Classifications

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  • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
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Definitions

• the present disclosure relates to combination therapies for the treatment of pathological conditions, such as cancer.
• the present disclosure relates to combination therapies comprising treatment with an Antibody Drug Conjugate (ADC) and a secondary agent.
• ADC Antibody Drug Conjugate
• ADC antibody-drug conjugates
• cytotoxic or cytostatic agents i.e. drugs to kill or inhibit tumour cells in the treatment of cancer
• Ax I is a member of the receptor tyrosine kinase sub-family. Although similar to other receptor tyrosine kinases, the Axl protein represents a unique structure of the
• Axl transduces signals from the extracellular matrix into the cytoplasm by binding growth factors like vitamin K-dependent protein growth-arrest-specific gene 6 (Gas6).
• the extracellular domain of Axl can be cleaved and a soluble extracellular domain of 65 kDa can be released. Cleavage enhances receptor turnover and generates a partially activated kinase (O'Bryan JP, eta/ (1995) J Bioi Chern. 270 (2): 551-557).
• Axl is involved in the stimulation of cell proliferation. Specifically, Axl is a chronic myelogenous leukaemia-associated oncogene, that is also associated with colon cancer and melanoma. It is in close vicinity to the bcl3 oncogene which is at 19q13.1-q13.2. The Axl gene is evolutionarily conserved among vertebrate species, and is expressed during development in the mesenchyme.
• Axl Upon interaction with the Gas6 ligand, Axl becomes autophosphorylated, and a cascade of signal transduction events takes place.
• PI3K, AKT, src, Bad, 14-3-3, PLC, ERK, S6K (mitogen-regulated kinase) and STAT are each known to be involved in this cascade.
• Gas6 has a region rich with y-carboxyglutamic acid (GLA domain) that allows for Ca++- dependent binding to membrane phospholipids.
• GLA domain y-carboxyglutamic acid
• Gas6 is a weak mitogen and has an anti- apoptotic effect in NIH3T3 fibroblasts subjected to stress by TNF-induced cytotoxicity, or growth factor withdrawal.
• TNF-induced cytotoxicity or growth factor withdrawal.
• the binding of Gas6 to Axl results in activation of P13K, AKT, src and Bad.
• Axl plays a number of different roles in tumour formation.
• Axl is a key regulator of angiogenic behaviours including endothelial cell migration, proliferation and tube formation.
• Axl is also required for human breast carcinoma cells to form a tumour in vivo, indicating that Axl regulates processes that are vital for both
• Axl receptor tyrosine kinase The activity of Axl receptor tyrosine kinase is positively correlated with tumour metastasis. More specifically, studies have shown that Axl enhances expression of MMP-9, which is required for Axl-mediated invasion. Axl promotes cell invasion by inducing MMP-9 activity through activation of NF-BK and Brg-1 (Tai, K-Y et a/, Oncogene (2008), 27, 4044-4055). Axl is overexpressed in human glioma cells and can be used to predict poor prognosis in patients with Glioblastoma Multiforme (GBM) (Vajkoczy P. et a/, PNAS, April 1 1 , 2006, val 103, no.
• GBM Glioblastoma Multiforme
• Axl is also relatively overexpressed in highly invasive lung cancer cell lines compared to their minimally invasive counterparts (Shieh, Y-S eta/, Neoplasia, val 7, no. 12, Dec 2005, 1058-1064). Axl is therefore believed to play an important role in tumour invasion and progression.
• Axl is expressed in highly invasive breast cancer cells, but not in breast cancer cells of low invasivity. More specifically, inhibition of Axl signalling (by dominant-negative Axl mutant, an antibody against the extracellular domain of Axl, or by short hairpin RNA knockdown of Axl) decreased the mobility and invasivity of highly invasive breast cancer cells. Small molecule Axl inhibitors interfered with motility and invasivity of breast cancer cells. Thus, Axl is understood to be a critical element in the signalling network that governs the motility/invasivity of breast cancer cells (Zhang, Y-X et al., Cancer Res 2008; 68 (6), March 15, 2008).
• Gas6 was found to have a mitogenic effect, indicative of a possible role in the progression of glomerulosclerosis.
• Evidence has suggested that the Gas6/Axl pathway also plays a role in glomerulonephritis (Yanagita M. at a/, The Journal of Clinical Investigation; 2002, 1 10 (2) 239-246).
• Further studies have shown that Gas6 promotes the survival of endothelial cells in a model for arterial injury.
• Angiotensin II via its AT1 receptor, was shown to increase Ax I mRNA and protein receptor in vascular smooth muscle cells (Melaragno M. G. eta/, Circ Res., 1998, 83 (7): 697- 704).
• Axl has also been shown to be involved in cellular adhesion, cell proliferation and regulation of homeostasis in the immune system (Lu Q., 2001 ) Science 293 (5528): 306 31 1 ). Following Axl activation, the following phenomena have been observed: inhibition of apoptosis, increase in "normal" cell (non-transformed) survival of fibroblasts and endothelial cells, migration of Vascular Smooth Muscle Cell (VSMC) (inactivation of the Axl kinase blocks migration), enhancement of neointima formation in blood vessel wall (Melaragno M.G. eta/, Trends Cardiovasc Med., 1999, (Review) 9 (8): 250-253) and involvement in lesion formation and the progression of atherosclerosis. Therapeutic uses of anti-AXL ADCs
• an Antibody Drug Conjugate comprising an anti-AXL antibody (an anti-AXL-ADC) in the treatment of, for example, cancer has been established - see, for example, WO2016/166297, WO2016/166302, GB1702029.8, GB1719906.8. and PCT/EP2018/053163.
• the disclosure provides a method for treating a disorder in an individual, the method comprising administering to the individual an effective amount of an ADC and secondary agent.
• the disorder may be a proliferative disease, for example a cancer.
• Cancers include metastatic cancers and metastatic cancer cells, such as circulating tumour cells, which may be found circulating in body fluids such as blood or lymph.
• Cancers of particular interest include, but are not limited to, breast, lung, gastric, head and neck, colorectal, renal, pancreatic, uterine, hepatic, bladder, endometrial and prostate cancers as well as lymphomas (e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acute myeloid leukemia, AML).
• lymphomas e.g., non-Hodgkin's lymphoma, NHL
• leukemia particularly acute myeloid leukemia, AML.
• disorders of interest include any condition in which Axl is overexpressed, or wherein Axl antagonism will provide a clinical benefit.
• These include immune disorders, cardiovascular disorders, thrombosis, diabetes, immune checkpoint disorders, or fibrotic disorders (fibrosis) such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal fibrosis, cancer, and atherosclerosis.
• the proliferative disease may be characterised by the presence of a neoplasm comprising both AXL+ve and AXL-ve cells.
• the proliferative disease may be characterised by the presence of a neoplasm composed of AXL-ve neoplastic cells, optionally wherein the AXL-ve neoplastic cells are associated with AXL+ve non-neoplastic cells.
• the target neoplasm or neoplastic cells may be all or part of a solid tumour.
• Solid tumor herein will be understood to include solid haematological cancers such as lymphomas (Hodgkin's lymphoma or non-Hodgkin's lymphoma) which are discussed in more detail herein.
• lymphomas Hodgkin's lymphoma or non-Hodgkin's lymphoma
• Solid tumors may be neoplasms, including non-haematological cancers, comprising or composed of AXL+ve neoplastic cells.
• Solid tumors may be neoplasms, including non- haematological cancers, infiltrated with AXL+ve cells, such as AXL+ve immune suppressive dendritic cells, NK cells, or macrophages; such solid tumours may lack expression of AXL (that is, comprise or be composed of AXL-ve neoplastic cells).
• the solid tumour may be a tumour with high levels of infiltrating AXL+ve cells, such as infiltrating dendritic cells, NK cells, or macrophages (Paolino, M., et al., Cancers 2016, 8, 97; doi:10.3390/cancers8100097).
• the solid tumour may be pancreatic cancer, breast cancer, colorectal cancer, gastric and oesophageal cancer, leukemia and lymphoma, melanoma, non-small cell lung cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, and head and neck cancer.
• the ADC may be anti-AXL-ADC, such as ADCxAXL described herein.
• the secondary agent may be a PD1 antagonist, a PD-L1 antagonist, a GITR agonist, an OX40 agonist, a CTLA-4 antagonist, Fludarabine or Cytarabine, a hypomethylating agent, a PARP inhibitor (PARPi), an agent that upregulates HER2 expression, an AXL inhibitor (AXLi), a BRAF inhibitor (BRAFi), or a MEK inhibitor (MEKi).
• PARPi PARP inhibitor
• AXL inhibitor AXL inhibitor
• BRAFi BRAF inhibitor
• MEKi MEK inhibitor
• the individual may be human.
• the individual may have cancer, or may have been determined to have cancer.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a PD-L1 + cancer.
• the ADC may be administered before the secondary agent, simultaneous with the secondary agent, or after the secondary agent.
• the disclosed methods may comprise administering a further chemotherapeutic agent to the individual.
• the disclosure provides a first composition comprising an ADC for use in a method of treating a disorder in an individual, wherein the treatment comprises administration of the first composition in combination with a second composition comprising a secondary agent.
• a first composition comprising a secondary agent for use in a method of treating a disorder in an individual, wherein the treatment comprises administration of the first composition in combination with a second composition comprising an ADC.
• the disorder may be a proliferative disease, for example a cancer.
• Cancers include metastatic cancers and metastatic cancer cells, such as circulating tumour cells, which may be found circulating in body fluids such as blood or lymph.
• Cancers of particular interest include, but are not limited to, breast, lung, gastric, head and neck, colorectal, renal, pancreatic, uterine, hepatic, bladder, endometrial and prostate cancers as well as lymphomas (e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acute myeloid leukemia, AML).
• lymphomas e.g., non-Hodgkin's lymphoma, NHL
• leukemia particularly acute myeloid leukemia, AML.
• Other disorders of interest include any condition in which Ax I is overexpressed, or wherein Ax I antagonism will provide a clinical benefit.
• fibrotic disorders such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal fibrosis, cancer, and atherosclerosis.
• fibrosis fibrotic disorders
• strabmisus such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal
• the proliferative disease may be characterised by the presence of a neoplasm comprising both AXL+ve and AXL-ve cells.
• the proliferative disease may be characterised by the presence of a neoplasm composed of AXL-ve neoplastic cells, optionally wherein the AXL-ve neoplastic cells are associated with AXL+ve non-neoplastic cells.
• the target neoplasm or neoplastic cells may be all or part of a solid tumour.
• Solid tumor herein will be understood to include solid haematological cancers such as lymphomas (Hodgkin's lymphoma or non-Hodgkin's lymphoma) which are discussed in more detail herein.
• Solid tumors may be neoplasms, including non-haematological cancers, comprising or composed of AXL+ve neoplastic cells.
• Solid tumors may be neoplasms, including non- haematological cancers, infiltrated with AXL+ve cells, such as AXL+ve immune suppressive dendriti cells, NK cells, or macrophages; such solid tumours may lack expression of AXL (that is, comprise or be composed of AXL-ve neoplastic cells).
• the ADC may be anti-AXL-ADC, such as ADCxAXL described herein.
• the secondary agent may be a PD1 antagonist, a PD-L1 antagonist, a GITR agonist, an OX40 agonist, a CTLA-4 antagonist, Fludarabine or Cytarabine, a hypomethylating agent, a PARP inhibitor (PARPi), an agent that upregulates HER2 expression, an AXL inhibitor (AXLi), a BRAF inhibitor (BRAFi), or a MEK inhibitor (MEKi).
• PARPi PARP inhibitor
• AXL inhibitor AXL inhibitor
• BRAFi BRAF inhibitor
• MEKi MEK inhibitor
• the individual may be human.
• the individual may have cancer, or may have been determined to have cancer.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a PD-L1 + cancer.
• the first composition may be administered before the second composition, simultaneous with the second composition, or after the second composition.
• the treatment may comprise administering a further chemotherapeutic agent to the individual.
• the disclosure provides the use of n ADC in the manufacture of a medicament for treating a disorder in an individual, wherein the medicament comprises an ADC, and wherein the treatment comprises administration of the medicament in combination with a composition comprising secondary agent.
• Also provided by this aspect is the use of secondary agent in the manufacture of a medicament for treating a disorder in an individual, wherein the medicament comprises a secondary agent, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an ADC.
• the disorder may be a proliferative disease, for example a cancer.
• Cancers include metastatic cancers and metastatic cancer cells, such as circulating tumour cells, which may be found circulating in body fluids such as blood or lymph.
• Cancers of particular interest include, but are not limited to, breast, lung, gastric, head and neck, colorectal, renal, pancreatic, uterine, hepatic, bladder, endometrial and prostate cancers as well as lymphomas (e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acute myeloid leukemia, AML).
• lymphomas e.g., non-Hodgkin's lymphoma, NHL
• leukemia particularly acute myeloid leukemia, AML.
• Other disorders of interest include any condition in which Ax I is overexpressed, or wherein Axl antagonism will provide a clinical benefit.
• fibrotic disorders such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal fibrosis, cancer, and atherosclerosis.
• fibrosis fibrotic disorders
• strabmisus such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal
• the proliferative disease may be characterised by the presence of a neoplasm comprising both AXL+ve and AXL-ve cells.
• the proliferative disease may be characterised by the presence of a neoplasm composed of AXL-ve neoplastic cells, optionally wherein the AXL-ve neoplastic cells are associated with AXL+ve non-neoplastic cells.
• the target neoplasm or neoplastic cells may be all or part of a solid tumour.
• Solid tumor herein will be understood to include solid haematological cancers such as lymphomas (Hodgkin's lymphoma or non-Hodgkin's lymphoma) which are discussed in more detail herein.
• lymphomas Hodgkin's lymphoma or non-Hodgkin's lymphoma
• Solid tumors may be neoplasms, including non-haematological cancers, comprising or composed of AXL+ve neoplastic cells.
• Solid tumors may be neoplasms, including non- haematological cancers, infiltrated with AXL+ve cells, such as AXL+ve immune suppressive dendritic cells, NK cells, or macrophages; such solid tumours may lack expression of AXL (that is, comprise or be composed of AXL-ve neoplastic cells).
• the ADC may be anti-AXL-ADC, such as ADCxAXL described herein.
• the secondary agent may be a PD1 antagonist, a PD-L1 antagonist, a GITR agonist, an OX40 agonist, a CTLA-4 antagonist, Fludarabine or Cytarabine, a hypomethylating agent, a PARP inhibitor (PARPi), an agent that upregulates HER2 expression, an AXL inhibitor (AXLi), a BRAF inhibitor (BRAFi), or a MEK inhibitor (MEKi).
• PARPi PARP inhibitor
• the individual may be human.
• the individual may have cancer, or may have been determined to have cancer.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a PD-L1 + cancer.
• the medicament may be administered before the composition, simultaneous with the composition, or after the composition.
• the treatment may comprise administering a further chemotherapeutic agent to the individual.
• a first medicament comprising an ADC
• a second medicament comprising a secondary agent
• kits comprising a medicament comprising an ADC and a package insert comprising instructions for administration of the medicament to an individual in combination with a composition comprising a secondary agent for the treatment of a disorder.
• kit comprising a medicament comprising a secondary agent and a package insert comprising instructions for administration of the medicament to an individual in combination with a composition comprising an ADC for the treatment of a disorder.
• the disorder may be a proliferative disease, for example a cancer.
• Cancers include metastatic cancers and metastatic cancer cells, such as circulating tumour cells, which may be found circulating in body fluids such as blood or lymph.
• Cancers of particular interest include, but are not limited to, breast, lung, gastric, head and neck, colorectal, renal, pancreatic, uterine, hepatic, bladder, endometrial and prostate cancers as well as lymphomas (e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acute myeloid leukemia, AML).
• lymphomas e.g., non-Hodgkin's lymphoma, NHL
• leukemia particularly acute myeloid leukemia, AML.
• disorders of interest include any condition in which Ax I is overexpressed, or wherein Axl antagonism will provide a clinical benefit.
• fibrotic disorders such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal fibrosis, cancer, and atherosclerosis.
• fibrosis fibrotic disorders
• fibrosis such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic
• the proliferative disease may be characterised by the presence of a neoplasm comprising both AXL+ve and AXL-ve cells.
• the proliferative disease may be characterised by the presence of a neoplasm composed of AXL-ve neoplastic cells, optionally wherein the AXL-ve neoplastic cells are associated with AXL+ve non-neoplastic cells.
• Solid tumor herein will be understood to include solid haematological cancers such as lymphomas (Hodgkin's lymphoma or non-Hodgkin's lymphoma) which are discussed in more detail herein.
• Solid tumors may be neoplasms, including non-haematological cancers, comprising or composed of AXL+ve neoplastic cells.
• Solid tumors may be neoplasms, including non- haematological cancers, infiltrated with AXL+ve cells, such as AXL+ve immune suppressive dendritic cells, NK cells, or macrophages; such solid tumours may lack expression of AXL (that is, comprise or be composed of AXL-ve neoplastic cells).
• the ADC may be anti-AXL-ADC, such as ADCxAXL described herein.
• the secondary agent may be a PD1 antagonist, a PD-L1 antagonist, a GITR agonist, an OX40 agonist, a CTLA-4 antagonist, Fludarabine or Cytarabine, a hypomethylating agent, a PARP inhibitor (PARPi), an agent that upregulates HER2 expression, an AXL inhibitor (AXLi), a BRAF inhibitor (BRAFi), or a MEK inhibitor (MEKi).
• PARPi PARP inhibitor
• AXL inhibitor AXL inhibitor
• BRAFi BRAF inhibitor
• MEKi MEK inhibitor
• the individual may be human.
• the individual may have cancer, or may have been determined to have cancer.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a PD-L1 + cancer.
• the medicament or composition comprising the ADC may be administered before the medicament or composition comprising the secondary agent, simultaneous with the medicament or composition comprising the secondary agent, or after the medicament or composition comprising the secondary agent.
• the treatment may comprise administering a further chemotherapeutic agent to the individual.
• the disclosure provides a composition comprising an ADC and a secondary agent.
• Also provided in this aspect of the disclosure is a method of treating a disorder in an individual, the method comprising administering to the individual an effective amount of the composition comprising an ADC and a secondary agent.
• composition comprising an ADC and a secondary agent for use in a method of treating a disorder in an individual.
• composition comprising an ADC and a secondary agent in the manufacture of a medicament for treating a disorder in an individual.
• a kit comprising composition comprising an ADC and a secondary agent and a set of instructions for administration of the medicament to an individual for the treatment of a disorder.
• the disorder may be a proliferative disease, for example a cancer.
• Cancers include metastatic cancers and metastatic cancer cells, such as circulating tumour cells, which may be found circulating in body fluids such as blood or lymph.
• Cancers of particular interest include, but are not limited to, breast, lung, gastric, head and neck, colorectal, renal, pancreatic, uterine, hepatic, bladder, endometrial and prostate cancers as well as lymphomas (e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acute myeloid leukemia, AML).
• lymphomas e.g., non-Hodgkin's lymphoma, NHL
• leukemia particularly acute myeloid leukemia, AML.
• disorders of interest include any condition in which Ax I is overexpressed, or wherein Axl antagonism will provide a clinical benefit.
• fibrotic disorders such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic steatohepatitis (NASH), other types of liver fibrosis, primary biliary cirrhosis, renal fibrosis, cancer, and atherosclerosis.
• fibrosis fibrotic disorders
• fibrosis such as strabmisus, scleroderma, keloid, Nephrogenic systemic fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), systemic sclerosis, cardiac fibrosis, non-alcoholic
• the proliferative disease may be characterised by the presence of a neoplasm comprising both AXL+ve and AXL-ve cells.
• the proliferative disease may be characterised by the presence of a neoplasm composed of AXL-ve neoplastic cells, optionally wherein the AXL-ve neoplastic cells are associated with AXL+ve non-neoplastic cells.
• the target neoplasm or neoplastic cells may be all or part of a solid tumour.
• Solid tumor herein will be understood to include solid haematological cancers such as lymphomas (Hodgkin's lymphoma or non-Hodgkin's lymphoma) which are discussed in more detail herein.
• Solid tumors may be neoplasms, including non-haematological cancers, comprising or composed of AXL+ve neoplastic cells.
• Solid tumors may be neoplasms, including non- haematological cancers, infiltrated with AXL+ve cells, such as AXL+ve immune suppressive dendritic cells, NK cells, or macrophages; such solid tumours may lack expression of AXL (that is, comprise or be composed of AXL-ve neoplastic cells).
• the ADC may be anti-AXL-ADC, such as ADCxAXL described herein.
• the secondary agent may be a PD1 antagonist, a PD-L1 antagonist, a GITR agonist, an OX40 agonist, a CTLA-4 antagonist, Fludarabine or Cytarabine, a hypomethylating agent, a PARP inhibitor (PARPi), an agent that upregulates HER2 expression, an AXL inhibitor (AXLi), a BRAF inhibitor (BRAFi), or a MEK inhibitor (MEKi).
• the individual may be human.
• the individual may have cancer, or may have been determined to have cancer.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a AXL+ cancer or AXL+ tumour-associated non-tumour cells.
• the individual may have, or have been determined to have, a PD-L1 + cancer.
• the treatment may comprise administering a further chemotherapeutic agent to the individual.
• ADCs Antibody Drug Conjugates
• the present disclosure relates to the improved efficacy of combinations of an ADC and a secondary agent.
• the ADC of present disclosure provides a PBD dimer with a linker connected through the N10 position on one of the PBD moieties conjugated to an antibody as defined below.
• the present disclosure is suitable for use in providing a PBD compound to a preferred site in a subject.
• the conjugate allows the release of an active PBD compound that does not retain any part of the linker. There is no stub present that could affect the reactivity of the PBD compound. Thus the conjugate of formula (I) would release the compound
• the specified link between the PBD dimer and the antibody in the present invention is preferably stable extracellularly.
• the antibody-drug conjugate (ADC) is preferably stable and remains intact, i.e. the antibody remains linked to the drug moiety.
• the linkers are stable outside the target cell and may be cleaved at some efficacious rate inside the cell.
• An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e.
• Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the
• the disclosure also particularly relates treatment with an anti-AXL ADC disclosed in GB1702029.8, GB1719906.8, PCT/EP2018/053163, and as herein described.
• anti-AXL ADCs disclosed in GB1702029.8, GB1719906.8, PCT/EP2018/053163, and as herein described.
• AXL-ADC refers to an ADC in which the antibody component is an anti-AXL antibody.
• PBD-ADC refers to an ADC in which the drug component is a pyrrolobenzodiazepine (PBD) warhead.
• anti-AXL-ADC refers to an ADC in which the antibody component is an anti-AXL antibody, and the drug component is a PBD warhead.
• the ADC may comprise a conjugate of formula (I):
• Ab is an antibody that binds to AXL
• X is selected from the group comprising: a single bond, -CH2- and -C2H.1-;
• n is from 1 to 8;
• n 0 or 1 ;
• R 7 is either methyl or phenyl
• R 2 is selected the group consisting of:
• R 25a and R 25b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and
• R 24 is selected from: H; C1-3 saturated alkyi; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
• R 26a and R 26b are independently selected from H, F, C1-4 saturated alkyi, C2-3 alkenyl, which alkyi and alkenyl groups are optionally substituted by a group selected from C1-4 alkyi amido and C1-4 alkyi ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a CM alkyi ester;
• R 12 is selected the group consisting of:
• R 35a and R 35b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if) , where R 24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;
• R 12 is where R 36a and R 36b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R 36a and R 36b is H, the other is selected from nitrile and a CM alkyl ester;
• anti-AXL-ADC may include any embodiment described in GB1702029.8.
• the ADC may have the chemical structure:
• Ab is an antibody that binds to AXL
• the Ab is an anti-AXL antibody.
• DL may be conjugated to the antibody through the sidechain of an antibody asparagine residue, for example Asn297 according to the numbering system of Kabat.
• the structure of the linkage to the antibody may be N-[sugar]-DL, wherein N is the asparagine residue, and [sugar] represents a sugar residue, such as a GlcNAc residue, p may be 1 to 4, preferably 2.
• Ab is an antibody that binds to AXL, the antibody comprising:
• X is a single bond.
• X is -CH 2 -.
• X is -C2H4-.
• n 1 to 4.
• n 1 .
• n is 2.
• n 4
• R 7 is methyl
• R 7 is phenyl
• R 2 is selected from:
• each of R 21 , R 22 and R 23 are independently selected from H, Ci- 3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R 2 group is no more than 5;
• R 25a and R 25b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl; and (f) , where R 24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl.
• R 2 is a C5-10 aryl group, it may be a C5-7 aryl group.
• a C5-7 aryl group may be a phenyl group or a C5-7 heteroaryl group, for example furanyl, thiophenyl and pyridyl.
• R 2 is preferably phenyl.
• R 12 is preferably thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.
• R 2 is a C5- 1 0 aryl group, it may be a Ce-io aryl, for example a quinolinyl or isoquinolinyl group.
• the quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position.
• the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred.
• the isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.
• R 2 When R 2 is a C5-10 aryl group, it may bear any number of substituent groups. It preferably bears from 1 to 3 substituent groups, with 1 and 2 being more preferred, and singly substituted groups being most preferred.
• the substituents may be any position.
• R 2 is C5-7 aryl group
• a single substituent is preferably on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it is preferably ⁇ or ⁇ to the bond to the remainder of the compound. Therefore, where the C5-7 aryl group is phenyl, the substituent is preferably in the meta- or para- positions, and more preferably is in the para- position.
• R 2 is a Cs-io aryl group, for example quinolinyl or isoquinolinyl, it may bear any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).
• R 2 substituents, when R 2 is a C5- 10 aryl group
• R 2 when R 2 is a C5-10 aryl group is halo, it is preferably F or CI, more preferably CI.
• R 2 when R 2 is a C5-10 aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C1-7 alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C5-7 aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy).
• the alkoxy group may itself be further substituted, for example by an amino group (e.g.
• R 2 when R 2 is a C5-10 aryl group is C1-7 alkyl, it may preferably be a C1-4 a Iky I group (e.g. methyl, ethyl, propryl, butyl).
• R 2 when R 2 is a C5-10 aryl group is C3-7 heterocyclyl, it may in some embodiments be Ce nitrogen containing heterocyclyl group, e.g. morpholino,
• thiomorpholino piperidinyl, piperazinyl.
• These groups may be bound to the rest of the PBD moiety via the nitrogen atom.
• These groups may be further substituted, for example, by C alkyl groups. If the Ce nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.
• R 2 when R 2 is a C5-10 aryl group is bis-oxy-Ci-3 alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.
• R 2 when R 2 is a C5-10 aryl group is ester, this is preferably methyl ester or ethyl ester.
• R 2 is a C5-10 aryl group
• substituents when R 2 is a C5-10 aryl group include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl- thiophenyl.
• Other particularly preferred substituent for R 2 are dimethylaminopropyloxy and carboxy.
• Particularly preferred substituted R 2 groups when R 2 is a C5-10 aryl group include, but are not limited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4- fluoro-phenyl, 4-chloro-phenyl, 3.4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4- cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.
• R 2 group is 4-nitrophenyl.
• R 2 groups of particular interest include 4-(4- methylpiperazin-1 -yl)phenyl and 3,4-bisoxymethylene-phenyl.
• R 2 is C1-5 saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl.
• it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.
• R 2 is C3-6 saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.
• each of R 21 , R 22 and R 23 are independently selected from H, Ci-
• R 21 , R 22 and R 23 is H, with the other two groups being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
• two of R 21 , R 22 and R 23 are H, with the other group being selected from H, C1-3 saturated alkyl, C2-3 alkenyl, C2-3 alkynyl and cyclopropyl.
• the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl. In some embodiments, R 21 is H.
• R 22 is H.
• R 23 is H.
• R 21 and R 22 are H.
• R 21 and R 23 are H.
• R 22 and R 23 are identical to R 22 and R 23.
• a R 2 group of particular interest is:
• R 25a and R 25b are H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl.
• the group which is not H is optionally substituted phenyl.
• the phenyl optional substituent is halo, it is preferably fluoro.
• the phenyl group is unsubstituted.
• R 24 is selected from: H; C1-3 saturated alkyl; C2-3 alkenyl; C2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo methyl, methoxy; pyridyl; and thiophenyl. If the phenyl optional substituent is halo, it is preferably fluoro. In some embodiment, the phenyl group is unsubstituted. In some embodiments, R 24 is selected from H, methyl, ethyl, ethenyl and ethynyl. In some of these embodiments, R 24 is selected from H and methyl.
• R 26a and R 26b are independently selected from H, F, C1-4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by group selected from C1-4 alkyl amido and C1-4 alkyl ester; or, when one of R 26a and R 26b is H, the other is selected from nitrile and a C1.4 alkyl ester.
• R 26a and R 26b are both H.
• R 26a and R 26b are both methyl.
• R 26a and R 26b are H, and the other is selected from C1.4 saturated alkyl, C2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
• the group which is not H is selected from methyl and ethyl.
• the above DL may be preferably comprised in an ADC having the formula Ab - (DL) P , wherein Ab is an antibody that binds to AXL.
• DL may be conjugated to the antibody through the sidechain of an antibody asparagine residue, for example Asn297 according to the numbering system of Kabat.
• the structure of the linkage to the antibody may be N-[sugar]-DL, wherein N is the asparagine residue, and [sugar] represents a sugar residue, such as a GlcNAc residue, p may be 1 to 4, for example 2.
• the invention provides a conjugate having the formula:
• Ab is an antibody comprising:
• DL is the drug-iinker described immediately above.
• the antibody component of the anti-AXL ADC is the antibody component of the anti-AXL ADC
• the antibody is an antibody that binds to AXL.
• the antibody comprises a VH domain having a VH CDR3 with the amino acid sequence of SEQ ID NO.7. In some embodiments the VH domain further comprises a VH CDR2 with the amino acid sequence of SEQ ID NO.6, and/or a VH
• the antibody comprises a VH domain having a VH CDR1 with the amino acid sequence of SEQ ID NO.5, a VH CDR2 with the amino acid sequence of SEQ ID NO.6, and a VH CDR3 with the amino acid sequence of SEQ ID NO.7.
• the antibody comprises a VH domain having the sequence according to SEQ ID NO. 1 .
• the antibody may further comprise a VL domain.
• the antibody comprises a VL domain having a VL CDR3 with the amino acid sequence of SEQ ID NO.10.
• the VL domain further comprises a VL CDR2 with the amino acid sequence of SEQ ID NO.9, and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.8.
• the antibody comprises a VL domain having a VL CDR1 with the amino acid sequence of SEQ ID NO.8, a VL CDR2 with the amino acid sequence of SEQ ID NO.9, and a VL CDR3 with the amino acid sequence of SEQ ID NO.10.
• the antibody comprises a VL domain having the sequence according to SEQ ID NO. 2.
• the antibody comprises a VH domain and a VL domain.
• VH comprises the sequence of SEQ ID N0.1 and the VL domain comprises the sequence of SEQ ID NO.2.
• VH and VL domain(s) may pair so as to form an antibody antigen binding site that binds AXL.
• the antibody is an intact antibody comprising a VH domain paired with a VL domain, the VH and VL domains having sequences of SEQ ID N0.1 paired with SEQ ID NO.2.
• the antibody comprises a heavy chain having the sequence of SEQ ID NO. 3 paired with a light chain having the sequence of SEQ ID NO.4.
• the antibody is an intact antibody comprising two heavy chains having the sequence of SEQ ID NO.3, each paired with a light chain having the sequence of SEQ ID NO.4.
• the antibody is an antibody as described herein which has been modified (or further modified) as described below.
• the antibody is a humanised, deimmunised or resurfaced version of an antibody disclosed herein.
• the antibody is a fully human monoclonal lgG1 antibody, preferably IgGI . K.
• the antibody is an antibody as described herein which has been modified (or further modified) as described below.
• the antibody is a humanised, deimmunised or resurfaced version of an antibody disclosed herein.
• anti-AXL-ADC for use with the aspects of the present disclosure is ADCxAXL, as described herein below. 5F1 1
• the antibody comprises a VH domain having a VH CDR3 with the amino acid sequence of SEQ ID NO.15. In some embodiments the VH domain further comprises a VH CDR2 with the amino acid sequence of SEQ ID NO.14, and/or a VH CDR1 with the amino acid sequence of SEQ ID NO.13. In some embodiments the the antibody comprises a VH domain having a VH CDR1 with the amino acid sequence of SEQ ID NO.13, a VH CDR2 with the amino acid sequence of SEQ ID NO.14, and a VH CDR3 with the amino acid sequence of SEQ ID NO.15.
• the antibody comprises a VH domain having the sequence according to SEQ ID NO. 1 1 . In some embodiments the antibody comprises a VH domain having the sequence according to SEQ ID NO. 19. In some embodiments the antibody comprises a VH domain having the sequence according to SEQ ID NO. 20. In some embodiments the antibody comprises a VH domain having the sequence according to SEQ ID NO. 21.
• the antibody may further comprise a VL domain.
• the antibody comprises a VL domain having a VL CDR3 with the amino acid sequence of SEQ ID NO.18.
• the VL domain further comprises a VL CDR2 with the amino acid sequence of SEQ ID NO.17, and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.16.
• the antibody comprises a VL domain having a VL CDR1 with the amino acid sequence of SEQ ID NO.16, a VL CDR2 with the amino acid sequence of SEQ ID NO.17, and a VL CDR3 with the amino acid sequence of SEQ ID NO.18.
• the antibody comprises a VL domain having the sequence according to SEQ ID NO. 22.
• the antibody comprises a VH domain and a VL domain.
• the VH comprises a VH CDR1 with the amino acid sequence of SEQ ID NO.13, a VH CDR2 with the amino acid sequence of SEQ ID NO.14, and a VH CDR3 with the amino acid sequence of SEQ ID NO.15; and the VL domain comprises a VL CDR1 with the amino acid sequence of SEQ ID NO.16, a VL CDR2 with the amino acid sequence of SEQ ID NO.17, and a VL CDR3 with the amino acid sequence of SEQ ID NO.18.
• the antibody comprises a VH domain having the sequence of SEQ ID NO.19 and the VL domain having the sequence of SEQ ID NO.22.
• the antibody comprises a VH domain having the sequence of SEQ ID NO.20 and the VL domain having the sequence of SEQ ID NO.22. In some embodiments the antibody comprises a VH domain having the sequence of SEQ ID NO.21 and the VL domain having the sequence of SEQ ID NO.22.
• the antibody is an antibody as described herein which has been modified (or further modified) as described below.
• the antibody is a humanised, deimmunised or resurfaced version of an antibody disclosed herein.
• the antibody is a fully human monoclonal lgG1 antibody, preferably lgG1 , K.
• the antibody is an antibody as described herein which has been modified (or further modified) as described below.
• the antibody is a humanised, deimmunised or resurfaced version of an antibody disclosed herein.
• the antibodies disclosed herein may be modified. For example, to make them less immunogenic to a human subject. This may be achieved using any of a number of techniques familiar to the person skilled in the art. Some of these techniques are described in more detail below.
• a “humanized antibody” refers to a polypeptide comprising at least a portion of a modified variable region of a human antibody wherein a portion of the variable region, preferably a portion substantially less than the intact human variable domain, has been substituted by the corresponding sequence from a non-human species and wherein the modified variable region is linked to at least another part of another protein, preferably the constant region of a human antibody.
• the expression “humanized antibodies” includes human antibodies in which one or more complementarity determining region (“CDR”) amino acid residues and/or one or more framework region (“FW" or "FR”) amino acid residues are substituted by amino acid residues from analogous sites in rodent or other non-human antibodies.
• humanized antibody also includes an immunoglobulin amino acid sequence variant or fragment thereof that comprises an FR having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
• “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Or, looked at another way, a humanized antibody is a human antibody that also contains selected sequences from non-human (e.g. murine) antibodies in place of the human sequences.
• a humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biologic activity.
• Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.
• CDR grafting There are a range of humanisation techniques, including 'CDR grafting', 'guided selection', 'deimmunization', 'resurfacing' (also known as 'veneering'), 'composite antibodies', 'Human String Content Optimisation' and framework shuffling.
• CDR grafting includes 'CDR grafting', 'guided selection', 'deimmunization', 'resurfacing' (also known as 'veneering'), 'composite antibodies', 'Human String Content Optimisation' and framework shuffling.
• the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties (in effect, the non-human CDRs are 'grafted' onto the human framework).
• donor antibody such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties (in effect, the non-human CDRs are 'grafted' onto the human framework.
• framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues (this may happen when, for example, a particular FR residue has significant effect on antigen binding).
• humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• a humanized antibody will comprise all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
• the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin.
• the method consists of combining the VH or Vi domain of a given non-human antibody specific for a particular epitope with a human VH or V L library and specific human V domains are selected against the antigen of interest. This selected human VH is then combined with a VL library to generate a completely human VHxVL combination. The method is described in Nature Biotechnology (N.Y.) 12, (1994) 899-903.
• two or more segments of amino acid sequence from a human antibody are combined within the final antibody molecule. They are constructed by combining multiple human VH and VL sequence segments in combinations which limit or avoid human T cell epitopes in the final composite antibody V regions. Where required, T cell epitopes are limited or avoided by, exchanging V region segments contributing to or encoding a T cell epitope with alternative segments which avoid T cell epitopes. This method is described in US 2008/0206239 A1 .
• This method involves the removal of human (or other second species) T-cell epitopes from the V regions of the therapeutic antibody (or other molecule).
• the therapeutic antibodies V-region sequence is analysed for the presence of MHC class II- binding motifs by, for example, comparison with databases of MHC-binding motifs (such as the "motifs" database hosted at www.wehi.edu.au).
• MHC class II- binding motifs may be identified using computational threading methods such as those devised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, consecutive overlapping peptides from the V-region sequences are testing for their binding energies to MHC class II proteins. This data can then be combined with information on other sequence features which relate to successfully presented peptides, such as
• T-cell epitopes Once potential second species (e.g. human) T-cell epitopes have been identified, they are eliminated by the alteration of one or more amino acids.
• the modified amino acids are usually within the T-cell epitope itself, but may also be adjacent to the epitope in terms of the primary or secondary structure of the protein (and therefore, may not be adjacent in the primary structure). Most typically, the alteration is by way of substitution but, in some circumstances amino acid addition or deletion will be more appropriate. All alterations can be accomplished by recombinant DNA technology, so that the final molecule may be prepared by expression from a recombinant host using well established methods such as Site Directed Mutagenesis. However, the use of protein chemistry or any other means of molecular alteration is also possible. Resurfacing
• This method involves:
• variable region of the non-human (e.g. rodent) antibody (or fragment thereof) by constructing a three-dimensional model of the non-human antibody variable region
• step (c) defining for the non-human antibody to be humanized, a set of heavy and light chain surface exposed amino acid residues using the set of framework positions generated in step (b);
• step (d) identifying from human antibody amino acid sequences a set of heavy and light chain surface exposed amino acid residues that is most closely identical to the set of surface exposed amino acid residues defined in step (c), wherein the heavy and light chain from the human antibody are or are not naturally paired; (e) substituting, in the amino acid sequence of the non-human antibody to be humanized, the set of heavy and light chain surface exposed amino acid residues defined in step (c) with the set of heavy and light chain surface exposed amino acid residues identified in step (d);
• step (f) constructing a three-dimensional model of the variable region of the non-human antibody resulting from the substituting specified in step (e);
• step (h) changing any residues identified in step (g) from the human to the original non- human amino acid residue to thereby define a non-human antibody humanizing set of surface exposed amino acid residues; with the proviso that step (a) need not be conducted first, but must be conducted prior to step (g).
• the method compares the non-human sequence with the functional human germline gene repertoire. Those human genes encoding canonical structures identical or closely related to the non-human sequences are selected. Those selected human genes with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these human FRs. This method is described in patent WO
• This method compares the non-human (e.g. mouse) sequence with the repertoire of human germline genes and the differences are scored as Human String Content (HSC) that quantifies a sequence at the level of potential MHC T-cell epitopes.
• HSC Human String Content
• the target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).
• the CDRs of the non-human antibody are fused in-frame to cDNA pools encompassing all known heavy and light chain human germline gene frameworks. Humanised antibodies are then selected by e.g. panning of the phage displayed antibody library. This is described in Methods 36, 43-60 (2005).
• the antibody may prepared for conjugation with the drug linker through a three step process:
• ADCxAXL is an antibody drug conjugate composed of a humanized antibody against human AXL attached to a pyrrolobenzodiazepine (PBD) warhead via a cleavable linker.
• the mechanism of action of ADCxAXL depends on AXL binding.
• the AXL specific antibody targets the antibody drug conjugate (ADC) to cells expressing AXL.
• ADC antibody drug conjugate
• the ADC internalizes and is transported to the Iysosome, where the protease sensitive linker is cleaved and free PBD dimer is released inside the target cell.
• the released PBD dimer inhibits transcription in a sequence-selective manner, due either to direct inhibition of RNA polymerase or inhibition of the interaction of associated transcription factors.
• the PBD dimer produces covalent crosslinks that do not distort the DNA double helix and which are not recognized by nucleotide excision repair factors, allowing for a longer effective period (Hartley 201 1
• Ab is an antibody that binds to AXL, the antibody comprising:
• DL may be conjugated to the antibody through the sidechain of the asparagine at position 302 of SEQ ID NO.3.
• the structure of the linkage to the antibody may be N-[GlcNAc]-DL, wherein N is the asparagine residue, and [GlcNac] represents a GlcNAc residue, p may be up to 2, and is typically greater than 1.9.
• the "first target protein” (FTP) as used herein may be AXL.
• binds AXL is used to mean the antibody binds AXL with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1 GL3336842, record update date: Jan 7, 201 1 02:30 PM).
• BSA Bovine Serum Albumin
• the antibody binds AXL with an association constant (K A ) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 4 , 10 5 or 10 6 -fold higher than the antibody's association constant for BSA, when measured at physiological conditions.
• K A association constant
• the antibodies of the invention can bind AXL with a high affinity.
• the antibody can bind AXL with a KD equal to or less than about 10- 6 M, such as 1 x 10 "6 , 10 “7 , 10 10- 9 , 10 "10 , 10 “11 , 10 “12 , 10- 13 or 10 "14 .
• binds AXL is used to mean the antibody binds AXL with a higher affinity than a non-specific partner such as Bovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1 GL3336842, record update date: Jan 7, 201 1 02:30 PM).
• BSA Bovine Serum Albumin
• the antibody binds AXL with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 104, 105 or 106-fold higher than the antibody's association constant for BSA, when measured at physiological conditions.
• the antibodies of the invention can bind AXL with a high affinity.
• the antibody can bind AXL with a KD equal to or less than about 10-6 M, such as 1 x 10-6, 10-7, 10-8, 10-9, 10-10, 10-1 1 , 10-12, 10-13 or 10- 14.
• AXL is member of the human TAM family of receptor tyrosine kinases.
• the AXL polypeptide corresponds to Genbank accession no. AAH32229, version no. AAH32229.1 Gl:21619004, record update date: March 6, 2012 01 :18 PM (SEQ ID NO.9).
• the nucleic acid encoding AXL polypeptide corresponds to Genbank accession no. M76125, version no. M76125.1 Gl:292869, record update date: Jun 23, 2010 08:53 AM.
• the AXL polypeptide has the sequence of SEQ ID NO.23.
• substituted refers to a parent group which bears one or more substituents.
• substituted is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group.
• substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
• C1-12 alkyl refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
• C1-4 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated).
• alkyl includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.
• saturated alkyl groups include, but are not limited to, methyl (Ci ), ethyl (C2), propyl (C 3 ), butyl (C 4 ), pentyl (C 5 ), hexyl (C 6 ) and heptyl (C 7 ).
• saturated linear alkyl groups include, but are not limited to, methyl (Ci ), ethyl (C 2 ), n-propyl (C 3 ), n-butyl (C 4 ), n-pentyl (amyl) (C 5 ), n-hexyl (C 6 ) and n-heptyl (C 7 ).
• saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C 4 ), sec-butyl (C 4 ), tert-butyl (C 4 ), iso-pentyl (C.3 ⁇ 4), and neo-pentyl (C5).
• C2-12 Alkenyl The term "C2-12 alkenyl" as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.
• C2-12 alkynyl refers to an alkyl group having one or more carbon-carbon triple bonds.
• unsaturated alkynyl groups include, but are not limited to, ethynyl (-C ⁇ CH) and 2-propynyl (propargyl, -CH 2 -C ⁇ CH).
• C3-12 cycloalkyl refers to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.
• cycloalkyl groups include, but are not limited to, those derived from:
• C3-20 heterocyclyl refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms.
• each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.
• the prefixes e.g. C3-20, C3-7, C5-6, etc.
• the term "Cs-eheterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.
• monocyclic heterocyclyl groups include, but are not limited to, those derived from:
• Ni aziridine (C3), azetidine (C 4 ), pyrrolidine (tetrahydropyrrole) (Cs), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (Cs), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (Cs), piperidine (Ce), dihydropyridine (Ce), tetrahydropyridine (Ce), azepine (C 7 );
• O1 oxirane (C3), oxetane (C 4 ), oxolane (tetrahydrofuran) (Cs), oxole (dihydrofuran) (Cs), oxane (tetrahydropyran) (Ce), dihydropyran (Ce), pyran (Ce), oxepin (C 7 );
• O2 dioxolane (Cs), dioxane (Ce), and dioxepane (C 7 );
• N 2 imidazolidine (Cs), pyrazolidine (diazolidine) (Cs), imidazoline (Cs), pyrazoline
• N1O1 tetrahydrooxazole (Cs), dihydrooxazole (Cs), tetrahydroisoxazole (Cs),
• Cs dihydroisoxazole
• morpholine Ce
• tetrahydrooxazine Ce
• dihydrooxazine Ce
• oxazine Ce
• N1S1 thiazoline (Cs), thiazolidine (Cs), thiomorpholine (Ce);
• N2O1 oxadiazine (Ce);
• O1S1 oxathiole (Cs) and oxathiane (thioxane) (Ce); and,
• N1O1S1 oxathiazine (Ce).
• substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, fura noses (Cs), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (Ce), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose,
• C5-20 aryl refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms.
• C5-7 aryl pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 7 ring atoms and the term “C5-10 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 5 to 10 ring atoms.
• each ring has from 5 to 7 ring atoms.
• the prefixes e.g. C3-20, C5-7, C5-6, C5-10, etc.
• the term "Cs-e aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.
• the ring atoms may be all carbon atoms, as in "carboaryl groups".
• carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (Ce), naphthalene (C10), azulene (C10), anthracene (CM), phenanthrene (CM), naphthacene (Cie), and pyrene (Cie).
• aryl groups which comprise fused rings include, but are not limited to, groups derived from indane (e.g. 2.3-dihydro-1 H- indene) (C9), indene (Cg), isoindene (Cg), tetraline (1 ,2,3,4-tetrahydronaphthalene (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), and aceanthrene (de).
• indane e.g. 2.3-dihydro-1 H- indene
• Cg indene
• isoindene Cg
• tetraline (1 ,2,3,4-tetrahydronaphthalene (C10) acenaphthene
• fluorene C13
• phenalene C13
• acephenanthrene C15
• aceanthrene de
• the ring atoms may include one or more heteroatoms, as in "heteroaryl groups".
• heteroaryl groups include, but are not limited to, those derived from:
• Ni pyrrole (azole) (Cs), pyridine (azine) (Ce);
• CM furan (oxole) (Cs);
• N1O1 oxazole (C5), isoxazole (Cs), isoxazine (Ce);
• N2O1 oxadiazole (furazan) (Cs);
• N3O1 oxatriazole (Cs);
• N1S1 thiazole (Cs), isothiazole (Cs);
• N2 imidazole (1 ,3-diazole) (Cs), pyrazole (1 ,2-diazole) (Cs), pyridazine (1 ,2-diazine) (Ce), pyrimidine (1.3-diazine) (Ce) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (Ce); N3: triazole (Cs), triazine (Ce); and,
• heteroaryl which comprise fused rings, include, but are not limited to:
• Ni isoindole
• indolizine Ni
• indoline Ni
• isoindoline Ni
• purine N4 (e.g., adenine, guanine), benzimidazole (N?), indazole (N2), benzoxazole (N1O1 ), benzisoxazole (N 1 O1 ), benzodioxole (O2), benzofurazan (N 2 Oi), benzotriazole (N 3 ), benzothiofuran (Si), benzothiazole (N1S1 ), benzothiadiazole (N2S);
• Ci3 (with 3 fused rings) derived from carbazole (Ni ), dibenzofuran (O1 ), dibenzothiophene (Si ), carboline (N 2 ), perimidine (N2), pyridoindole (N 2 ); and,
• CM (with 3 fused rings) derived from acridine (Ni), xanthene (O1 ), thioxanthene (Si), oxanthrene (0 2 ), phenoxathiin (O1S1 ), phenazine (N 2 ), phenoxazine (N1O1 ), phenothiazine (N 1S1 ), thianthrene (S 2 ), phenanthridine (Ni), phenanthroline (N 2 ), phenazine (N 2 ).
• Ether -OR, wherein R is an ether substituent, for example, a C1-7 aSkyi group (also referred to as a C 1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a d./alkyl group.
• R is an ether substituent, for example, a C1-7 aSkyi group (also referred to as a C 1-7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C5-20 aryl group (also referred to as a C5-20 aryloxy group), preferably a d./alkyl group.
• Alkoxy -OR, wherein R is an alkyl group, for example, a Ci-/ alky! group.
• C1-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).
• Carboxy (carboxylic acid): -C( 0)OH.
• R 1 and R 2 are independently amino substituents, for example, hydrogen, a C1-7 alkyl group (also referred to as Ci-/ alkylamino or di-C alkylamino), a C3-20 heterocyclyl group, or a C5-2o aryl group, preferably H or a Ci-? alkyl group, or, in the case of a "cyclic" amino group, R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
• Amino groups may be primary (-NH2), secondary (-NHR 1 ), or tertiary (-NHR 1 R 2 ), and in cationic form, may be quaternary (- + NR 1 R 2 R 3 ).
• amino groups include, but are not limited to, -NH 2 , -NHCH3, -NHC(CH 3 ) 2 , -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , and -NHPh.
• Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
• Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C( 0)NR R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
• the drug loading is the average number of PBD drugs per antibody, e.g. antibody.
• the average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis.
• the quantitative distribution of ADC in terms of p may also be determined.
• ELISA the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 1 1 :843-852).
• the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA.
• ELISA assay for detection of antibody- drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some instances, separation, purification, and characterization of
• homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.
• p is limited by the number of attachment sites on the antibody, i.e. the number of azide groups.
• the antibody may have only one or two azide groups to which the drug linker may be attached.
• the loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug- linker intermediate (D-L) or linker reagent relative to antibody, and (ii) limiting the conjugation reaction time or temperature. Where more than one nucleophilic or electrophilic group of the antibody reacts with a drug-iinker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1 , 2, 3, etc.
• Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value.
• Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.
• antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.
• the average number of dimer pyrrolobenzodiazepine groups per antibody is in the range 1 to 8. In some embodiments the range is selected from 1 to 4, 1 to 4, 2 to 4, and 1 to 3.
• a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ), a salt or solvate thereof, as well as conventional protected forms.
• a reference to an amino group includes the protonated form (-N + HR 1 R 2 ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group.
• a reference to a hydroxyl group also includes the anionic form (-0 ), a salt or solvate thereof, as well as conventional protected forms.
• a corresponding salt of the active compound for example, a pharmaceutically-acceptable salt.
• pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1 -19 (1977).
• a salt may be formed with a suitable cation.
• suitable inorganic cations include, but are not limited to, alkali metal ions such as Na * and K ⁇ alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Al' 3 .
• Suitable organic cations include, but are not limited to, ammonium ion (i.e. NH 4 + ) and substituted ammonium ions (e.g. NH3R", NH2R2 ', NHR3 * , NR 4 + ).
• suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine,
• ethanolamine diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
• amino acids such as lysine and arginine.
• An example of a common quaternary ammonium ion is N(CH3).t * .
• a salt may be formed with a suitable anion.
• suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
• Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acid and valeric.
• Suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
• solvate is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
• the invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R A OH, where R A is C1-4 alkyl):
• carbinolamine and carbinolamine ether forms of the PBD can be called the carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to R 10 above).
• the balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.
• Certain compounds of the invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L- forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti- forms; synclinal- and anticlinal-forms; a- and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers” (or "isomeric forms").
• chiral refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are
• stereoisomers refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
• “Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities.
• Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.
• Enantiomers refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
• the compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention.
• a specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
• a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no
• racemic mixture and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
• isomers are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space).
• a reference to a methoxy group, -OCH3 is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH2OH.
• a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl.
• a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
• keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
• tautomer or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier.
• proton tautomers also known as prototropic tautomers
• Valence tautomers include
• H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T); C may be in any isotopic form, including 12 C, 13 C, and 14 C; O may be in any isotopic form, including 16 0 and 18 0; and the like.
• isotopes examples include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to 2 H (deuterium, D), 3 H (tritium), 1 C, 13 C, 14 C, 16 N, 8 F, 31 P, 32 P, 35 S, 3H CI, and 125 l.
• isotopically labeled compounds of the present invention for example those into which radioactive isotopes such as 3H, 13C, and 14C are
• Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients.
• PET positron emission tomography
• SPECT single-photon emission computed tomography
• Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements.
• An 18F labeled compound may be useful for PET or SPECT studies.
• Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.
• substitution with heavier isotopes may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index.
• deuterium in this context is regarded as a substituent.
• concentration of such a heavier isotope, specifically deuterium may be defined by an isotopic enrichment factor.
• any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.
• a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.
• the ADC is well tolerated and active across a range of cancer types, and will likely be one component of combination therapies that increase the response rate and durability of treatment.
• the purpose of this disclosure is to combine the ADC with the secondary agent.
• a secondary agent as described herein may be an Immune-oncology (IO) drug.
• Immune-oncology (IO) drugs a type of cancer therapy relying on the body's immune system to help fight cancer, have shown enhanced durability of anti-tumor response.
• IO Immune-oncology
• There are different types of IO including but not limited to PD1 inhibitors, PD-L1 inhibitors, CLTL4 inhibitors, GITR agonists and OX40 agonists.
• Immunogenic cell death is a particular form of cell death that stimulates an immune response against dead-cell antigens (released by dying cells) and it is considered as one of the best way to induce an adaptive immune response and improve the efficacy of anticancer treatment.
• anti-neoplastic agents that can induce ICD such as various anthracyclines (including doxorubicin, epirubicin and idarubicin), alkylating agents (including oxaliplatin and cyclophosphamide), the topoisomerase II inhibitor mitoxantrone, and the proteasomal inhibitor Bortezomib.
• Antibody-drug conjugates including those with a PBD warhead, may be particularly suited as combination partners because they are more targeted compared to conventional chemotherapy and expected to offer an increased antigen presentation to infiltrating cells as has been shown for aurista tin-based ADCs.
• ADCs with IO therefore allows for dual benefits: on the one hand, the ADC will directly kill the tumor expressing the target, providing immediate anti-tumor activity, and on the other the immunogenic cell death induced by ADC mediated cell kill may boost a stronger and more durable adaptive immune response, as compared to when the IO is given as a single agent.
• the secondary agent may be:
• a PD1 antagonist such as pembrolizumab, nivolumab, MEDI0680, PDR001 (spartalizumab), Camrelizumab, AUNP12, Pidilizumab, Cemiplimab (REGN- 2810), AMP-224, BGB-A317 (Tisleizumab), or BGB-108;
• a PD-L1 antagonist such as atezolizumab (Tecentriq), BMS- 936559/MDX-1 105, durvalumab/MEDI4736, or MSB0010718C (Avelumab);
• GITR G I u cocorti coid- In d u ced TNFR-Related protein
• MEDI 1873, TRX518, GWN323, MK-1248, MK-4166, BMS-986156 or INCAGN1876 a GITR (G I u cocorti coid- In d u ced TNFR-Related protein) agonist, such as MEDI 1873, TRX518, GWN323, MK-1248, MK-4166, BMS-986156 or INCAGN1876;
• an OX40 agonist such as MEDI0562, MEDI6383, MOXR0916, RG7888, OX40mAb24, INCAGN1949, GSK3174998, or PF-04518600;
• CTLA-4 antagonist such as ipilimumab (brand name Yervoy) or Tremelimumab (Originally developed by Pfizer, now Medimmune); (f) Fludarabine or Cytarabine;
• a hypomethylating agent such as cytidine analogs - for example, 5- azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine); or
• PARPi PARP inhibitor
• Olaparib CEP-9722
• BMN-673/talazoparib Rucaparib
• lniparib/SAR24-550/BSI-201 Veliparib (ABT-888)
• an agent that upregulates HER2 expression such as gemcitabine and tamoxifen:
• AXLi an AXL-kinase inhibitor
• BGB324 bemcentinib
• TP0903 Gilteritinib
• ASP2215 Gilteritinib
• Cabozantinib XL184
• SGI7079 SGI7079
• erestinib amuvatinib
• MP-470 bosutinib
• MGCD265 MGCD265, and foretinib
• BRAFi BRAF inhibitor
• MEKi MEK inhibitor
• Programmed death receptor I is an immune-inhibitory receptor that is primarily expressed on activated T and B cells. Interaction with its ligands has been shown to attenuate T-cell responses both in vitro and in vivo. Blockade of the interaction between PD1 and one of its ligands, PD-L1 , has been shown to enhance tumor-specific CD8+ T- cell immunity and may therefore be helpful in clearance of tumor cells by the immune system.
• PD1 (encoded by the gene Pdcdl) is an Immunoglobulin superfamily member related to CD28, and CTLA-4. PD1 has been shown to negatively regulate antigen receptor signalling upon engagement of its ligands (PD-L1 and/or PD-L2). The structure of murine PD1 has been solved as well as the co-crystal structure of mouse PD1 with human PD-L1 (Zhang, X., et al., (2004) Immunity 20: 337-347; Lin, et al., (2008) Proc. Natl. Acad. Sci.
• PD1 and like family members are type I transmembrane glycoproteins containing an Ig Variable-type (V-type) domain responsible for ligand binding and a cytoplasmic tail that is responsible for the binding of signaling molecules.
• the cytoplasmic tail of PD1 contains two tyrosine-based signaling motifs, an ITIM (immunoreceptor tyrosine-based inhibition motif) and an ITSM (immunoreceptor tyrosine-based switch motif).
• PD1 on tumor infiltrating lymphocytes
• PD-L1 on tumor cells
• Such tissues include cancers of the lung, liver, ovary, cervix, skin, colon, glioma, bladder, breast, kidney, esophagus, stomach, oral squamous cell, urothelial cell, and pancreas as well as tumors of the head and neck (Brown, J. A., et al., (2003) J Immunol. I 70: I257-I266; Dong H., et al., (2002) Nat. Med. 8: 793-800; Wintterle, et al.,
• Antibody blockade effectively promoted tumor reactive CD8+ T cell infiltration into the tumor resulting in the up-regulation of antitumor effectors including IFN gamma, granzyme Band perforin. Additionally, the authors showed that PD1 blockade can be effectively combined with chemotherapy to yield a synergistic effect. In another study, using a model of squamous cell carcinoma in mice, antibody blockade of PD1 or PD-L1 significantly inhibited tumor growth (Tsushima, F., et al., (2006) Oral Oneal. 42: 268-274).
• PD1 antagonist means any chemical compound or biological molecule that stimulates an immune reaction through inhibition of PD1 signalling.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20- fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• ADC which targets a first target protein (FTP) with PD1 inhibitors is advantageous, because on the one hand, the ADC will directly kill the FTP positive tumor cells, while on the other hand the PD1 inhibitor will engage the patient's own immune system to eliminate the cancer cells.
• FTP first target protein
• FTP negative tumor cells in close proximity to FTP(+) tumor cells will potentially be killed by the bystander mechanism of the PBD-dimer released after cell kill of CD19(+) or CD22(+) cells. Hence, the ADC will directly kill the tumor cells.
• PD1 programmed cell death protein 1
• TILs tumour infiltrating lymphocytes
• Blockade of the PD1 pathway may enhance antitumour immune responses against the antigens released from the tumors killed by the ADC by diminishing the number and/or suppressive activity of intratumoral TReg cells.
• the major function of PD1 is to limit the activity of T-cells at the time of an antiinflammatory response to infection and to limit autoimmunity.
• PD1 expression is induced when T-cells become activated, and binding of one of its own ligands inhibits kinases involved in T-cell activation. Hence, in the tumor environment this may translate into a major immune resistance, because many tumours are highly infiltrated with TReg cells that probably further suppress effector immune responses. This resistance mechanism is alleviated by the use of PD1 inhibitors in combination with the ADC.
• PD1 antagonists suitable for use as secondary agents in the present disclosure include: a) a PD1 antagonist which inhibits the binding of PD1 to its ligand binding partners. b) a PD1 antagonist which inhibits the binding of PD1 to PD- L1 .
• a PD1 antagonist which inhibits the binding of PD-1 to PDL2.
• a PD1 antagonist which inhibits the binding of PD-1 to both PDLI and PDL2.
• Specific PD1 antagonists suitable for use as secondary agents in the present disclosure include:
• pembrolizumab brand name Keytruda
• PD1 polypeptide corresponds to Genbank accession no. AAC51773, version no. AAC51773.1 , record update date: Jun 23, 2010 09:24 AM.
• the nucleic acid encoding PD1 polypeptide corresponds to Genbank accession no. U64863, version no. U64863.1 , record update date: Jun 23, 2010 09:24 AM.
• PD1 polypeptide corresponds to Uniprot/Swiss-Prot accession No. Q151 16.
• PD-L1 antagonist means any chemical compound or biological molecule that stimulates an immune reaction through inhibition of PD-L1 signalling.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20- fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• an ADC which targets a first target protein (FTP) positive lymphomas and leukemias with PD-L1 inhibitors is advantageous because, on the one hand, the ADC will directly kill the FTP positive tumor cells while, on the other hand, the PD-L1 inhibitor will engage the patient's own immune system to eliminate the cancer cells.
• FTP first target protein
• target negative tumor cells in close proximity to FTP(+) tumor cells will potentially be killed by the bystander mechanism of the PBD-dimer released after cell kill of FTP(+) cells.
• the ADC will directly kill the tumor cells.
• the resulting release of tumor associated antigens from cells that are killed with the PBD dimer will trigger the immune system, which will be further enhanced by the use of programmed cell death protein 1 ligand inhibitors (PD-L1 , aka B7-H1 or CD274 ).
• PD-L1 is commonly upregulated on the tumour cell surface from many different human tumours. Interfering with the PD1 ligand expressed on the tumor will avoid the immune inhibition in the tumor microenvironment and therefore blockade of the PD1 pathway using PDL1 inhibitors may enhance antitumour immune responses against the antigens released from the tumors killed by the ADC.
• PDL1 inhibitors may enhance antitumour immune responses against the antigens released from the tumors killed by the ADC.
• FTP first target protein
• FTP negative tumor cells in close proximity to FTP(+) tumor cells will potentially be killed by the bystander mechanism of the PBD-dimer released after cell kill of CD19(+) or CD22 (+) cells. Hence, the ADC will directly kill the tumor cells.
• PD-L1 antagonists suitable for use as secondary agents in the present disclosure include PD-L1 antagonists that:
• (e) are anti-PD-L1 antibodies.
• Specific PD-L1 antagonists suitable for use as secondary agents in the present disclosure include:
• VH CDR1 DYGFS
• VH CDR2 WITAYNGNTNYAQKLQG
• VH CDR3 DYFYGMDV
• VL CDR1 RASQSVSSYLV
• VL CDR2 DAS N RAT
• VL CDR3 QQRSNWPRT ii.
• Antibody having:
• VH CDR1 TYAIS
• VH CDR2 GIIPIFGKAHYAQKFQG
• VH CDR3 KFHFVSGSPFGMDV
• VL CDR1 RASQSVSSYLA
• VL CDR2 DASNRAT
• VL CDR3 QQRSNWPT
• Antibody having:
• VH CDR1 SYDVH
• VH CDR2 WLHADTGITKFSQKFQG
• VH CDR3 ERIQLWFDY
• VL CDR1 RASQGISSWLA
• VL CDR2 AASSLQS
• VL CDR3 QQYNSYPYT durvalumab/MEDI4736
• PD-L1 polypeptide corresponds to Genbank accession no. AAF25807, version no. AAF25807.1 , record update date: Mar 10, 2010 10:14 PM.
• the nucleic acid encoding PD1 polypeptide corresponds to Genbank accession no. AF177937, version no. AF177937.1 , record update date: Mar 10, 2010 10:14 PM.
• PD1 polypeptide corresponds to Uniprot/Swiss-Prot accession No. Q9NZQ7.
• glucose-induced TNF receptor (abbreviated herein as
• GITR also known as TNF receptor superfamily 18 (TNFRSF18, CD357), TEASR, and 312C2, as used herein, refers to a member of the tumor necrosis factor/nerve growth factor receptor family.
• GITR is a 241 amino acid type I transmembrane protein characterized by three cysteine pseudo-repeats in the extracellular domain and specifically protects T-cell receptorinduced apoptosis, although it does not protect cells from other apoptotic signals, including Fas triggering, dexamethasone treatment, or UV irradiation (Nocentini, G., et al. (1997) Proc. Natl. Acad. Sci. USA 94:6216-622).
• GITR activation increases resistance to tumors and viral infections, is involved in autoimmune/inflammatory processes and regulates leukocyte extravasation (Nocentini supra; Cuzzocrea, et al. (2004) J Leukoc. Biol. 76:933-940; Shevach, et al. (2006) Nat. Rev. Immunol. 6:613-618; Cuzzocrea, et al. (2006) J Immunol. I 77:631-641; and Cuzzocrea, et al. (2007) FASEB J 21 :l I 7-129).
• GITR human GITR
• hGITR human GITR
• GITR agonist means any chemical compound or biological molecule that stimulates an immune reaction through activation of GITR signalling.
• soluble GITR-L proteins a GITR binding partner.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20- fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• ADC which targets a first target protein (FTP) positive lymphomas and leukemias with GITR agonists is advantageous, because on the one hand the ADC will directly kill the FTP positive tumor cells, while on the other hand the GITR agonist will engage the patient's own immune system to eliminate the cancer cells.
• FTP first target protein
• GITR Glucocorticoid-lnduced TNFR-Related protein
• GITR ligation via its ligand GITRL stimulates both proliferation and function of both effector and regulatory CD4+ T cells. This promotes T- cell survival, and differentiation into effector cells, while abrogating suppression. Therefore it will be beneficial to target a FTP(+) tumor with the ADC, causing the antigenic cell death, while the GITR agonist induces a stronger, durable immune response.
• GITR agonists suitable for use as secondary agents in the present disclosure include:
• INCAGN1876 is an agonist antibody targeting the glucocorticoid-induced TNFR- related protein, or GITR. Discovered during a collaboration with Ludwig Cancer Research. INCAGN1876 is being co-developed with Incyte
• ⁇ VL comprising the sequence (CDR underline):
• ⁇ VH comprising the sequence (CDR underline):
• GWN323 an anti-GITR agonistic monoclonal antibody, which activates GITRs found on multiple types of T-cells. GWN323 is developed by Novartis
• MK-1248 a humanized lgG4 anti-human glucocorticoid-induced tumor necrosis factor receptor (GITR) agonistic monoclonal antibody (MoAb) with significantly reduced effector function
• MK-4166 a humanized IgG1 anti-human glucocorticoid-induced tumor necrosis factor receptor (GITR) agonistic monoclonal antibody (MoAb) with potential immunomodulating activity (see Sukumar et al., Cancer Res. 2017).
• GITR glucocorticoid-induced tumor necrosis factor receptor
• BMS-986156 An anti-human glucocorticoid-induced tumor necrosis factor receptor (GITR; tumor necrosis factor superfamily member 18; TNFRSF18;
• GITR polypeptide corresponds to Genbank accession no. AAD22635, version no. AAD22635.1 , record update date: Mar 10, 2010 09:42 PM.
• the nucleic acid encoding GITR polypeptide corresponds to Genbank accession no. AF 125304, version no. AF125304.1 , record update date: Mar 10, 2010 09:42 PM.
• GITR polypeptide corresponds to Uniprot/Swiss-Prot accession No. Q9Y5U5.
• OX40 (CD134; TNFRSF4) is a member of the TNFR super-family and is expressed by CD4 and CD8 T cells during antigen-specific priming. OX40 expression is largely transient following TCR/CD3 cross-linking, and by the presence of inflammatory cytokines. In the absence of activating signals, relatively few mature T cell subsets express OX40 at biologically relevant levels. Generating optimal "killer" CD8 T cell responses requires T cell receptor activation plus co-stimulation, which can be provided through ligation of OX40 using a OX40 agonist. This activating mechanism augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity. Therefore it will be beneficial to target a FTP(+) tumor with the ADC, causing the antigenic cell death, while the OX40 agonist induces a stronger, durable immune response.
• the OX40 agonist may be selected from the group consisting of an OX40 agonist antibody, an OX40L agonist fragment, an OX40 oligomeric receptor, and an OX40 immunoadhesin.
• the OX40 binding agonist is a trimeric OX40L-Fc protein.
• the OX40 binding agonist is an OX40L agonist fragment comprising one or more extracellular domains of OX40L.
• the OX40 binding agonist is an OX40 agonist antibody that binds human OX40.
• the 0X40 agonist antibody depletes cells that express human OX40.
• the 0X40 agonist antibody depletes cells that express human OX40 in vitro.
• the cells are CD4+ effector T cells.
• the cells are Treg cells.
• the depleting is by ADCC and/or phagocytosis.
• the depleting is by ADCC.
• the OX40 agonist antibody binds human OX40 with an affinity of less than or equal to about 1 nM.
• the OX40 agonist antibody increases CD4+ effector T cell proliferation
• the cytokine is gamma interferon.
• the OX40 agonist antibody increases memory T cell proliferation and/or increasing cytokine production by the memory cell.
• the cytokine is gamma interferon.
• the OX40 agonist antibody inhibits Treg function.
• the OX40 agonist antibody inhibits Treg suppression of effector T cell function.
• effector T cell function is effector T cell proliferation and/or cytokine production.
• the effector T cell is a CD4+ effector T cell.
• the OX40 agonist antibody increases 0X40 signal transduction in a target cell that expresses OX40.
• OX40 signal transduction is detected by monitoring NFkB downstream signalling.
• 0X40 agonist means any chemical compound or biological molecule that stimulates an immune reaction through iactivation of OX40 signalling.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2 -fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20- fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• ADC which targets a first target protein (FTP) positive lymphomas and leukemias with OX40 agonists is advantageous, because on the one hand the ADC will directly kill the FTP positive tumor cells, while on the other hand the OX40 agonist will engage the patient's own immune system to eliminate the cancer cells.
• FTP first target protein
• target negative tumor cells in close proximity to FTP(+) tumor cells will potentially be killed by the bystander mechanism of the PBD-dimer released after cell kill of FTP(+) cells.
• the ADC will directly kill the tumor.
• the resulting release of tumor associated antigens from cells killed with the PBD dimer will trigger the immune system, which will be further enhanced by the use of a OX40 agonist.
• OX40 agonists suitable for use as secondary agents in the present disclosure include:
• MEDI0562 (aka Tavolixizumab, Tavolimab)
• OX40mAb24 is a humanised version of 9B12.
• 9B12 is a murine IgGI, anti- OX40 mAb directed against the extracellular domain of human OX40
• an antibody comprising the sequences:
• PF-04518600 (PF-8600) is an investigational, fully human, monoclonal antibody (mAb) that targets OX40 protein
• OX40 polypeptide corresponds to Genbank accession no. CAA53576, version no. CAA53576.1 , record update date: Feb 2, 201 1 10:10 AM.
• the nucleic acid encoding OX40 polypeptide corresponds to Genbank accession no. X75962, version no. X75962.1 , record update date: Feb 2, 201 1 10:10 AM.
• OX40 polypeptide corresponds to Uniprot/Swiss-Prot accession No. P43489.
• CTLA4 (CD152) is expressed on activated T cells and serves as a co-inhibitor to keep T cell responses in check following CD28-mediated T cell activation.
• CTLA4 is believed to regulate the amplitude of the early activation of naive and memory T cells following TCR engagement and to be part of a central inhibitory pathway that affects both antitumor immunity and autoimmunity.
• CTLA4 is expressed exclusively on T cells, and the expression of its ligands CD80 (B7.1 ) and CD86 (B7.2), is largely restricted to antigen- presenting cells, T cells, and other immune mediating cells.
• Antagonistic anti-CTLA4 antibodies that block the CTLA4 signalling pathway have been reported to enhance T cell activation.
• ipilimumab was approved by the FDA in 201 1 for the treatment of metastatic melanoma.
• Another anti-CTLA4 antibody, tremelimumab was tested in phase III trials for the treatment of advanced melanoma, but did not significantly increase the overall survival of patients compared to the standard of care (temozolomide or dacarbazine) at that time.
• CTLA4 agonist means any chemical compound or biological molecule that stimulates an immune reaction through inhibition of CTLA4 signalling.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20- fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• an ADC which targets a first target protein (FTP) positive lymphomas and leukemias with CTLA4 inhibitors is advantageous, because on the one hand, the ADC will directly kill the FTP positive tumor cells, while on the other hand the CTLA4 inhibitor will engage the patient's own immune system to eliminate the cancer cells.
• FTP target protein
• target negative tumor cells in close proximity to FTP(+) tumor cells will potentially be killed by the bystander mechanism of the PBD-dimer released after cell kill of FTP(+) cells. Hence, the ADC will directly kill the tumor.
• CTLA4 tumour infiltrating lymphocytes
• CD28 tumour infiltrating lymphocytes
• Blockade of the CTLA4 pathway may therefore enhance enhancement of effector CD4+T cell activity, while it inhibits TReg cell- dependent immunosuppression. Therefore it will be beneficial to target a FTP(+) tumor with the ADC, causing the antigenic cell death, while the CTLA4 blockade induces a stronger immune, durable response.
• CTLA4 antagonists suitable for use as secondary agents in the present disclosure include:
• CTLA polypeptide corresponds to Genbank accession no. AAL07473, version no. AAL07473.1 , record update date: Mar 1 1 , 2010 01 :28 AM .
• the nucleic acid encoding CTLA4 polypeptide corresponds to Genbank accession no. AF414120, version no. AF414120.1 , record update date: Mar 1 1 , 2010 01 :28 AM .
• OX40 polypeptide corresponds to Uniprot Swiss- Prot accession No. P16410.
• Combination of agents with different action mechanisms is an established therapeutic principle for combating cancer. It can be a way of increasing anti-tumour activity when a synergic effect is shown and/or when reduced toxicity is observed.
• Antibody-drug conjugates including those with a PBD warhead, may be particularly suited as combination partners because they are more targeted compared to conventional chemotherapy.
• PBD dimers cross-link DNA in a covalent fashion, combining them with other agents that interfere with DNA synthesis via a different mechanism is likely to provide a benefit. Examples of such potential combinations are Fludarabine and Cytarabine.
• Fludarabine or fludarabine phosphate is a chemotherapy drug used in the treatment of hematological malignancies such as leukemias and lymphomas. It is a purine analog, which interferes with DNA by interfering with ribonucleotide reductase (RNAR) and DNA polymerase. It is active against both dividing and resting cells. Fludarabine has also been shown to suppress ERCC1 transcription and this may explain the observed synergy between Fludarabine and the PBD Dimer SJG136 (SG2000) against chronic lymphocytic leukaemia cells.
• CLAG/CLAG-M Cladribine is another purine analogue that inhibits RNR.
• the same panel of cell lines will be co-treated with a range of concentrations of Fludarabine and a non-targeted control ADC or with a range of concentration of the ADC and vehicle. After incubation, two parameters will be measured: the amount of surface FTP (as determined by flow cytometry) and the in vitro cytotoxicity of the combinations (as determined by CellTiter-Glo® or MTS assays). Cytotoxic synergy is calculated by transforming the cell viability data into fraction affected, and calculating the combination index using the CalcuSyn analysis program. CAS Number 21679-14-1
• Cytarabine or cytosine arabinoside is a antimetabolic chemotherapy drug used in the treatment of hematological malignancies such as acute myeloid leukemia (AML) and non-Hodgkin lymphoma. It is also known as ara-C (arabinofuranosyl cytidine). It kills cancer cells by interfering with DNA synthesis. It is actively metabolized to cytosine arabinoside triphosphate, which damages DNA when the cell cycle holds in the S phase (synthesis of DNA). Rapidly dividing cells, which require DNA replication for mitosis, are therefore most affected. Cytosine arabinoside also inhibits both DNA and RNA polymerases and nucleotide reductase enzymes needed for DNA synthesis.
• ADC which targets First Target Protein (FTP) positive lymphomas and leukemias
• Cytarabine is advantageous, because on the one hand, the ADC will directly kill the FTP positive tumor cells via a mechanisms depending on DNA cross- linking resulting in apoptosis, while on the other hand the Cytarabine will inhibit the cells RNA and DNA polymerase, while also suppressing DNA synthesis.
• FTP First Target Protein
• a panel of FTP(+) cell lines will be co-treated with a range of concentration of both the ADC and Cytarabine.
• the same panel of cell lines will be co-treated with a range of concentrations of Cytarabine and a non-targeted control ADC or with a range of concentration of the ADC and vehicle.
• two parameters will be measured: the amount of surface FTP (as determined by flow cytometry) and the in vitro cytotoxicity of the combinations (as determined by CellTiter-Glo® or MTS assays). Cytotoxic synergy is calculated by transforming the cell viability data into fraction affected, and calculating the combination index using the CalcuSyn analysis program (see example 4).
• hypomethylating agent refers to a class of compounds that interfere with DNA methylation which is the addition of a methyl group to the 5- position of the cytosine pyrimidine ring or the nitrogen in position 6 of the adenine purine ring.
• DNA methylation stably alters the gene expression pattern in cells i.e. decrease gene expression (i.e. for the Vitamin D receptor).
• Hypomethylating agent are compounds that can inhibit methylation, resulting in the expression of the previously hypermethylated silenced genes.
• Cytidine analogs such as 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine are the most commonly used Hypomethylating agent. These compounds work by binding to the enzymes that catalyse the methylation reaction, i.e. DNA methyltransferases.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule.
• Control samples are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• an ADC which targets a first target protein (FTP) positive lymphomas and leukemias with a hypomethylating agent is advantageous, because on the one hand the ADC will directly kill the FTP positive tumor cells, while on the other hand the a hypomethylating agent will interfere with DNA methylation. This interference is by way of causing demethylation in that sequence, which adversely affects the way that cell regulatory proteins are able to bind to the DNA/RNA substrate. This activity synergises with the ADC because PBD dimers cross-link DNA in a covalent fashion, so combining them with other agents that interfere with DNA synthesis via a different mechanism provides a benefit.
• FTP first target protein
• Specific Hypomethylating agents suitable for use as secondary agents in the present disclosure include:
• 5-azacytidine 4-Amino-1-3-D-ribofuranosyl-1 ,3,5-triazin-2(1 H)-one b) 5-aza-2'-deoxycytidine (decitabine)
• PARP-1 Poly (adenosine diphosphate [ADP]) ribose polymerase (PARP) are a family of enzymes involved in a wide range of cellular functions including DNA transcription, DNA damage response, genomic stability maintenance, cell cycle regulation, and cell death.
• PARP-1 is the most abundant and best characterised protein of this group. In oncology, its integral role in the repair of single-strand DNA breaks (SSBs) via the base excision repair (BER) pathway has been a focus of high interest and several PARP-1 inhibitors (PARPi) have been developed (including but not limited to Olaparib, CEP-9722, talazoparib, Rucaparib, Iniparib, Veliparib and Niraparib) and are tested clinically. In cancer therapeutics, PARPi work predominantly by preventing the repair of DNA damage, ultimately causing cell death.
• SSBs single-strand DNA breaks
• BER base excision repair
• PARPi PARP-1 inhibitors
• PARPi work predominantly by preventing
• PARP is composed of four domains of interest: a DNA-binding domain, a caspase- cleaved domain, an auto-modification domain, and a catalytic domain.
• the DNA-binding domain is composed of two zinc finger motifs. In the presence of damaged DNA (base pair-excised), the DNA-binding domain will bind the DNA and induce a conformational shift. It has been shown that this binding occurs independent of the other domains. This is integral in a programmed cell death model based on caspase cleavage inhibition of PARP.
• the auto-modification domain is responsible for releasing the protein from the DNA after catalysis. Also, it plays an integral role in cleavage-induced inactivation.
• PARP is found in the cell nucleus. The main role is to detect and initiate an immediate cellular response to metabolic, chemical, or radiation-induced single-strand DNA breaks (SSB) by signalling the enzymatic machinery involved in the SSB repair. Once PARP detects a SSB, it binds to the DNA, undergoes a structural change, and begins the synthesis of a polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) chain, which acts as a signal for the other DNA-repairing enzymes.
• Target enzymes include DNA ligase III (Liglll), DNA polymerase beta ( ⁇ ), and scaffolding proteins such as X- ray cross-complementing gene 1 (XRCC1 ).
• PARP Poly(ADP-ribose) glycohydrolase
• PARP enzymes are essential in a number of cellular functions, including expression of inflammatory genes: PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells, in response to TNF.
• PBDs are a class of naturally occurring anti-tumor antibiotics found in Streptomyces. PBD dimers exert their cytotoxic mode of action via cross-linking of two strands of DNA, which results in the blockade of replication and tumor cell death. Importantly, the cross-links formed by PBD dimers are relatively non-distorting of the DNA structure, making them hidden to DNA repair mechanisms, which are often impaired in human tumors as opposed to normal tissues.
• PBD-based ADCs with PARPi including but not limited to Olaparib, CEP- 9722, talazoparib, Rucaparib, Iniparib, Veliparib and Niraparib
• PARPi including but not limited to Olaparib, CEP- 9722, talazoparib, Rucaparib, Iniparib, Veliparib and Niraparib
• a panel of solid tumor-derived cell lines will be treated with a range of concentration of each ADC and a PARPi.
• Cytotoxic synergy is calculated by transforming the cell viability data into fraction affected, and calculating the combination index using the CalcuSyn analysis program.
• PARP inhibitor means any chemical compound or biological molecule reduces PARP activity.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Specific PARPi suitable for use in the present disclosure include: a) Olaparib
• Rucaparib 8-Fluoro-2- ⁇ 4-[(methylamino)methyl]phenyl ⁇ -1 ,3,4,5-tetrahydro- 6H-azepino[5,4,3-cd]indol-6-one e) lniparib/SAR24-550/BSI-201
• Niraparib 2-[4-[(3S)-3-Piperidyl]p enyl]indazole-7-carboxamide
• PARP polypeptide is PARP1 , which corresponds to Genbank accession no. AAA60137, version no. AAA60137.1 , record update date: Jun 23, 2010 08:48 AM.
• the nucleic acid encoding PARP1 polypeptide corresponds to Genbank accession no. M181 12, version no. M181 12.1 , record update date: Jun 23, 2010 08:48 AM.
• PARP1 polypeptide corresponds to Uniprot/Swiss-Prot accession No. P09874. Agents that uprequlate HER2 expression
• An agent that "upregulates HER2 expression” means any chemical compound or biological molecule that increase the amount of HER2 protein on a tumour cell surface.
• enhancement samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating agent and are compared to control samples treated with an inactive control molecule.
• Control samples are assigned a relative expression value of 100%. Activation is achieved when the expression value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10- fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• agents that upregulate HER2 expression suitable for use as secondary agents in the present disclosure include: a) gemcitabine
• Gemcitabine is the preferred agent that upregulated HER2.
• the secondary agent as described herein may be an AXL inhibitor.
• AXL inhibitor means any chemical compound or biological molecule that reduces AXL signalling.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• ADC will induce DNA damage in AXL-positive cancer cell lines, while on the other hand treatment with the AXLi will diminish the efficiency of the homologous recombination repair machinery making the cells more sensitive to the DNA damage induced by the PBD dimers hence resulting in accumulation of DNA damage leading to cancer cell death.
• AXL-positive cancer cell lines with ADC and the AXLi including but not limited to BGB324 and TP0903
• AXLi including but not limited to BGB324 and TP0903
• a panel of cell lines including, but not limited to MDA-MB-157 and SKLU1 will be co-treated with a range of concentration of both ADC and the AXLi BGB324 or TP- 093.
• the in vitro cytotoxicity of the combinations (as determined by CellTiter-Glo® or MTS assays) will be measured.
• Specific AXL inhibitors suitable for use as secondary agents in the present disclosure include:
• the secondary agent as described herein may be an BRAF inhibitor.
• BRAF inhibitor means any chemical compound or biological molecule that reduces BRAF activity.
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• B-Raf is a member of the Raf kinase family of growth signal transduction protein kinases. This protein plays a role in regulating the MAP kinase/ERKs signaling pathway, which affects cell division, differentiation, and secretion.
• B-Raf is a serine/threonine-specific protein kinase. As such, it catalyzes the phosphorylation of serine and threonine residues in a consensus sequence on target proteins by ATP, yielding ADP and a phosphorylated protein as products. Since it is a highly regulated signal transduction kinase, B-Raf must first bind Ras-GTP before becoming active as an enzyme.
• a conserved protein kinase catalytic core phosphorylates protein substrates by promoting the nucleophilic attack of the activated substrate serine or threonine hydroxy I oxygen atom on the ⁇ -phosphate group of ATP through bimolecular nucleophilic substitution.
• BRAF BRAF-associated fatty acid deficiency fatty acid fatty acid fatty acid fatty acid fatty acid fatty acid fatty acid fatty acid fatty acid fatty acid fatty acid sulfate, fatty acid fatty acid fatty acid sulfate, fatty acid fatty acid sulfate, fatty acid fatty acid sulfate, fatty acid sulfate, fatty acid sulfate, fatty acid, and adenocarcinoma of the lung, brain tumours including glioblastoma and pilocytic astrocytomas as well as inflammatory diseases like erdheim-chester disease. Mutation can lead to uncontrolled growth, especially in melanoma.
• the V600E mutation in B-RAF is known to drive cell proliferation in melanoma mutated gene. Such mutations makes the mutant BRAF gene constitutively active, driving proliferation of the melanoma.
• BRAF inhibitors such as vemurafenib and dabrafenib. These BRAF inhibitors inhibits the B-RAF protein directly.
• ADC which targets AXL positive tumors
• BRAFi BRAFi
• ADC works synergistically with BRAFi
• a panel of AXL (+) cell lines including, but not limited to MDA-MB231 , NCI-H1299 and SNU12 cells, will be co-treated with a range of concentrations of both ADC and BRAFi.
• the same panel of cell lines will be co-treated with a range of concentrations of MEKi or with a range of concentration of ADC and vehicle.
• the in vitro cytotoxicity of the combinations will be determined by an MTS assay.
• MTS assay MTS assay
• Cell viability is measured by adding MTS per well and incubating for 4 hours at 37 C. Percentage cell viability is calculated compared to the untreated control. Cytotoxic synergy is calculated by transforming the cell viability data into fraction affected, and calculating the combination index (Table 1 ) using the CalcuSyn analysis program.
• Specific BRAF inhibitors suitable for use as secondary agents in the present disclosure include:
• the secondary agent as described herein may be a MEK inhibitor.
• MEK inhibitor means any chemical compound or biological molecule that reduces MEK1 and/or MEK2 activity.
• MEK1 in humans is encoded by the MAP2K1 gene.
• MEK1 is a member of the dual- specificity protein kinase family that acts as a mitogen-activated protein (MAP) kinase kinase.
• MAP kinases also known as extracellular signal-regulated kinases (ERKs)
• ERKs extracellular signal-regulated kinases
• This protein kinase lies upstream of MAP kinases and stimulates the enzymatic activity of MAP kinases upon activation by a wide variety of extra- and intracellular signals.
• this kinase is involved in many cellular processes such as proliferation, differentiation, transcription regulation and development.
• MEK2 in humans is encoded by the MAP2K2 gene.
• the protein encoded by this gene is a dual specificity protein kinase that belongs to the MAP kinase kinase family. This kinase is known to play a critical role in mitogen growth factor signal transduction. It phosphorylates and thus activates MAPK1 /ERK2 and MAPK3/ERK1 .
• samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples treated with an inactive control molecule. Control samples are assigned a relative activity value of 100%.
• Inhibition is achieved when the activity value relative to the control is about 90% or less, typically 85% or less, more typically 80% or less, most typically 75% or less, generally 70% or less, more generally 65% or less, most generally 60% or less, typically 55% or less, usually 50% or less, more usually 45% or less, most usually 40% or less, preferably 35% or less, more preferably 30% or less, still more preferably 25% or less, and most preferably less than 20%.
• Activation is achieved when the activity value relative to the control is about 1 10%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.
• MEK inhibitors are Trametinib, Cobimetinib, Binimetinib and Selumetinib.
• a MEK inhibitor inhibits the mitogen-activated protein kinase kinase enzymes MEK1 and/or MEK2. Defects in the MAP/ERK pathway can lead uncontrolled growth, especially in melanoma.
• Some MEK inhibitors such as Trametinib, inhibit MEK1 and MEK2 and are approved for the treatment of patients with BRAF V600E mutated metastatic melanoma.
• the V600E mutation makes the mutant BRAF gene constitutively active, driven proliferation of the melanoma.
• MAP/ERK pathway By Inhibiting the MAP/ERK pathway, cell proliferation is blocked and apoptosis (controlled cell death) is induced.
• ADC which targets AXL positive tumors
• MEKi will interfere with cell proliferation through inhibition of the MAP/ERK cell signalling pathway.
• ADC works synergistically with MEKi
• a panel of AXL (+) cell lines including, but not limited to MDA-MB231 , H1299 and SNU12C cells, will be co-treated with a range of concentration of both ADC and MEKi.
• the same panel of cell lines will be co-treated with a range of concentrations of MEKi or with a range of concentration of ADC and vehicle.
• the in vitro cytotoxicity of the combinations will be determined by an MTS assay.
• MTS MTS assay
• Cell viability is measured by adding MTS per well and incubating for 4 hours at 37 C. Percentage cell viability is calculated compared to the untreated control. Cytotoxic synergy is calculated by transforming the cell viability data into fraction affected, and calculating the combination index using the CalcuSyn analysis program.
• MEK inhibitors suitable for use as secondary agents in the present disclosure include:
• BRAF polypeptide corresponds to Genbank accession no. AAA35609, version no. AAA35609.2, record update date: Jun 23, 2010 09:41 AM.
• the nucleic acid encoding BRAF polypeptide corresponds to Genbank accession no. M95712, version no. M95712.2, record update date: Jun 23, 2010 09:41 AM.
• BRAF polypeptide corresponds to Uniprot/Swiss-Prot accession No. P15056.
• MEK1 polypeptide corresponds to Genbank accession no. AAA36318, version no. AAA36318.1 , record update date: Jun 23, 2010 08:48 AM.
• the nucleic acid encoding MEK1 polypeptide corresponds to Genbank accession no. L05624, version no. L05624.1 , record update date: Jun 23, 2010 08:48 AM.
• MEK1 polypeptide corresponds to Uniprot/Swiss-Prot accession No. Q02750.
• MEK2 polypeptide corresponds to Genbank accession no. AAH00471 , version no. AAH00471 .1 , record update date: Sep 23, 2014 03:30 PM.
• the nucleic acid encoding MEK2 polypeptide corresponds to Genbank accession no. BC000471 , version no. BC000471.2, record update date: Sep 23, 2014 03:30 PM.
• MEK2 polypeptide corresponds to Uniprot/Swiss-Prot accession No. P36507.
• ADC and secondary agent when used as a single agent in isolation have demonstrated clinical utility - for example, in the treatment of cancer.
• combination of the ADC and secondary agent is expected to provide one or more of the following advantages over treatment with either ADC or secondary agent alone:
• Effective treatment of a broader range of cancers as used herein means that following treatment with the combination a complete response is observed with a greater range of recognised cancer types. That is, a complete response is seen from cancer types not previously reported to completely respond to either ADC or secondary agent alone.
• Effective treatment of a resistant, refractory, or relapsed forms as used herein means that following treatment with the combination a complete response is observed in individuals that are either partially or completely resistant or refractory to treatment with either ADC or secondary agent alone (for example, individuals who show no response or only partial response following treatment with either agent alone, or those with relapsed disorder).

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