoma, Mixed-cellularity subtype of Hodgkin lymphoma.
• Types of lung tumour include tumours of non-small-cell lung cancer (adenoca rcinoma, squamous-cell ca rcinoma and large-cell ca rcinoma) and small-cell lung carcinoma.
• 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 a high tumour mutational burden (i.e. TMB-high).
• MSI-H microsatellite instability high
• dMMR deficient mismatch repair
• the cancer may be a mesothelioma.
• the method of the invention comprises (a) administering to the subject a therapeutically effective amount of an antibody that specifically binds to CD137, and (b) administering to the subject a therapeutically effective amount of an further immunotherapeutic agent, wherein the further immunotherapeutic agent is a PD-1 inhibitor, optionally wherein the PD-1 inhibitor is administered systemically.
• Steps (a) and (b) may be carried out simultaneously. Alternatively steps (a) and (b) may be carried our sequentially provided step (a) precedes step (b).
• the anti- CD137 antibody is preferably administered systemically to the tumour and most preferably the anti-CD137 antibody is administered intravenously.
• terapéuticaally effective amount of a substance it is meant that a given substance is administered to a subject suffering from a condition, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. Effective amounts for a given purpose and a given agent will depend on the severity of the disease or injury as well as the weight and general state of the subject. As used herein, the term “subject” includes any mammal, preferably a human.
• the invention also provides: an antibody that specifically binds to CD137 for use in a method for treating a solid tumour in a subject, the method comprising (a) administering to the subject 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 carried out simultaneously. Alternatively steps (a) and (b) may be carried our sequentially provided step (a) precedes step (b).
• said anti-CD137 antibody is preferably administered locally to the tumour.
• an antibody that specifically binds to CD137 in the manufacture of a medicament for treating a solid tumour in a subject, wherein said treating comprises (a) administering to the tumour 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 carried out simultaneously. Alternatively steps (a) and (b) may be carried our sequentially provided step (a) precedes step (b).
• said anti-CD137 antibody is preferably administered systemically, and most preferably said antLCD137 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 tumour in a subject comprising (a) systemically administering to the tumour 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 carried out simultaneously. Alternatively steps (a) and (b) may be carried our sequentially provided step (a) precedes step (b).
• said anti-CD137 antibody is preferably administered locally to the tumour.
• steps (a) and (b) may be carried out sequentially (i.e. at different times), with step (a) being carried out before step (b).
• Steps (a) and (b) may be separated by an interval such that the combined anti-tumour effect is optimised.
• Step (b) may be conducted a sufficiently long interval after step (a) that at least one physiological effect of step (a) is at or close to its peak level.
• the anti-CD137 antibody will typically stimulate CD137 and activate T cells and/or other immune cells (e.g. to induce release of interferon gamma from CD8 + cell).
• the activated T cells may begin to express higher levels of immune system checkpoint molecules (such as PD-1) within around 24 hours of treatment with anti- CD137. These immune system checkpoint molecules may negatively regulate the anti tumour response.
• the further immunotherapeutic agent administered in step (b) is a PD-1 inhibitor and may thus preferably be an anti-PD-1 or anti-PDLl antibody which blocks or inhibits such the activity of PD-1.
• step (b) may be carried out a sufficiently long interval after step (a) such that the expression level of the immune system checkpoint molecule (such as PD-1) in cells in the subject, 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 in a healthy subject.
• step (b) may be conducted within 24 hours after step (a), between 24 hours and two weeks after step (a), between 24 hours and one week after step (a), between 24 hours and 72 hours after step (a), or between 24 hours and 48 hours after step (a).
• step (b) is conducted within 24 hours after step (a).
• step (b) may be conducted at a time point after step (a) where the expression level of the immune system checkpoint molecule (such as PD-1) in a cell of the subject, or the number of cells in the subject expressing said immune system checkpoint molecule, is determined to be elevated relative to said level or number in the subject prior to step (a), or relative to said level or number in in a healthy subject.
• the immune system checkpoint molecule such as PD-1
• the expression level of an immune system checkpoint molecule (such as PD-1) in a cell of a subject, or the number of cells in a subject expressing such a molecule, may be determined by any suitable means, for example by flow cytometric analysis of a sample taken from the subject.
• steps (a) and (b) are carried out on the same day. It may be preferable to carry out steps (a) and (b) simultaneously (i.e. at the same time), or within 24 hours of each other, such that both steps may be carried out on the same day or during the same visit to a treatment centre. This may be particularly advantageous where access to treatment centres is restricted.
• steps (a) and (b) may be carried out simultaneously, or may be carried out 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.
• steps (a) and (b) are carried out simultaneously or wherein step (b) is carried out between 24 hours and two weeks after step (a), between 24 hours and one week after step (a), between 24 and 72 hours after step (a), or between 24 and 48 hours after step (a).
• step (a) may be conducted on multiple further instances after the first instance. That is, the subject may receive a series of doses of anti-CD137 antibody. These doses are administered such that the subject has only intermittent exposure to the anti-CD137 antibody, preferably such that the immune cells of the subject do not become depleted and/or that the subject does not suffer from tachyphylaxia to the anti-CD137 antibody. At detection of either of these symptoms, the next administration of anti-CD137 antibody may be delayed or cancelled.
• step (b) is preferably conducted in a manner which, following initiation of step (b), permits continuous exposure of the subject to the further immunotherapeutic agent (PD-1 inhibitor) for the duration of the method, including during any second and further instances of step (a).
• the additional agent is an anti-PD-1 or anti-PDLl antibody which blocks or inhibits such the activity of the immune system checkpoint molecule, PD-1.
• Continuous receptor blockade may be particularly important for the therapeutic effects of such agents.
• step (a) is conducted on multiple separate occasions and step (b) is conducted such that exposure of the subject to the further immunotherapeutic agent is continuous for the duration of the method.
• Step (a) of the method concerns the local or systemic administration of an anti-CD137 antibody to a subject having a solid tumour.
• step (a) concerns the systemic administration of an anti-CD137 antibody, e.g. via intravenous or subcutaneous administration.
• step (a) involves intravenous administration of the anti-CD137 antibody.
• the anti-CD137 antibody or antigen binding fragment thereof is locally administered to a tumour site in a subject.
• Local administration to the tumour site includes peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracranial and intravesicle administration by any suitable means, such as injection. Local administration may also include intra cavity infusion and inhalation, depending on the site of the tumour.
• a high proportion of the anti-CD137 antibody may be retained at the tumour site in vivo, that is within the tumour microenvironment, for an extended period of time following administration of said antibody. That is, the antibody exhibits reduced leakage from the tumour site into vascular or lymphatic circulation, particularly when locally administered to the tumour site.
• Preferably at least 30% of an antibody dose administered to a tumour in accordance with the method is retained in the tumour site at four hours after administration, more preferably at least 40% of the dose is retained at four hours after administration and most preferably at least 50% of the dose is reta i ned at four hours after administration.
• the anti-CD137 antibody is released only slowly from the tumour area.
• Antibody retention in a tumour microenvironment can be studied by injecting the antibody into tumours in murine models and measuring the serum levels of the antibody over time after administration. Alternatively the distribution of an antibody can be measured using radiolabelled antibodies injected into tumours in murine models. Suitable techniques are known to the skilled person. For example, retention of the antibody at the tumour site may be assessed by monitoring serum levels of the antibody post-administration (see Mangsbo et al., 2014, Clin. Cancer Res. 21(5) : 1115-1126, the disclosure of which are incorporated herein by reference). For example, in one embodiment, the serum levels of anti-CD137 four hours following intratumoral injection of 30 pg of the antibody (in 60 mI_) are less than 1 pL/ml.
• Step (b) of the method concerns the systemic administration of a PD-1 inhibitor to a subject.
• Systemic administration of any agent described herein means administration into the circulatory system of the subject, including the vascular and/or lymphatic system. Such administration may be by any suitable route, but is typically parenteral.
• the PD-1 inhibitor is administered locally to a tumour site in a subject.
• the PD-1 inhibitor is administered to the subject systemically, e.g. intravenous or sub-cutaneous.
• the systemic administration of a PD-1 inhibitor is intravenous.
• parenteral administration means modes of administration other than enteral and topical administration, and is typically achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intra peritoneal, subcutaneous, spinal, intracerebral, intrathecal, intraosseous or other parenteral routes of administration.
• Kits and pharmaceutical compositions The invention also provides a kit for treating a cancer, preferably a solid tumour in a subject, the kit comprising a combination therapy as defined above.
• the kit may comprise (a) a therapeutically effective amount of an antibody that specifically binds to CD137 and optionally that is retained at the tumour site following 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 a tumour site.
• kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out.
• reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions) and means to administer the anti-CD137 antibody and/or the PD- 1 inhibitor (such as a vessel or an instrument comprising a needle).
• the anti-CD137 antibody and the PD-1 inhibitor used in the methods of the invention, or provided in the kits of the invention, may each be provided as a separate pharmaceutical composition formulated together with a pharmaceutically acceptable carrier.
• pharmaceutically 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 are also compatible with the required routes of administration.
• the carrier for the anti-CD137 antibody and the PD-1 inhibitor may be suitable for systemic administration, which as defined above means administration into the circulatory system of the subject, including the vascular and/or lymphatic system. Such administration may be by any suitable route, but is typically parenteral.
• parenteral administration as used herein means modes of administration other than enteral and topical administration, and is typically achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intra peritoneal, subcutaneous, spinal or other parenteral routes of administration.
• the carrier for the anti-CD137 antibody is preferably suitable for local administration, which as defined above includes peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracranial and intravesicle administration by any suitable means, such as injection. Local administration may also include intra cavity infusion and inhalation, depending on the site of the tumour.
• the antibody and/or the agent may be coated in a material to protect the antibody from the action of acids and other natural conditions that may inactivate or denature the antibody and/or agent.
• Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents.
• aqueous carriers examples include water, buffered water and saline.
• suitable aqueous carriers include water, buffered water and saline.
• suitable aqueous 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 the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• the antibody components of the combination therapies of the present invention are typically provided in the form of one or more pharmaceutical compositions, each containing a therapeutically-effective amount of the antibody component(s) together with a pharmaceutically-acceptable buffer, excipient, diluent or carrier.
• chelating agents such as EDTA, citrate, EGTA or glutathione.
• pharmaceutically acceptable we mean a non-toxic material that does not decrease the effectiveness of the CD137-binding activity of the antibody polypeptide of the invention.
• pharmaceutically 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).
• a 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
• This is a predetermined quantity of active antibody calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle.
• diluent i.e. a carrier or administration vehicle.
• a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host.
• the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
• a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
• a pharmaceutical composition may include a pharmaceutically acceptable anti-oxidant. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminium monostearate and gelatin.
• adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol,
• compositions typically must be sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
• Sterile injectable solutions can be prepared by incorporating the active agent (e.g. antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
• dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• compositions may comprise additional active ingredients as well as those mentioned above.
• Suitable pharmaceutically acceptable buffers, diluents, carriers and excipients are well- known in the art (see Remington's Pharmaceutical Sciences, 18 th edition, A.R 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).
• buffer is intended to include an aqueous solution containing an acid-base mixture with the purpose of stabilising pH
• buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AM PD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.
• diluent is intended to include an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation.
• the diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).
• adjuvant is intended to include any compound added to the formulation to increase the biological effect of the agent of the invention.
• the adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition.
• the adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
• cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
• the excipient may be one or more of carbohydrates, polymers, lipids and minerals.
• carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation.
• polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation.
• lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers.
• minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.
• the active antibody-based agents of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.
• the pharmaceutical compositions of the invention may be in the form of a liposome, in which the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals.
• Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
• Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example US 4,235,871 and in EP 0 213 303, the disclosures of which are incorporated herein by reference.
• compositions of the invention may also be in the form of biodegradable microspheres.
• Aliphatic polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. Preparations of such microspheres can be found in US 5,851,451 and in EP 0 213 303, the disclosures of which are incorporated herein by reference.
• compositions of the invention are 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 WO 2011/128642, the disclosures of which are incorporated herein by reference. It will be appreciated by persons skilled in the art that one or more of the active components of the combination therapies of the present invention may be formulated in separate nanoparticles, or both active components may be formulated in the same nanoparticles.
• compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose ca rboxy methy lcel I u lose, hyd roxy p ropy I methyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the agent.
• the polymers may also comprise gelatin or collagen.
• the agents 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.
• oils such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil
• tragacanth gum and/or various buffers.
• compositions of the invention may include ions and a defined pH for potentiation of action of the active agent. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.
• compositions according to the invention may be administered via any suitable route known to those skilled in the art.
• routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, parenteral, vaginal and rectal. Also administration from implants is possible.
• the pharmaceutical composition is suitable for administration at or near the site of a tumour, e.g. intra-tumourally or peri-tumourally.
• the pharmaceutical composition is suitable for parenteral administration, for example the pharmaceutical composition is preferably suitable for administration intravenously, intracerebroventricularly, intraarticularly, intra arterially, intraperitoneally, intratheca I ly, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or by infusion techniques.
• Methods for formulating an antibody into a pharmaceutical composition such as a pharmaceutical composition suitable for parenteral administration, will be well-known to those skilled in the arts of medicine and pharmacy. Preferred compositions are described in the accompanying Examples.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) 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.
• compositions of the invention are particularly suitable for parenteral, e.g. intravenous, administration.
• the combination therapy of the invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections.
• An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
• delivery is performed intra-muscularly (i.m.) and/or sub-cutaneously (s.c.) and/or intravenously (i.v.).
• the combination therapy of the invention can be administered by a surgically implanted device that releases the drug directly to the required site.
• Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis.
• the direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.
• Electroporation therapy (EFT) systems can also be employed for the administration of the combination therapy of the invention.
• a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
• the combination therapy of the invention can also be delivered by electro-incorporation (El).
• El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin.
• the particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
• ReGel injectable system that is thermo-sensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active substance is delivered over time as the biopolymers dissolve.
• the combination therapy of the invention can also be delivered orally.
• the process employs a natural process for oral upta ke of vitamin B12 and/or vitamin D in the body to co-deliver proteins and peptides.
• the agents, medicaments and pharmaceutical compositions of the invention can move through the intestinal wall.
• Complexes are synthesised between vitamin B12 analogues and/or vitamin D analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B12 portion/vitamin D portion of the complex and significant bioactivity of the active substance of the complex.
• IF intrinsic factor
• the combination therapy of the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al. (1998), Trends Cell Biol. 8, 84-87.
• the combination therapy of the invention is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.
• the combination therapy of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
• the compositions may be administered at varying doses.
• the combination therapy of the 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.
• the combination therapy of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
• the agents, medicaments and pharmaceutical compositions of the invention may also be administered via intra cavernosa I injection.
• Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disinteg rants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxy propyl methylcel I ulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
• excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
• disinteg rants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmel
• Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
• Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
• the agents, medicaments and pharmaceutical compositions of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
• the combination therapy of the invention can be administered parenterally, for example, intravenously, intra-arterially, intra peritonea I ly, intra-thecally, intraventricularly, intrasterna I ly, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
• the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 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.
• Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the medicaments 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 (lyophilised) 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.
• compositions of the invention are particularly suitable for parenteral, e.g. intravenous, administration.
• the combination therapy of the invention can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
• a lubricant e.g. sorbitan trioleate.
• Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of an agent of the invention and a suitable powder base such as lactose or starch.
• 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 overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
• the combination therapy of the invention can be administered in the form of a suppository or pessary, or they 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 invention may also be transdermally administered, 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.
• the combination therapy of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
• a preservative such as a benzylalkonium chloride.
• they may be formulated in an ointment such as petrolatum.
• the combination therapy of the invention can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following : mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water.
• they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following : mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
• Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
• lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth
• pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia
• mouth-washes comprising the active ingredient in a suitable liquid carrier.
• local administration of the combination therapy of the invention at or near the site of a tumour is the preferred route, in particular intra-tumoural or peri-tumoural administration.
• compositions will be administered to a patient in a pharmaceutically effective dose / a therapeutically effective amount, as described above.
• a therapeutically effective amount of the active component is provided.
• a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
• the administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a prolonged period.
• the antibody polypeptides can be formulated at various concentrations, depending on the efficacy/toxicity of the polypeptide being used.
• the formulation may comprise the active antibody polypeptide at a concentration of between 0.1 pM and 1 mM, more preferably between 1 pM and 500 pM, between 500 pM and 1 mM, between 300 pM and 700 pM, between 1 pM and 100 pM, between 100 pM and 200 pM, between 200 pM and 300 pM, between 300 pM and 400 pM, between 400 pM and 500 pM, between 500 pM and 600 pM, between 600 pM and 700 pM, between 800 pM and 900 pM or between 900 pM and 1 mM.
• the formulation comprises the active antibody polypeptide at a concentration of between 300 pM and 700 pM.
• the therapeutic dose of the antibody polypeptide (with or without a therapeutic moiety) in a human patient will be in the range of 100 pg to 1 g per administration (based on a body weight of 70kg, e.g. between 300 pg to 700 mg per administration).
• the maximum therapeutic dose may be in the range of 0.1 to 10 mg/ kg per administration, e.g. between 1 and 10 mg/ kg or between 0.1 and 5 mg/ kg or between 1 and 5 mg/ kg or between 0.1 and 2 mg/kg.
• the therapeutic dose is between 1 and 10 mg/kg, optionally between 2.5 and 7.5 mg/kg.
• a dose may be administered at different intervals, as determined by the oncologist/physician; for example, a dose may be administered daily, twice-weekly, weekly, bi-weekly or monthly.
• a dose may be administered daily, twice-weekly, weekly, bi-weekly or monthly.
• the polypeptides and pharmaceutical formulations of the present invention have utility in both the medical and veterinary fields.
• the methods of the invention may be used in the treatment of both human and non-human animals (such as horses, dogs and cats).
• the patient is human.
• the combination therapy of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
• the invention also provides a kit for treating a solid tumour 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 an further immunotherapeutic agent that is suitable for systemic administration to a subject.
• the further immunotherapeutic agent is a PD-1- inhibitor, optionally as defined in the first aspect.
• the antibody that specifically binds to CD137 is preferably provided in a form suitable for local administration to a tumour.
• the invention also provides a kit comprising a first isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 (or a component polypeptide chain thereof) and:
• nucleic acid molecule we include DNA (e.g. genomic DNA or complementary DNA) and mRNA molecules, which may be single- or double-stranded.
• isolated we mean that the nucleic acid molecule is not located or otherwise provided within a cell.
• the first and/or second nucleic acid molecule(s) is/are cDNA molecule(s).
• the first and/or second isolated nucleic acid molecules encode an antibody heavy chain or variable region thereof and/or encode an antibody light chain or variable region thereof.
• the first nucleic acid molecule comprises one or more nucleotide sequence selected from either SEQ ID NO: 9 and/or SEQ ID NO: 10, reproduced below.
• the first nucleic acid molecule comprises one or more nucleotide sequence selected from either SEQ ID NO: 27 and/or SEQ ID NO: 28, reproduced below.
• the first nucleic acid molecule may be codon-optimised for expression of the antibody polypeptide in a particular host cell, e.g. for expression in human cells (for example, see Angov, 2011, Biotechnol. J. 6(6) :650-659, the disclosures of which are incorporated herein by reference).
• the invention also provides a kit comprising a vector comprising a first isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 or a component polypeptide chain thereof, and :
• the vector is an expression vector.
• the first and/or second isolated nucleic acid may be as described earlier.
• the invention also provides a kit comprising a host cell comprising a first isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to CD137 or a component polypeptide chain thereof, and:
• the host cell may be a mammalian cell (e.g. a human cell, or Chinese hamster ovary cell, e.g. CHOK1SV cells), a bacterial cell or a yeast cell.
• the first and/or second isolated nucleic acid may be as described earlier.
• the first and/or second isolated nucleic acid may be comprised in a vector, such as an expression vector.
• SEQ ID NO: 1 is the amino acid sequence of VH region of "1630".
• SEQ ID NO: 2 is the amino acid sequence of VL region of "1631”.
• SEQ ID NO: 3 is the amino acid sequence of HCDR 1 of "1630”.
• SEQ ID NO: 4 is the amino acid sequence of HCDR 2 of "1630".
• SEQ ID NO: 5 is the amino acid sequence of HCDR 3 of "1630".
• SEQ ID NO: 6 is the amino acid sequence of LCDR 1 of "1631".
• SEQ ID NO: 7 is the amino acid sequence of LCDR 2 of "1631”.
• SEQ ID NO: 8 is the amino acid sequence of LCDR 3 of "1631”.
• SEQ ID NO: 9 is the nucleotide sequence encoding VH region of "1630".
• SEQ ID NO: 10 is the nucleotide sequence encoding VL region of "1631".
• SEQ ID NO: 11 is the amino acid sequence of human CD137 sequence (amino acids 66 to 107 correspond to domain 2 of human CD137).
• SEQ ID NO: 12 is the amino acid sequence of IgGl heavy chain constant region.
• SEQ ID NO: 13 is the amino acid sequence of modified IgG4 constant region.
• SEQ ID NO: 14 is the amino acid sequence of modified IgG4 constant region.
• SEQ ID NO 15 is the amino acid sequence of wild-type IgG4 constant region.
• SEQ ID NO 16 is the amino acid sequence of kappa chain constant region.
• SEQ ID NO 17 is the full amino acid sequence of heavy chain of "1630”.
• SEQ ID NO 18 is the full amino acid sequence of the light chain of "1631”.
• SEQ ID NO 19 is the amino acid sequence of the VH region of "2674”.
• SEQ ID NO 20 is the amino acid sequence of the VL region of "2675”.
• SEQ ID NO 21 is the amino acid sequence of HCDR 1 of "2674”.
• SEQ ID NO 22 is the amino acid sequence of HCDR 2 of "2674".
• SEQ ID NO 23 is the amino acid sequence of HCDR 3 of "2674".
• SEQ ID NO 24 is the amino acid sequence of LCDR 1 of "2675”.
• SEQ ID NO 25 is the amino acid sequence of LCDR 2 of "2675).
• SEQ ID NO 26 is the amino acid sequence of LCDR 3 of "2675).
• SEQ ID NO 27 is the nucleotide sequence encoding VH region of "2674”.
• SEQ ID NO 28 is the nucleotide sequence encoding VL region of "2675”.
• SEQ ID NO 29 is the full amino acid sequence of the heavy chain "2674”.
• SEQ ID NO 30 is the full amino acid sequence of the light chain of "2675”.
• SEQ ID NO 31 is the heavy chain amino acid sequence of Nivolumab.
• SEQ ID NO 32 is the light chain amino acid sequence of Nivolumab.
• SEQ ID NO 33 is the heavy chain amino acid sequence of Pembrolizumab.
• SEQ ID NO 34 is the light chain amino acid sequence of Pembrolizumab.
• SEQ ID NO 35 is the amino acid sequence of the human PD-1 sequence.
• SEQ ID NO 36 is the amino acid sequence of the human PD-L1 sequence.
• SEQ ID NO 37 is the heavy chain sequence of Pidilizumab.
• SEQ ID NO 38 is the light chain sequence of Pidilizumab.
• SEQ ID NO 39 is the heavy chain sequence of Cemiplimab.
• SEQ ID NO 40 is the light chain sequence of Cemiplimab.
• SEQ ID NO 41 is the heavy chain sequence of Spartalizumab.
• SEQ ID NO 42 is the light chain sequence of Spartalizumab.
• SEQ ID NO 43 is the heavy chain sequence of Camrelizumab.
• SEQ ID NO 44 is the light chain sequence of Camrelizumab.
• SEQ ID NO 45 is the heavy chain sequence of Tislelizumab.
• SEQ ID NO 46 is the light chain sequence of Tislelizumab.
• SEQ ID NO 47 is the heavy chain sequence of Toripalimab.
• SEQ ID NO 48 is the light chain sequence of Toripalimab.
• SEQ ID NO 49 is the heavy chain sequence of Dostarlimab.
• SEQ ID NO 50 is the light chain sequence of Dostarlimab.
• SEQ ID NO 51 is the heavy chain sequence of INCMGA00012.
• SEQ ID NO: 52 is the light chain sequence of INCMGA00012.
• SEQ ID NO: 53 is the heavy chain se uence of Atezolizumab.
• SEQ ID NO: 54 is the light chain sequence of Atezolizumab.
• SEQ ID NO: 55 is the heavy chain sequence of Durvalumab.
• SEQ ID NO: 56 is the light chain sequence of Durvalumab.
• SEQ ID NO: 57 is the heavy chain sequence of Avelumab.
• SEQ ID NO: 58 is the light chain sequence of Avelumab.
• SEQ ID NO: 59 is the heavy chain sequence of CK-301.
• SEQ ID NO: 60 is the light chain sequence of CK-301.
• SEQ ID NO: 61 is the heavy chain sequence of JTX-4014
• SEQ ID NO: 62 is the light chain sequence of JTX-4014.
• Embodiments of the invention include, but are not limited to, the following: A. A method for treating a solid tumour in a subject, the method comprising
• the solid tumour is an adenoma, a blastoma, a carcinoma, a desmoid tumour, a desmopolastic small round cell tumour, an endocrine tumour, a germ cell tumour, a lymphoma, a sarcoma, a Wilms tumour, a lung tumour, a colon tumour, a lymph tumour, a breast tumour or a melanoma.
• the solid tumour is a lung tumour (such as a non-small cell lung cancer or a small cell lung cancer); a head and/or neck tumour, a gastric tumour, oesophageal tumour, renal tumour, urothelial tumour, MSI-high tumour, dMMR tumour,
• a lung tumour such as a non-small cell lung cancer or a small cell lung cancer
• a head and/or neck tumour such as a gastric tumour, oesophageal tumour, renal tumour, urothelial tumour, MSI-high tumour, dMMR tumour,
• TMB-high tumour breast tumour, cervical tumour, prostate tumour, or a melanoma, preferably wherein the solid tumour is metastatic.
• the antibody of step (a) comprises at least one CDR selected from SEQ ID NOs 3, 4, 5, 6, 7 and 8.
• 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.
• the antibody of step (a) comprises the light chain variable region of SEQ ID NO: 2 and/or the heavy chain variable region of SEQ ID NO: 1.
• step (a) comprises the light chain constant region of SEQ ID NO: 16 and/or the heavy chain constant region of SEQ ID NO: 13.
• step (a) competes for binding to human CD137 with an antibody which comprises the light chain variable region of SEQ ID NO: 2 and the heavy chain variable region of SEQ ID NO: 1.
• step (a) comprises at least one CDR selected from 21, 22, 23, 24, 25 and 26, 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.
• step (a) comprises the light chain variable region of SEQ ID NO: 20 and/or the heavy chain variable region of SEQ ID NO: 19.
• step (a) comprises the light chain constant region of SEQ ID NO: 16 and/or the heavy chain constant region of SEQ ID NO: 13.
• step (a) competes for binding to human CD137 with an antibody which comprises the light chain variable region of SEQ ID NO: 20 and the heavy chain variable region of SEQ ID NO: 19.
• step (a) and (b) are carried out simultaneously or wherein step (b) is carried out within 24 hours after step (a), between 24 hours and two weeks after step (a), between 24 hours and one week after step (a), between 24 and 72 hours after step (a), or between 24 and 48 hours after step (a), preferably wherein step
• step (b) is carried out within 24 hours after step (a),
• step (a) comprises systemic administration of the antibody to the subject, optionally wherein the antibody is formulated as a composition suitable for system administration with at least one pharmaceutically acceptable diluent or carrier.
• step (a) The method according to any one of the preceding Embodiments, wherein at least 30% of the amount of antibody administered in step (a) is retained at the tumour site at four hours after administration, preferably wherein at least 40% of the said amount is retained at the tumour site at four hours after administration.
• step (b) The method according to any one of the preceding Embodiments wherein the further immunotherapeutic agent of step (b) is formulated as a composition suitable for systemic administration with at least one pharmaceutically acceptable diluent or carrier.
• step (a) is conducted on multiple separate occasions and step (b) is conducted such that exposure of the subject to the further immunotherapeutic agent is continuous for the duration of the method.
• kits for treating a solid tumour in a subject comprising (a) a therapeutically effective amount of an antibody that specifically binds to CD137 and preferably is retained at the tumour site following administration and optionally (b) a therapeutically effective amount of a further immunotherapeutic agent that is suitable for systemic administration to a subject, wherein the further immunotherapeutic agent is a PD-1 inhibitor.
• the solid tumour is an adenoma, a blastoma, a carcinoma, a desmoid tumour, a desmopolastic small round cell tumour, an endocrine tumour, a germ cell tumour, a lymphoma, a sarcoma, a Wilms tumour, a lung tumour, a colon tumour, a lymph tumour, a breast tumour or a melanoma.
• the solid tumour is a lung tumour (such as a non small cell lung cancer or a small cell lung cancer); a head and/or neck tumour, a gastric tumour, oesophageal tumour, renal tumour, urothelial tumour, MSI- high tumour, d MR tumour, TMB-high tumour, breast tumour, cervical tumour, prostate tumour, or a melanoma, preferably wherein the solid tumour is metastatic.
• a lung tumour such as a non small cell lung cancer or a small cell lung cancer
• a head and/or neck tumour a gastric tumour, oesophageal tumour, renal tumour, urothelial tumour, MSI- high tumour, d MR tumour, TMB-high tumour, breast tumour, cervical tumour, prostate tumour, or a melanoma, preferably wherein the solid tumour is metastatic.
• An antibody, or antigen binding portion thereof, according to any one of Embodiments U to X comprising at least one CDR selected from SEQ ID NOs 3, 4, 5, 6, 7 and 8.
• Embodiments U to Y comprising at least one CDR selected from SEQ ID NOs 21, 22, 23, 24, 25 and 26.
• AA An antibody, or antigen binding portion thereof, according to any one of Embodiments U to Z 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.
• An antibody, or antigen binding portion thereof, according to any one of Embodiments U to AA comprising the light chain variable region of SEQ ID NO: 2 and/or the heavy chain variable region of SEQ ID NO: 1 or comprising the light chain variable region of SEQ ID NO: 20 and/or the heavy chain variable region of SEQ ID NO: 19.
• Figure 1 shows the effect of administration of an anti-CD137 antibody (ATOR-1017) and/or a PD-1 inhibitor (RMP1-14 clone) on tumour volume in female h4-lBBtg mice i nfected with MC38 colon adenoca rcinoma cells.
• Figure 2 shows the effect of administration of an anti-CD137 antibody (ATOR-1017) and/or a PD-1 inhibitor (RMP1-14 clone) on survival of female h4-lBBtg mice infected with MC38 colon adenocarcinoma cells.
• Figure 3 shows the effect of administration of an anti-CD137 antibody (ATOR-1017) and/or a PD-1 inhibitor (RMP1-14 clone) on tumour growth inhibition in female h4- lBBtg mice infected with MC38 colon adenocarcinoma cells.
• Figure 4 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (Nivolumab) in mixed lymphocyte reaction (MLR) assay.
• Figure 5 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (Pembrolizumab) in mixed lymphocyte reaction (LR) assay.
• Figure 6 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (Nivolumab) in mixed lymphocyte reaction (MLR) assay.
• Figure 7 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (Atezolizumab) in mixed lymphocyte reaction (MLR) assay.
• Figure 8 shows T cell activation by an anti-CD137 antibody (ATOR-1017) in combination with a PD-1 inhibitor (Nivolumab) in mixed lymphocyte reaction (MLR) assay with exhausted CD4+ T cells.
• Eruslanov EB Bhojnagarwala PS, Quatromoni JG et al. Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer. J Clin Invest. 2014 Dec; 124(12): 5466-80. Eruslanov E, Neuberger M, Daurkin I et al. Circulating and tumor-infiltrating myeloid cell subsets in patients with bladder cancer. Int J Cancer. 2012 Mar 1;130(5): 1109- 19.
• Tumour-associated macrophages are related to progression in patients with metastatic melanoma following interleukin-2 based immunotherapy. Acta Oncol. 2006;45(4) :400-5.
• Pancreatic adenoca rcinoma induces bone marrow mobilization of myeloid-derived suppressor cells which promote primary tumor growth. Cancer Immunol Immunother. 2012 Sep;61(9): 1373-85.
• Example 1 Anti-tumor efficacy of anti-CDl 37 antibody in combination with a PD-1 inhibitor in MC38 colon carcinoma turn or-bearing h4-lBB transgenic mice
• ATOR-1017 a human anti-4-lBB (anti-CD137) agonist IgG4 antibody, triggers potent dose-dependent anti-tumor efficacy in MC38 colon carcinoma tumor-bearing mice, transgenic for human 4-1BB (h4-lBBtg). This is demonstrated by reduced tumor volume, improved tumour growth inhibition and induction of complete responders, as well as prolonged survival of mice.
• an anti-CD137 antibody such as ATOR-1017
• a PD-1 inhibitor surrogate anti-PD-1 antibody clone RPM1-14
• mice Female h4-lBBtg mice, 8 weeks of age, were inoculated with 5xl0 5 MC38 colon adenocarcinoma cells subcutaneously (s.c.) in the right front flank in a volume of 100 pi. On day 7 post inoculation, mice were randomized into six study groups, and treated with the anti-CD137 antibody ATOR-1017 intraperitoneally (i.p.) given at two doses, 0.5 mg/ kg or 5 mg/kg, with or without the additional treatment with 5 mg/ kg surrogate anti-PD-1 antibody (clone RMP1-14; Cat no: BE0146 supplied by BioXCell, 39 Labombard Rd, Riverside, IMH 03766, USA).
• clone RMP1-14 Cat no: BE0146 supplied by BioXCell, 39 Labombard Rd, Riverside, IMH 03766, USA.
• Controls received 5 mg/ kg human IgG4 isotype control antibody. Treatments were given twice weekly for three weeks, summing up to six dosing occasions in tota I . Tumor growth was measured twice weekly, and mice sacrificed once tumor volumes approached the ethical limit of 3000 mm 3 .
• the tumor volume is further reduced when compared to the corresponding ATOR-1017 monotherapy.
• mice receiving a combination therapy comprising 10 pg/mouse ATOR-1017 and 100 pg/mouse anti-PD-1 antibody was markedly increased in comparison to the individual monotherapies (Figure 2B),
• Figure 3 demonstrates the effect of treatment with a combination of 100 pg/mouse anti-CD137 antibody, ATOR-1017, and 100 pg/mouse of a surrogate anti-PD-1 antibody, clone RPM1-14, on tumour growth inhibition (TGI, %) compared to the corresponding monotherapies.
• the inventors have surprisingly identified that the effect on TGI is further increased in mice receiving the combination therapy, compared to the corresponding ATOR-1017 monotherapy.
• the number of complete responders was monitored (Table 3) and is increased in mice receiving the combination of ATOR-1017 and the anti-PD-1 antibody.
• Example 2 T cell activation of ATOR-1017 in combination with PD-1 inhibitor in mixed lymphocyte reaction (MLR) assay
• 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), where both targets (4-1BB and PD-1) are endogenously expressed.
• MLR mixed lymphocyte reaction
• Human primary CD4+ T cells were isolated from leukocyte concentrates using a Human CD4+ T Cell Isolation Kit (130-096-533, Miltenyi Biotec Ltd) according to the manufacturer's instructions.
• DynabeadsTM Human T-Activator CD3/CD28 (11131D, Gibco) were used in the presence of 50 IU/mL recombinant human IL-2 (202-IL, R&D) with 1: 1 bead to cell ratio to expand cells for 7 days.
• Dynabeads were removed and CD4+ T cells were rested overnight with reduced 10 IU/mL recombinant human IL-2.
• Monocytes were isolated from human PBMCs using a Human CD14 Isolation Kit, (130- 050-201, Miltenyi Biotec Ltd, UK) following the manufacturer's instructions. Monocytes were differentiated to monocyte derived DCs (Mo-DCs) using Mo-DC differentiation media 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 Ii- ⁇ b (130-093-563, Miltenyi), IL-6 (130-095-352, Miltenyi), TNFo (130-094-023, Miltenyi) and PGE2 (P0409-1MG, Merck Millipore).
• the PD-1 inhibitors tested by the present 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 Figure 5).
• Nivolumab (SEQ ID NO: 31): QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFY
• Example 2B T cell activation of ATOR-1017 in combination with PD-1 inhibitor in mixed lymphocyte reaction (MLR) assay
• Example 2A was repeated, with the same materials and methods as described for Example 2A but using the anti-PD-1 antibody Nivolumab ( Figure 6) or the anti-PD-Ll antibody Atezolizumab (Tecentriq ® , Roche) ( Figure 7) as the PD-1 inhibitor and using a variable concentration of the anti-F(ab)2 used for crosslinking.
• SEQ ID NO: 53 is the heavy chain sequence of Atezolizumab
• SEQ ID NO: 54 is the light chain sequence of Atezolizumab.
• Anti-F(ab)2 was administered at a ratio of the ATOR-1017 dose (anti-F(ab)2/ ATOR-1017 ratio of 5: 1), instead of a fixed concentration of anti-F(ab)2.
• the dosages investigated for the monotherapy and combination studies were: 0.005, 0.02, 0.09, 0.35, 1.4, 5.62 and 22.5 nM.
• concentration of that monotherapy is 5.62nM.
• each element (ATOR-1017 and the PD-1 inhibitor) is present at 5.62nM.
• a dose of 22.5nM in the MLR assay corresponds approximately to a dose of O.lmg/kg when administered to a human patient.
• a synergistic improvement of the combination therapy was observed at a high concentration (22.5 nM) as shown in Figure 6B (Nivolumab) and Figure 7B (Atezolizumab).
• the dotted lines shown on Figures 6B and 7B indicate the additive effect of the ATOR-1017 and PD-1 inhibitor monotherapies, demonstrating the synergistic effect of the combination.
• Example 3 Exhausted T cell activation of ATOR-1017 in combination with anti -PD-1 antibody in mixed lymphocyte reaction (MLR) assay
• 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.
• MLR mixed lymphocyte reaction
• mMo-DCs monocyte derived dendritic cells
• CD4+ T cells were expanded for 8 days as described for the generation of expanded CD4+ T cells previously, with the exception that every second day, for a total of 3 times during the 8 days expansion period, fresh CD3/CD28 Dynabeads were added to the CD4+ T cells. After 8 days, exhausted CD4+ T cells were characterized as having an increased expression of PD-1, TIM-3 and LAG-3 with flow cytometry.
• Mature mo-DC were generated as described previously for differentiation of mMo-DCs.
• the dosages investigated for the monotherapy and combination studies were: 0.005, 0.02, 0.09, 0.35, 1.4, 5.62 and 22.5 nM.
• concentration of that monotherapy is 5.62nM.
• concentration of that monotherapy is 5.62nM.
• each element (ATOR-1017 and the PD-1 inhibitor) is present at 5.62nM.
• a dose of 22.5nM in the MLR assay corresponds approximately to a dose of O.lmg/kg when administered to a human patient.

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