WO2022155311A1 - Methods and systems for analysis of drug target engagement and treatment of cancer - Google Patents

Methods and systems for analysis of drug target engagement and treatment of cancer Download PDF

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Publication number
WO2022155311A1
WO2022155311A1 PCT/US2022/012284 US2022012284W WO2022155311A1 WO 2022155311 A1 WO2022155311 A1 WO 2022155311A1 US 2022012284 W US2022012284 W US 2022012284W WO 2022155311 A1 WO2022155311 A1 WO 2022155311A1
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kinase inhibitor
glucose consumption
consumption level
cancer
subject
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PCT/US2022/012284
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French (fr)
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Peter M. Clark
Chiara GHEZZI
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The Regents Of The University Of California
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Publication of WO2022155311A1 publication Critical patent/WO2022155311A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • aspects of this disclosure relate, generally, to at least the fields of cancer biology and medicine and, more specifically, to methods of measuring biomarkers of drug target engagement in cancer.
  • Metabolic reprogramming is an emerging hallmark of cancer, and most aggressive human cancers present with elevated glucose consumption relative to surrounding tissue. 18 F- FDG PET measures this elevated glucose consumption and has become an important tool in the care of cancer patients.
  • aspects of the present disclosure address needs in the art by providing methods for treating subjects with cancer (e.g., lung cancer) and methods for evaluating the efficacy of a kinase inhibitor by comparing glucose consumption levels in a tumor of the subject before and after treatment with the cancer therapy.
  • methods for treating a subject with cancer comprising measuring a first glucose consumption level in a tumor of the subject before administration of a dose of a kinase inhibitor; administering the kinase inhibitor dose to the subject; measuring a second glucose consumption level in the tumor after administration of the kinase inhibitor dose; and continuing or modifying the therapy based on the second glucose consumption level relative to the first glucose consumption level.
  • the second glucose consumption level is reduced relative to the first glucose consumption level, and administration of the kinase inhibitor is continued.
  • the second glucose level is unchanged or is not reduced relative to the first glucose consumption level, and administration of the therapy is modified.
  • modification of the therapy comprises an increase in the dose of the cancer therapy.
  • modification of the therapy comprises administration of an alternative cancer therapy different from the kinase inhibitor previously administered.
  • the kinase inhibitor is a Fms Related Receptor Tyrosine Kinase 3 (FLT3) inhibitor.
  • the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
  • Embodiments of the disclosure include methods for treating a subject having cancer, methods for evaluating the efficacy of a cancer therapy used to treat a subject having cancer, methods for predicting a subject ’s response to a cancer therapy, methods for identifying a subject with cancer as a candidate for a cancer therapy, and methods and compositions for treating a subject having lung cancer.
  • Methods of the disclosure can include 1, 2, 3, 4, 5, 6, or more of the following steps: measuring a first glucose consumption level in a subject, providing a first dose of a cancer therapy to a subject, measuring a second glucose consumption level in a subject, comparing a first glucose consumption level measured in a subject to a second glucose consumption level measured in the subject, providing a second dose of a cancer therapy to a subject, providing three or more doses of a cancer therapy a subject, providing a combination cancer therapy to a subject, providing an alternative therapy to a subject, determining a subject to have cancer, providing two or more types of cancer therapy to a subject, identifying one or more cancer therapy as being in need of evaluation of efficacy, evaluating the efficacy of a cancer therapy, identifying a subject as being a candidate for a cancer therapy, and predicting a subject’s response to a cancer therapy. Certain embodiments of the disclosure may exclude one or more of the preceding elements and/or steps.
  • a method of treating a subject for cancer comprising administering a dose of a kinase inhibitor to the subject, wherein a first glucose consumption level and a second glucose consumption level were measured in a tumor of the subject, wherein a previous dose of the kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was reduced relative to the first glucose consumption level.
  • a method of treating a subject for cancer comprising administering a cancer therapy to the subject, wherein a first glucose consumption level and a second glucose consumption level were measured in a tumor of the subject, wherein a dose of a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, wherein the second glucose consumption level was not reduced relative to the first glucose consumption level, and wherein the cancer therapy does not comprise the kinase inhibitor.
  • a method for evaluating efficacy of a kinase inhibitor in a subject having cancer comprising: (a) measuring a first glucose consumption level in a tumor of the subject; (b) administering a dose of the kinase inhibitor to the subject; and (c) measuring a second glucose consumption level in the tumor at most 48 hours after administering the kinase inhibitor to the subject.
  • the method further comprises identifying the kinase inhibitor as effective, wherein the second glucose consumption level is reduced compared to the first glucose consumption level.
  • the method further comprises (d) administering an additional dose of the kinase inhibitor to the subject, wherein the second glucose consumption level is reduced compared to the first glucose consumption level. In some embodiments, the method further comprises identifying the kinase inhibitor as ineffective, wherein the second glucose consumption level is not reduced compared to the first glucose consumption level. In some embodiments, the method further comprises (d) administering an alternative cancer therapy, wherein the second glucose consumption level is not reduced compared to the first glucose consumption level.
  • a method for evaluating efficacy of a kinase inhibitor in a subject having cancer comprising: (a) administering the kinase inhibitor to the subject; and (b) measuring a glucose consumption level in a tumor of the subject at most 48 hours after administering the kinase inhibitor.
  • a reference glucose consumption level was measured in the tumor prior to administering the kinase inhibitor to the subject.
  • the method further comprises administering an additional dose of the kinase inhibitor to the subject, wherein the glucose consumption level is reduced relative to the reference glucose consumption level.
  • the method further comprises administering an alternative cancer therapy to the subject, wherein the glucose consumption level is not reduced relative to the reference glucose consumption level.
  • a method of treating a subject for cancer comprising: (a) selecting a subject for treatment, wherein a first glucose consumption level and a second glucose consumption level were measured in the subject, wherein a first dose of a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was reduced relative to the first glucose consumption level; and (b) administering to the subject a second dose of the kinase inhibitor.
  • a method of treating a subject for cancer comprising: (a) selecting a subject for treatment, wherein a first glucose consumption level and a second glucose consumption level were measured in the subject, wherein a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was not reduced compared to the first glucose consumption level; and (b) administering an alternative cancer therapy to the subject, wherein the alternative cancer therapy does not comprise the kinase inhibitor.
  • a method of treating a subject for cancer comprising: (a) measuring a first glucose consumption level in a tumor of the subject; (b) administering to the subject a first dose of a kinase inhibitor; (c) subsequent to (b), measuring a second glucose consumption level in the tumor; and (d) administering to the subject a second dose of the kinase inhibitor, wherein the second glucose consumption level is reduced relative to the first glucose consumption level.
  • a method of treating a subject for cancer comprising: (a) measuring a first glucose consumption level in a tumor of the subject; (b) administering to the subject a kinase inhibitor; (c) subsequent to (b), measuring a second glucose consumption level in the tumor; and (d) administering to the subject an alternative cancer therapy, wherein the second glucose consumption level is not reduced relative to the first glucose consumption level, wherein the alternative cancer therapy does not comprise the kinase inhibitor.
  • the second glucose consumption level was measured at most 48 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments of the methods disclosed herein, the second glucose consumption level was measured at most 36 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments, the second glucose consumption level was measured at most 24 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments, the second glucose consumption level was measured at most 18 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor.
  • the second glucose consumption level was measured at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours (or any range or value derivable therein) after administering the previous dose, the dose, or the first dose of the kinase inhibitor.
  • measuring the glucose consumption level comprises positron emission tomography (PET).
  • PET positron emission tomography
  • the first glucose consumption level and the second glucose consumption level were measured using PET.
  • measuring the first glucose consumption level comprises PET.
  • measuring the second glucose consumption level comprises PET.
  • measuring the glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • measuring the first glucose consumption level and the second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • measuring the first glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • measuring the second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • the kinase inhibitor is a FLT3 inhibitor.
  • the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
  • the kinase inhibitor is afatinib.
  • the kinase inhibitor is buparlisib.
  • the kinase inhibitor is cabozantinib.
  • the kinase inhibitor is ceritinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the kinase inhibitor is dovitinib. In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is pacritinib. In some embodiments, the kinase inhibitor is ponatinib. In some embodiments, the kinase inhibitor is trametinib. In some embodiments, the kinase inhibitor is vemurafenib. In some embodiments, the kinase inhibitor is quizartinib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is TCS 359.
  • the second or additional dose of the kinase inhibitor when the second glucose consumption level is reduced compared to or relative to the first or reference glucose consumption level, is the same amount as the previous or first dose of the kinase inhibitor. In some embodiments, the second or additional dose of the kinase inhibitor is a lower amount than the previous or first dose of the kinase inhibitor.
  • the cancer therapy or alternative cancer therapy is an alternative kinase inhibitor.
  • the cancer therapy or alternative cancer therapy is chemotherapy, radiotherapy, or immunotherapy.
  • the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is squamous cell lung carcinoma.
  • “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the results of treatment can be determined by methods known in the art, such as determination of reduction of pain as measured by reduction of requirement for administration of opiates or other pain medication, determination of reduction of tumor burden, determination of restoration of function, or other methods known in the art.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristics of the disclosure.
  • any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
  • FIG. 1 illustrates some methods for studying glucose consumption by cells.
  • FIG. 2 illustrates a luminescence-based method for measuring cellular glucose consumption.
  • FIG. 3 illustrates a high-throughput method for measuring cellular glucose consumption.
  • FIG. 4 provides an overview of lung cancer cell lines and small molecule libraries screened for new regulators of glucose consumption by cells using the high throughput method illustrated in FIG. 3.
  • FIGs. 5A-5C show glucose consumption in A549 (FIG. 5A), H460 (FIG. 5B) and HCC827 (FIG. 5C) lung cancer cells after treatment with various kinase inhibitors.
  • FIGs. 6A-6B show glucose consumption of H460 (FIG. 6A) and HCC827 (FIG. 6B) lung cancer cells after treatment with kinase inhibitors.
  • FIG. 7 provides the structure of milciclib and IC50 values for inhibition of CDK and TRK proteins by milciclib.
  • FIGs. 8A-8C show glucose consumption by H460 lung cancer cells in culture and in vivo after treatment with milciclib.
  • FIG. 8A Normalized glucose consumption by H460 cells as a function of milciclib concentration.
  • FIG. 8B Pre- and post-treatment 18 F-FDG PET imaging of glucose consumption by H460 tumors in mice treated with either vehicle (top) or milciclib (bottom).
  • FIG. 8C Quantification of milciclib-induced changes in glucose consumption in H460 cells (left panel) and the brain, heart, liver, and muscle of mice (right panel) pre- and post-treatment with vehicle or milciclib.
  • FIGs. 9A-9B show GLUT1, GLUT3, Hexokinase 1, and Hexokinase 2 mRNA (FIG. 9A) and protein (FIG. 9B) levels in H460 lung cancer cells after milciclib treatment compared to vehicle treatment.
  • FIG. 10 illustrates a method of assessing glucose transport and hexokinase activity by H460 lung cancer cells using a fluorescent nanosensor.
  • FIGs. 11A-11B show glucose transport and hexokinase activity in H460 lung cancer cells as measured using the fluorescent nanosensor illustrated in FIG. 10.
  • FIG. 11A Change in fluorescence after vehicle treatment (left) or milciclib treatment (right).
  • FIG. 11B Quantification of the results in FIG. 11A.
  • the graph on the left shows that milciclib treatment does not affect glucose phosphorylation, which is measured by the fluorescent nanosensor when the cells are treated with glucose and Cytochalasin B.
  • the graph on the right shows that milciclib treatment decreases glucose transport, which is measured by the fluorescent nanosensor when the cells are treated with glucose only and when glucose phosphorylation activity is unaffected.
  • FIGs. 11A Change in fluorescence after vehicle treatment (left) or milciclib treatment (right).
  • FIG. 11B Quantification of the results in FIG. 11A.
  • the graph on the left shows that milciclib treatment does not affect glucose phosphorylation, which is measured by the fluorescent nanosensor when the cells are treated with glucose and
  • FIG. 12A-12B show glucose consumption by H460 lung cancer cells overexpressing CDK7 before and after treatment with milciclib.
  • FIG. 12A Glucose consumption in H460 cells overexpressing CDK7.
  • FIG. 12B Glucose consumption in H460 cells overexpressing CDK7 after treatment with milciclib.
  • FIG. 13A-13B show glucose consumption by lung cancer cell lines and following overexpression of CDK7 before and after treatment with CDK7 inhibitors THZ1 (FIG. 13A) and LDC4297 (FIG. 13B).
  • FIGs. 14A-14B show glucose consumption by H460 and HCC827 overexpressing PTEN or PIK3CA.
  • FIG. 14A Phosphorylated CDK7, CDK7, and GLUT1 protein levels in H460 and HCC827 overexpressing PTEN or PIK3CA was measured by Western blot.
  • FIG. 14B Glucose consumption by H460 and HCC827 overexpressing PTEN or PIK3CA after treatment with milciclib.
  • FIGs. 15A-15C show glucose consumption and cell growth data for H460 lung cancer cells overexpressing GLUT1 after treatment with milciclib.
  • FIG. 15A GLUT1 protein levels in H460 cells was measured by Western blot.
  • FIG. 15B Glucose consumption by H460 cells overexpressing GLUT1 up to 24 hours post- milciclib treatment.
  • FIG. 15C Growth of H460 cells overexpressing GLUT1 up to 48 hours post-milciclib treatment.
  • FIG. 16 provides an overview of lung cancer cell lines that model the genetics of human lung cancer and small molecule inhibitors that can target many of the oncogenic drivers of lung cancer.
  • FIG. 17 provides an overview of the experimentally-tested lung cancer cell lines and small molecule inhibitors for which glucose consumption was measured 24 hours posttreatment and cell growth was measured 72 hours post-treatment.
  • FIG. 18 shows glucose consumption by nine different lung cancer cell lines 24 hours after treatment with afatinib.
  • FIG. 19 shows glucose consumption by nine different lung cancer cell lines 24 hours after treatment with erlotinib.
  • FIGs. 20A-20B show glucose consumption by nine different lung cancer cell lines 24 hours after treatment with buparlisib.
  • FIG. 20B shows the levels of phosphorylated AKT and total AKT in A549 lung cancer cells (top) and the ratio of phosphorylated AKT to total AKT in six different lung cancer cell lines after treatment with buparlisib (bottom).
  • FIG. 21 shows growth of nine different cancer cell lines 72 hours after treatment with afatinib.
  • FIG. 22 shows growth of nine different cancer cell lines 72 hours after treatment with erlotinib.
  • FIG. 23 shows a receiver operating characteristic (ROC) curve plotting the sensitivity and specificity of using early inhibition of glucose consumption as a predictor of the inhibition of cell growth (defined as >40% growth inhibition) as a function of decrease in glucose consumption. The results show that early inhibition of glucose consumption is a strong predictor of drug therapeutic efficacy.
  • ROC receiver operating characteristic
  • FIG. 24 shows glucose consumption by and growth of H460 (left), A549 (middle), and H2229 (right) cancer cells after treatment with dovitinib.
  • FIGs. 25A-25C show in vivo glucose consumption by and growth of PC9 lung cancer cells treated with dovitinib or erlotinib.
  • FIG. 25A Pre- and post-treatment 18 F-FDG PET imaging of glucose consumption by PC9 tumors in mice treated with vehicle (top), dovitinib (middle), or erlotinib (bottom).
  • FIG. 25B Quantification of drug-induced changes in glucose consumption by PC9 tumors in mice.
  • FIG. 25C Quantification of drug-induced changes in PC9 tumor size for up to 12 days of treatment with kinase inhibitors.
  • FIGs. 26A-26D show glucose consumption and cell growth data for lung cancer cell lines overexpressing GLUT1 after treatment with kinase inhibitors.
  • H460 lung cancer cells were treated with dovitinib (FIG. 26A) or buparlisib (FIG. 26B) and HCC827 lung cancer cells were treated with buparlisib (FIB. 26C) or trametinib (FIG. 26D).
  • FIGs. 31A-31G show glucose consumption by nine different lung cancer cell lines 24 hours after treatment with cabozatinib (FIG. 31A), ceritinib (FIG. 31B), crizotinib (FIG. 31C), dovitinib (FIG. 31D), ponatinib (FIG. 31E), trametinib (FIG. 31F), and vemurafenib (FIG. 31G).
  • the present disclosure is based, at least in part, on the surprising discovery that certain kinase inhibitors, including afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, and vemurafenib, decrease glucose consumption within 24 hours of drug treatment specifically in cancer cells for which the drug has a longterm therapeutic effect.
  • a decrease in glucose consumption in a tumor indicates that that the kinase inhibitor has pharmacodynamically engaged its target in the tumor and is having a biological effect on the tumor, which suggests that the treatment regimen with the kinase inhibitor should be continued.
  • a subject for cancer comprising measuring a first glucose consumption level in a tumor of the subject before administration of a dose of a kinase inhibitor; administering the kinase inhibitor dose to the subject; measuring a second glucose consumption level in the tumor after administration of the kinase inhibitor dose; and continuing or modifying administration of the therapy based on the second glucose consumption level relative to the first glucose consumption level.
  • the second glucose consumption level is reduced relative to the first glucose consumption level, and administration of the kinase inhibitor is continued.
  • the second glucose level is unchanged or is not reduced relative to the first glucose consumption level, and administration of the therapy is modified.
  • modification of the therapy comprises an increase in the dose of the kinase inhibitor.
  • modification of the cancer therapy comprises administration of an alternative cancer therapy different from the kinase inhibitor previously administered.
  • Certain aspects of the present disclosure are related to measurement of glucose consumption by cells using 18 F-FDG PET. Cancer treatment and outcome depend largely on the accurate diagnosis and staging of disease. There is extensive data in the literature indicating the importance of 18 F-FDG PET imaging in accurately characterizing disease, as well as determining stage and sites of recurrent disease in many cancer types. For these indications, functional imaging with PET provides unique information which is not available from standard medical imaging modalities such as ultrasound, X-ray, computerized tomography (CT) or magnetic resonance imaging (MRI).
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • measuring glucose consumption levels comprises positron emission tomography (PET).
  • PET positron emission tomography
  • a first glucose consumption level and a second glucose consumption level are measured using PET.
  • measuring the first glucose consumption level comprises PET.
  • measuring the second glucose consumption level comprises PET.
  • a glucose consumption level, as measured herein is a signal level from a PET measurement.
  • measuring glucose consumption levels comprises administering fluorodeoxyglucose to the subject.
  • measuring a first glucose consumption level and a second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • measuring the first glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • measuring the second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
  • Positron emission tomography is a nuclear medicine procedure based on the measurement of positron emission from radiolabelled tracer molecules. These radiotracers allow biologic processes to be measured and whole body images to be obtained, which demonstrates sites of radiotracer accumulation.
  • a common radiotracer in use today is fluorodeoxyglucose, also known as 18 F, fluorodeoxyglucose Fl 8, 18F-fluorodeoxyglucose, [ 18 F]FDG, 18 F-FDG, or FDG), a radiolabelled sugar (glucose) molecule.
  • 18 F-FDG is 2-deoxy-2-[ 18 F]fluoro-D-glucose.
  • F-FDG is a glucose analog and is formed by replacing one of the 2-hydrogens of 2-deoxy-D-glucose (2-DG) with the positron-emitting isotope fluorine- 18, which emits paired gamma rays, allowing distribution of the tracer to be imaged by external gamma camera(s). More specifically, the positron-emitting radionuclide fluorine- 18 is substituted for the normal hydroxyl group at the C-2 position of a glucose molecule.
  • 18 F-FDG as a glucose analog, is taken up by high-glucose-using cells such as brain, brown adipocytes, kidney, and cancer cells, where phosphorylation prevents the glucose from being released again from the cell once it has been taken up by the cell.
  • the 2-hydroxyl group (-OH) in normal glucose is needed for further glycolysis, or glucose metabolism, but 18 F-FDG is missing this 2-hydroxyl.
  • 18 F-FDG cannot be further metabolized in cells.
  • the [ 18 F]FDG-6-phosphate formed when 18 F-FDG enters the cell thus cannot move out of the cell before radioactive decay.
  • the distribution of 18 F-FDG is a good reflection of the distribution of glucose uptake and metabolism by cells in the body.
  • Another fraction of 18 F-FDG representing about 20% of the total fluorine- 18 activity of an injection, is eliminated renally by two hours after a dose of 18 F-FDG, with a rapid half-life of about 16 minutes.
  • This portion makes the renal-collecting system and bladder prominent in a normal PET scan and indicates that the 20% portion of the total fluorine- 18 tracer activity is eliminated pharmacokinetic ally much more quickly than the isotope itself can decay.
  • All radioactivity of 18 F-FDG both the 20% portion which is rapidly excreted in urine in the first several hours after administration of the 18 F-FDG and the 80% portion which remains in the patient, decays with a half-life of 110 minutes.
  • the uptake of 18 F-FDG by tissues is a marker for the tissue uptake of glucose, which in turn is closely correlated with certain types of tissue metabolism.
  • 18 F-FDG is taken up by cells, phosphorylated by hexokinase, and retained by tissues with high metabolic activity, such as most types of malignant tumors.
  • imaging with 18 F-FDG PET is used to determine sites of abnormal glucose metabolism and can be used to characterize and localize many types of tumors and/or to diagnose, stage, and monitor treatment of cancers.
  • a dose of 18 F-FDG in solution (for example, 5 to 10 millicuries or 200 to 400 MBq) is injected rapidly into a saline drip running into a vein of a subject who has been fasting for at least six hours and who has a suitably low blood sugar.
  • the subject then waits about an hour for the sugar to distribute and be taken up into organs which use glucose.
  • physical activity should be kept to a minimum in order to minimize uptake of the radioactive sugar into muscles, which can cause unwanted artifacts in the scan.
  • Tumor 18 F-FDG uptake is analyzed in terms of Standardized Uptake Value (SUV).
  • SUV Standardized Uptake Value
  • a protein kinase inhibitor is a type of enzyme inhibitor that blocks the action of one or more protein kinases.
  • Protein kinases are enzymes that chemically add the terminal y-phosphate group of adenosine triphosphate to a protein, in some cases modulating function of the protein and/or regulating biological processes involving the protein. More specifically, phosphorylation results in a functional change of the target protein (substrate) by regulating signaling pathways by amplification or cellular location, or by interactions with regulatory proteins.
  • Human cells have many different kinases, and they help control important functions, such as cell signaling, metabolism, division, and survival.
  • phosphate groups added by kinases are usually added to serine, threonine, or tyrosine amino acids on the protein. Most kinases act on both serine and threonine, while tyrosine kinases act on tyrosine, and a number of kinases act on all three. There are also protein kinases that phosphorylate other amino acids, including histidine kinases that phosphorylate histidine residues.
  • the kinase inhibitor is imatinib, buparlisib, dovitinib, pacritinib, nilotinib, dasatinib, bosutinib, ponatinib, gefitinib, gilteritinib, quizartinib, crenolanib, erlotinib, afatinib, osimertinib, lapatinib, neratinib, sorafenib, sunitinib, pazopanib, axitinib, lenvatinib, cabozatinib, vandetanib, regorafenib, vemurafenib, dabrafenib, trametinib, cobimetinib, crizotinib, certinib, alectinib, brigatinib, lorlatinib, crizotinib, certin
  • kinase inhibitors inhibit Bcr- Abl tyrosine kinases, Epidermal Growth Factor receptor tyrosine kinases, Vascular Endothelial Growth Factor Receptor (VEGFR) tyrosine kinases, BRAF kinases, mitogen-activated protein kinases, anaplastic lymphoma kinase, Src family kinases like Bruton tyrosine kinase, Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FET3), Janus family kinases like JAK 2, lipid kinases like phosphoinositide 3-kinase, and/or cyclin dependent kinases, for example.
  • FMS Feline McDonough Sarcoma
  • FET3 Feline McDonough Sarcoma
  • JAK 2 lipid kinases like phosphoinositide 3-kinase
  • the kinase inhibitor is a FET3 inhibitor.
  • the FET3 inhibitor is midostaurin, gilteritinib, quizartinib (AC220), crenolanib, sorafenib, sunitinib, cabozatinib (XE184), or TCS 359.
  • FET3 and various examples of FET3 inhibitors are discussed in, for example, Gebru MT, Wang HG. “Therapeutic targeting of FET3 and associated drug resistance in acute myeloid leukemia,” J Hematol Oncol. 2020 Nov 19; 13(1): 155., incorporated by reference herein in its entirety.
  • the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
  • the kinase inhibitor is afatinib.
  • the kinase inhibitor is buparlisib.
  • the kinase inhibitor is cabozantinib.
  • the kinase inhibitor is ceritinib.
  • the kinase inhibitor is crizotinib. In some embodiments, the kinase inhibitor is dovitinib. In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is pacritinib. In some embodiments, the kinase inhibitor is ponatinib. In some embodiments, the kinase inhibitor is trametinib. In some embodiments, the kinase inhibitor is vemurafenib. In some embodiments, the kinase inhibitor is quizartinib. In some embodiments, the kinase inhibitor is cabozatinib. In some embodiments, the kinase inhibitor is TCS 359.
  • kinase inhibitors can decrease glucose consumption within 24 hours of treatment with the inhibitors specifically in cancer cells for which the inhibitors have a long-term therapeutic effect.
  • a decrease in glucose consumption in a tumor may indicate that that the kinase inhibitor has pharmacodynamically engaged its target in the tumor and is having a biological effect on the tumor. This suggests that the current treatment regimen with the kinase inhibitor should be continued.
  • no change or an increase in glucose consumption in a tumor indicates that the kinase inhibitor has not pharmacodynamically engaged its target or the target itself is not important for the tumor.
  • the kinase inhibitor treatment regimen should be altered by administering more of the inhibitor and/or an alternative cancer therapy, for example, a different kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy.
  • a dose of the kinase inhibitor is administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level.
  • the second glucose consumption level is measured at most 18 to 48 hours after administering the dose of the kinase inhibitor to the subject.
  • the second glucose consumption level is measured at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 48 hours, or any range or value derivable therein, after administering the dose of the kinase inhibitor to the subject.
  • the second glucose consumption level is measured at most 48 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 36 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 24 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 18 hours after administering the dose of the kinase inhibitor to the subject.
  • Administration of the kinase inhibitor may comprise administration of at least 1, 2, 3, 4, 5, or more kinase inhibitors. Any of these therapies may also be excluded. Combinations of these therapies may also be administered.
  • compositions and methods of administering therapeutically effective amounts one or more cancer therapies to a subject or patient in need thereof before and after measuring glucose consumption levels in a tumor of the subject may be used for in vivo, in vitro, or ex vivo administration.
  • the route of administration of the composition may be, for example, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, through inhalation, or through a combination of two or more routes of administration.
  • the cancer therapies may be administered via the same or different routes of administration.
  • cancer may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer.
  • the cancer may originate in the blood, bladder, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • the cancer is non-small cell lung cancer.
  • the cancer is squamous cell lung carcinoma.
  • the cancer therapy comprises a local cancer therapy. In some embodiments, the cancer therapy comprises a systemic cancer therapy. In some embodiments, the cancer therapy excludes a systemic cancer therapy. In some embodiments, the cancer therapy excludes a local cancer therapy.
  • a radiotherapy such as ionizing radiation
  • ionizing radiation means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons).
  • ionizing radiation is an x-radiation.
  • Means for delivering x-radiation to a target tissue or cell are well known in the art.
  • the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT).
  • the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy.
  • the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy.
  • the radiotherapy is administered to a primary tumor.
  • the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein).
  • the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein).
  • the does may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.
  • the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses.
  • the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each.
  • the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each.
  • the total dose of radiation is at least, at most, or about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
  • the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein).
  • At least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.
  • the cancer therapy comprises a cancer immunotherapy.
  • Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer.
  • Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor- associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates).
  • TAAs tumor- associated antigens
  • Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs.
  • Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.
  • Various immunotherapies are known in the art, and examples are described below.
  • the cancer immunotherapy comprises immune checkpoint inhibitors, examples of which are further described below.
  • checkpoint inhibitor therapy also “immune checkpoint blockade therapy”, “immune checkpoint therapy”, “ICT,” “checkpoint blockade immunotherapy,” or “CBI”
  • ICT immune checkpoint therapy
  • CBI checkpoint blockade immunotherapy
  • PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD- 1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity.
  • Alternative names for “PD-1” include CD279 and SLEB2.
  • Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H.
  • Alternative names for “PDL2” include B7- DC, Btdc, and CD273.
  • PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
  • the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PDL1 and/or PDL2.
  • a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners.
  • PDL1 binding partners are PD-1 and/or B7-1.
  • the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners.
  • a PDL2 binding partner is PD-1.
  • the inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference.
  • Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US 2014/022021, and US2011/0008369, all incorporated herein by reference.
  • the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab.
  • the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g. , an Fc region of an immunoglobulin sequence).
  • the PDL1 inhibitor comprises AMP- 224.
  • Nivolumab also known as MDX-1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/114335.
  • Pidilizumab also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009/101611.
  • AMP-224 also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
  • Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.
  • the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof.
  • the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies.
  • the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD152 cytotoxic T-lymphocyte-associated protein 4
  • the complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006.
  • CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells.
  • CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells.
  • CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells.
  • CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
  • Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
  • Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-CTLA-4 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used.
  • the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used.
  • a humanized CTLA-4 antibody is described in International Patent Application No. WO200 1/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
  • a further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01/14424).
  • the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab.
  • the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. c. LAG3
  • LAG3 lymphocyte-activation gene 3
  • CD223 lymphocyte activating 3
  • LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells.
  • LAG3 ’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function.
  • LAG3 also helps maintain CD8+ T cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection.
  • LAG3 is also known to be involved in the maturation and activation of dendritic cells.
  • Inhibitors of the disclosure may block one or more functions of LAG3 activity.
  • the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-LAG3 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-LAG3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-LAG3 antibodies can be used.
  • the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. d.
  • TIM-3 Another immune checkpoint that can be targeted in the methods provided herein is the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366.
  • HAVCR2 hepatitis A virus cellular receptor 2
  • TIM-3 is found on the surface IFNy- producing CD4+ Thl and CD8+ Tel cells.
  • the extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane.
  • TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates the T-cell exhaustion.
  • TIM-3 has also been shown as a CD4+ Thl -specific cell surface protein that regulates macrophage activation.
  • Inhibitors of the disclosure may block one or more functions of TIM- 3 activity.
  • the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-TIM-3 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e.g., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-TIM-3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-TIM-3 antibodies can be used.
  • anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein.
  • MBG453, TSR-022 also known as Cobolimab
  • LY3321367 can be used in the methods disclosed herein.
  • These and other anti-TIM-3 antibodies useful in the claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015/0218274, and US 2016/0200815.
  • the teachings of each of the aforementioned publications are hereby incorporated by reference.
  • Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.
  • the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. 2. Activation of co-stimulatory molecules
  • the cancer immunotherapy comprises an activator of a costimulatory molecule.
  • the activator comprises an agonist of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof.
  • Activators include activating antibodies, polypeptides, compounds, and nucleic acids.
  • the cancer immunotherapy comprises dendritic cell therapy.
  • Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen.
  • Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting.
  • APCs antigen presenting cells
  • cellular cancer therapy based on dendritic cells is sipuleucel-T.
  • One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses.
  • adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).
  • Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.
  • Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body.
  • the dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.
  • Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets. 4. CAR-T cell therapy
  • the cancer immunotherapy comprises chimeric immunoreceptors.
  • Chimeric antigen receptors CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors
  • CARs are engineered receptors that combine a new specificity with an immune cell to target cancer cells.
  • these receptors graft the specificity of a monoclonal antibody onto a T cell.
  • the receptors are called chimeric because they are fused of parts from different sources.
  • CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.
  • CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions.
  • the general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells.
  • scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells.
  • CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells.
  • the extracellular ligand recognition domain is usually a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • Example CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).
  • the cancer immunotherapy comprises cytokine therapy.
  • Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.
  • Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNk).
  • Interleukins have an array of immune system effects.
  • IE-2 is an example interleukin cytokine therapy. 6.
  • the cancer immunotherapy comprises adoptive T-cell therapy.
  • Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell’s surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.
  • APCs antigen presenting cells
  • T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.
  • TILs tumor sample
  • Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.
  • a cancer treatment may exclude any of the cancer treatments described herein.
  • embodiments of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein.
  • the patient is one that has been determined to be resistant to a therapy described herein.
  • the patient is one that has been determined to be sensitive to a therapy described herein.
  • the cancer therapy comprises an oncolytic virus.
  • An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy D. Polysaccharides
  • the cancer therapy comprises polysaccharides.
  • Certain compounds found in mushrooms primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties.
  • beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.
  • the cancer therapy comprises neoantigen administration.
  • Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy.
  • the presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden.
  • the level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.
  • the cancer therapy comprises a chemotherapy.
  • chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and
  • nitrogen mustards e.g.
  • Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg/m2 to about 20 mg/m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments.
  • the amount of cisplatin delivered to the cell and/or subject in conjunction with the construct comprising an Egr-1 promoter operatively linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.
  • chemotherapeutic agents include antimicro tubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”).
  • Paclitaxel e.g., Paclitaxel
  • doxorubicin hydrochloride doxorubicin hydrochloride
  • Doxorubicin is absorbed poorly and is preferably administered intravenously.
  • appropriate intravenous doses for an adult include about 60 mg/m2 to about 75 mg/m2 at about 21 -day intervals or about 25 mg/m2 to about 30 mg/m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg/m2 once a week.
  • the lowest dose should be used in elderly patients, when there is prior bone- marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
  • Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure.
  • a nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and/or ifosfamide, melphalan (E-sarcolysin), and chlorambucil.
  • Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent.
  • Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day
  • intravenous doses include, for example, initially about 40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day.
  • the intravenous route is preferred.
  • the drug also sometimes is administered intramuscularly, by infiltration or into body cavities.
  • chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR).
  • 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg/m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.
  • Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.
  • the amount of the chemotherapeutic agent delivered to the patient may be variable.
  • the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct.
  • the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent.
  • the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent.
  • chemotherapeutic s of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages.
  • suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc.
  • In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.
  • the cancer therapy comprises surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs ’ surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment, for example.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti- hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
  • aspects of the present disclosure are directed to methods comprising treatment of a subject suffering from, or suspected of having, cancer.
  • the cancer is lung cancer.
  • the cancer is non-small cell lung cancer.
  • the cancer is squamous cell lung carcinoma.
  • the disclosed methods comprise treating a subject suffering from a cancer with a kinase inhibitor.
  • the kinase inhibitor is a FLT3 inhibitor.
  • the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
  • the kinase inhibitor is afatinib.
  • the kinase inhibitor is buparlisib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is ceritinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the kinase inhibitor is dovitinib. In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is pacritinib. In some embodiments, the kinase inhibitor is ponatinib. In some embodiments, the kinase inhibitor is trametinib.
  • the kinase inhibitor is vemurafenib. In some embodiments, the kinase inhibitor is quizartinib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is TCS 359.
  • the kinase inhibitor is administered subsequent to measurement of a first or reference glucose consumption level in a tumor of the subject, and a second glucose consumption level is measured subsequent to administration of the kinase inhibitor.
  • changes in glucose consumption following administration of a cancer therapy for example, a kinase inhibitor
  • a cancer therapy for example, a kinase inhibitor
  • changes in glucose consumption following administration of a cancer therapy can be used to predict the long-term therapeutic effect of the therapy, with decreases in glucose consumption in a tumor indicating that the therapy has pharmacodynamically engaged its target in the tumor and is having a biological effect on the tumor, and no changes or increases in glucose consumption in a tumor indicating that the therapy has not pharmacodynamically engaged its target or the target itself is not important for the tumor.
  • Pharmacodynamic engagement of a kinase inhibitor target by the kinase inhibitor suggests that the treatment regimen with the kinase inhibitor should be continued, whereas lack of pharmacodynamic engagement of the kinase inhibitor target by the kinase inhibitor suggests that the treatment regimen should be altered by administering more of the kinase inhibitor and/or a different cancer therapy.
  • a subject for cancer comprising measuring a first glucose consumption level in a tumor of the subject before administration of a dose of a cancer therapy; administering the cancer therapy dose to the subject; measuring a second glucose consumption level in the tumor after administration of the cancer therapy dose; and continuing or modifying administration of the cancer therapy based on the second glucose consumption level relative to the first glucose consumption level.
  • the second glucose consumption level is reduced relative to the first glucose consumption level, and administration of the cancer therapy is continued.
  • the second glucose level is unchanged or is not reduced relative to the first glucose consumption level, and administration of the cancer therapy is modified.
  • modification of the cancer therapy comprises an increase in the dose of the cancer therapy.
  • modification of the cancer therapy comprises administration of an alternative cancer therapy different from the cancer therapy previously administered.
  • the cancer therapy comprises a kinase inhibitor.
  • the method comprises administering a dose of a kinase inhibitor to the subject.
  • the cancer therapy comprises a cancer therapy other than the kinase inhibitor, or an alternative cancer therapy.
  • the method comprises administering a cancer therapy other than the kinase inhibitor, or an alternative cancer therapy, to the subject.
  • a first glucose consumption level and a second glucose consumption level are measured in a tumor of the subject.
  • the dose of the kinase inhibitor is administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level. If the second glucose consumption level is reduced relative to the first glucose consumption level, then a second dose of the kinase inhibitor is administered to the subject.
  • the second dose of the kinase inhibitor can be the same as or a lower amount than the previous or first dose of the kinase inhibitor. If the second glucose consumption level is not reduced relative to the first glucose consumption level, then a cancer therapy other than the kinase inhibitor is administered to the subject.
  • the cancer therapy other than the kinase inhibitor, or alternative cancer therapy can be an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy.
  • a method for evaluating the efficacy of a kinase inhibitor in a subject having cancer comprising administering a dose of the kinase inhibitor to the subject and measuring a second glucose consumption level in the tumor at most 48 hours after administering the kinase inhibitor to the subject.
  • the method further comprises measuring a first or reference glucose consumption level in a tumor of the subject prior to administration of the dose of the kinase inhibitor to the subject.
  • the kinase inhibitor is identified as effective when the second glucose consumption level is reduced compared to the first or reference glucose consumption level, and the method further comprises administering an additional dose of the kinase inhibitor to the subject.
  • the additional dose of the kinase inhibitor can be the same as or a lower amount that the first or previous dose of the kinase inhibitor.
  • the kinase inhibitor is identified as ineffective when the second glucose consumption level is unchanged or not reduced compared to the first or reference glucose consumption level, and an alternative cancer therapy comprising an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy can be administered to the subject.
  • the disclosed methods comprise identifying one or more subjects as being candidates for treatment with a kinase inhibitor or an alternative cancer therapy other than the kinase inhibitor and/or identifying one or more subjects as subjects as having increased sensitivity to a kinase inhibitor or an alternative cancer therapy other than the kinase inhibitor.
  • a method of treating a subject for cancer comprising selecting a subject for treatment with a second dose of a kinase inhibitor when a second glucose consumption level measured in the subject after administration of a first dose of the kinase inhibitor is reduced relative to a first glucose consumption level measured in the subject before administration of a first dose of the kinase inhibitor.
  • the second dose of the kinase inhibitor can be the same as or a lower amount that the first dose of the kinase inhibitor.
  • a method of treating a subject for cancer comprising selecting a subject for treatment with an alternative cancer therapy when a second glucose consumption level measured in the subject after administration of a dose of a kinase inhibitor is unchanged or not reduced relative to a first glucose consumption level measured in the subject before administration of a dose of the kinase inhibitor.
  • the alternative cancer therapy can comprise an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy.
  • drug target engagement in tumors can be used as a pharmacodynamic biomarker to allow for stratification and treatment of cancer patients having increased sensitivity to drugs.
  • Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions.
  • the one or more cancer therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions.
  • the one or more cancer therapies are administered in a separate composition.
  • the one or more cancer therapies are in the same composition.
  • Various combinations of the agents may be employed.
  • compositions according to the present invention can be prepared according to standard techniques and may comprise water, buffered water, saline, glycine, dextrose, iso- osmotic sucrose solutions and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like.
  • the preparation of compositions that contains the cancer therapies will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
  • the cancer therapies of the disclosure may be administered by the same route of administration or by different routes of administration.
  • the cancer therapy is administered intraarterially, intravenously, intraperitoneally, subcutaneously, intramuscularly, intratumorally, topically, orally, transdermally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
  • the appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual ’s clinical history and response to the treatment, and the discretion of the attending physician.
  • the treatments may include various “unit doses.”
  • Unit dose is defined as containing a predetermined-quantity of the therapeutic composition.
  • the quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • a unit dose comprises a single administrable dose.
  • the therapy provided herein comprises administration of one or more cancer therapies.
  • the one or more cancer therapies comprise a kinase inhibitor.
  • the one or more therapies comprise an alternative cancer therapy to the kinase inhibitor, and the alternative cancer therapy can comprise an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy.
  • the one or more cancer therapies are administered sequentially, with the kinase inhibitor administered before the alternative cancer therapy.
  • the one or more cancer therapies are administered within 1 week, within 2 weeks, within 3 weeks, or within 1 month after administration of a first dose of a kinase inhibitor.
  • the one or more cancer therapies are administered within 1 week after administration of a first dose of a kinase inhibitor. In some embodiments, the one or more cancer therapies are administered within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, or within 6 days after administration of a first dose of a kinase inhibitor.
  • the one or more cancer therapies are administered within 48 hours, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 38 hours, 37 hours, 36 hours, 35 hours, 34 hours, 33 hours, 32 hours, 31 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour after administration of a first dose of a kinase inhibitor.
  • a single dose of the one or more cancer therapies are administered.
  • multiple doses of the one or more second cancer therapies are administered.
  • the method comprises administering multiple doses of the one or more cancer therapies, and the multiple doses are administered on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive days.
  • the method comprises administering multiple doses of the one or more cancer therapies, and the multiple doses are administered on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 non-consecutive days.
  • Administration of the multiple doses on consecutive or non-consecutive days can comprise a cycle, and the cycle may be repeated once a month for one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve consecutive or non-consecutive months, or once a year for one, two, three, four, or five consecutive or non-consecutive years.
  • the quantity to be administered depends on the treatment effect desired.
  • An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents.
  • doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein.
  • doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
  • the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM.
  • the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein).
  • the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent.
  • the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
  • dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein. Examples
  • FIG. 2 Methods for studying glucose consumption such as those illustrated by FIG. 1 are slow, labor intensive, expensive, and difficult to scale up.
  • luminescence-based methods were used for measuring cellular glucose consumption, illustrated by FIG. 2.
  • FIG. 3 these luminescence-based methods were employed by the inventors in a high-throughput method for measuring cellular glucose consumption by cells to quickly, easily, and cheaply identify potential regulators of glucose consumption by the cells.
  • the lung cancer cell lines and small molecule libraries screened using this high- throughput method are illustrated by FIG. 4.
  • Glucose consumption by the cells was measured by the inventors 24 hours postsmall molecule treatment. As shown in FIG. 5, several new regulators of glucose consumption were identified by the high-throughput screen of small molecule libraries in A549 (FIG. 5A), H460 (FIG. 5B), and HCC827 (FIG. 5C) lung cancer cell lines. The inventors also measured glucose consumption as a function of inhibitor concentration, as shown in FIG. 6, and found that the drugs that inhibit glucose consumption in H460 (FIG. 6A) and HCC827 (FIG. 6B) lung cancer cells do so across a range of concentrations.
  • Milciclib inhibits CDK2, CDK4, CDK7, and TRKA. It is also known to limit the growth of a wide range of xenograft models, including ovarian, colon, pancreatic, and prostate cancer.
  • FIG. 7 provides the structure of milciclib and IC50 values for inhibition of several CDK and TRK proteins by milciclib.
  • FIG. 8 shows that milciclib selectively inhibited glucose consumption by H460 lung cancer cells in culture and in vivo. Specifically, as the concentration of milciclib increased, glucose consumption by H460 cells decreased, as shown in FIG. 8A. The inventors also showed that milciclib treatment decreased glucose consumption in H460 tumors in mice. Pre- and post-treatment 18 F-FDG PET imaging of glucose consumption by H460 tumors in mice treated with either vehicle or milciclib was performed and quantified, as shown in FIG. 8B and FIG. 8C, respectively.
  • the inventors also determined changes in GLUT1 protein levels as a result of milciclib treatment. As shown in FIG. 9, milciclib treatment decreased both GLUT1 mRNA (FIG. 9A) and protein (FIG. 9B) levels in H460 lung cancer cells compared to vehicle treatment.
  • FIG. 11A shows a quantification of the results in FIG. 11A.
  • Milciclib has no effect on the rate of change of the fluorescent signal compared to vehicle in the cells treated with glucose and Cytochalasin B (left), demonstrating that Milciclib has no effect on glucose phosphorylation.
  • Milciclib decreases the rate of change of the fluorescent signal compared to vehicle in the cells treated with glucose alone (right). Combined with the results from the first graph on the left, this data demonstrates that Milciclib blocks glucose transport.
  • CDK7 is a CDK- activating kinase and part of the TFIIH transcription complex.
  • CDK7 overexpression increased GLUT1 protein levels and increased glucose consumption in H460 cells (FIG. 12A), and treatment of H460 cells overexpressing CDK7 with milciclib resulted in a smaller decrease in glucose consumption by the cells (FIG. 12B).
  • CDK7 inhibitors THZ1 (FIG. 13A) and LDC4297 FIG.
  • FIG. 14 shows glucose consumption by H460 and HCC827 overexpressing PTEN or PIK3CA.
  • Phosphorylated CDK7, CDK7, and GLUT1 protein levels in H460 and HCC827 overexpressing PTEN or PIK3CA were measured by Western blot (FIG. 14A), and treatment of H460 and HCC827 overexpressing PTEN or PIK3CA with milciclib resulted in a decrease in glucose consumption by the cells (FIG. 14B).
  • the inventors confirmed that all of the results related to CDK7 inhibitors and glucose consumption were reproducible in H1975 cells, a NSCLC line having a PIK3CA mutation.
  • the inventors also measured glucose consumption and cell growth data for H460 lung cancer cells overexpressing GLUT1 before and after treatment with milciclib, as shown in FIG. 15.
  • GLUT1 overexpression in H460 cells was confirmed by Western blot (FIG. 15A), and GLUT1 overexpression increased glucose consumption by H460 cells up to 24 hours post- milciclib treatment (FIG. 15B).
  • GLUT1 overexpression increased growth of H460cells up to 48 hours post-milciclib treatment (FIG. 15C).
  • the inventors identified: (1) a targetable protein that selectively activates glucose consumption in NSCLC; (2) the genetic driver of this effect; (3) an inhibitor of this protein that blocks glucose consumption sufficiently to limit cancer cell growth; and (4) a noninvasive clinical assay ( 18 F-FDG PET) to measure drug pharmacodynamics.
  • the inventors sought to use 18 F-FDG PET as predictive biomarker of the efficacy of a broad range of kinase inhibitors. Further, the inventors aimed to probe the link between inhibition of glucose consumption and inhibition of cell growth to determine whether the drug concentration necessary to block glucose consumption is also sufficient to block cancer cell growth. Finally, the inventors investigated the functional relevancy of glucose consumption inhibition and its contribution to cancer cell growth inhibition.
  • the inventors selected lung cancer as a model system based due in part to the number lung cancer cell lines that model the genetics of human lung cancer and the number of readily-available small molecule inhibitors that can target many of the oncogenic drivers of lung cancer. These lung cancer cell lines and small molecule inhibitors are described in FIG. 16, while FIG. 17 provides an overview of the experimentally-tested lung cancer cell lines and small molecule inhibitors. Glucose consumption was measured 24 hours post-treatment of the cell lines, and cell growth was measured 72 hours post-treatment of the cell lines.
  • the inventors measured glucose consumption by nine lung cancer cell lines after treatment with three different kinase inhibitors.
  • the shown lung cancer cell lines were treated with increasing concentrations of afatinib, and glucose consumption by the cell lines was measured 24 hours after treatment.
  • Glucose consumption was decreased in H1229, H1734, H1993, H3122, HCC827, and PC9 cells.
  • the shown lung cancer cell lines were treated with increasing concentrations of erlotinib, and glucose consumption by the cell lines was measured 24 hours after treatment. Glucose consumption was decreased in H1734, HCC827, and PC9 cells.
  • FIG. 19 the shown lung cancer cell lines were treated with increasing concentrations of erlotinib, and glucose consumption by the cell lines was measured 24 hours after treatment.
  • Glucose consumption was decreased in H1734, HCC827, and PC9 cells.
  • FIG. 20A the shown lung cancer cell lines were treated with increasing concentrations of buparlisib, and glucose consumption by the cell lines was measured 24 hours after treatment. Glucose consumption was decreased in H1734, H1993, H3122, H460, HCC827, and PC9 cells.
  • FIG. 20B shows that treatment with increasing concentrations of buparlisib resulted in decreased phosphorylation of AKT in six different lung cancer cell lines.
  • the shown lung cancer cell lines were treated with increasing concentrations of cabozantinib (FIG. 31A), ceritinib (FIG. 31B), crizotinib (FIG. 31C), dovitinib (FIG.
  • Glucose consumption was decreased in H1734, H1993, and H3122 cells in response to cabozantinib (FIG. 31A).
  • Glucose consumption was decreased in H1229, H1993, H2228, H3122, HCC827, and PC9 cells in response to ceritinib (FIG. 31B).
  • Glucose consumption was decreased in H1229, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells in response to crizotinib (FIG. 31C).
  • Glucose consumption was decreased in H1229, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells in response to dovitinib (FIG. 31D).
  • Glucose consumption was decreased in A549, H1229, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells in response to ponatinib (FIG. 31E).
  • Glucose consumption was decreased in H1229, H1734, H1993, H3122, H460, HCC827, and PC9 cells in response to trametinib (FIG. 31F).
  • Glucose consumption was decreased in A549, H1734, H3122, H460, HCC827, and PC9 cells in response to vemurafenib (FIG. 31G). These experiments demonstrate that kinases are strong drivers of glucose consumption.
  • the inventors measured the growth of the nine lung cancer cell lines after treatment with kinase inhibitors.
  • the lung cancer cell lines were treated with increasing concentrations of afatinib, and cell growth was measured 72 hours after treatment. Cell growth was decreased for A549, H1229, H1734, HCC827, and PC9 cells.
  • the lung cancer cell lines were treated with increasing concentrations of erlotinib, and cell growth was measured 48 hours after treatment. Cell growth was decreased for HCC1745, HCC827 and PC9 cells.
  • FIG. 24 shows that glucose consumption by and growth of H460 cancer cells are decreased after treatment with dovitinib.
  • FIGs. 25A-25C show that in vivo glucose consumption by and growth of PC9 lung cancer cells are decreased after treatment with dovitinib or erlotinib.
  • pre- and post-treatment 18 F- FDG PET imaging of glucose consumption by PC9 tumors in mice treated with vehicle (top), dovitinib (middle), or erlotinib (bottom) showed significant drug-induced decreases in glucose consumption by PC9 tumors in mice (FIG. 25B).
  • FIG. 25C quantification of drug-induced changes in PC9 tumor size after up to 12 days of treatment with kinase inhibitors revealed a decrease in PC9 tumor size.
  • the inventors established that early decreases in cancer cell glucose consumption in response to a kinase inhibitor are a strong predictor of eventual changes in cell growth across kinase inhibitors, drug concentrations, and lung cancer genetics in culture and in vivo. Further, early decreases in cancer cell glucose consumption in response to a kinase inhibitor is necessary for the inhibitor to inhibit cancer cell growth. These studies provide a strong rationale for use of early 18 F-FDG PET scans to predict therapeutic efficacy.
  • Pacritinib reduces glucose consumption in a subset of lung squamous cell carcinoma by inhibiting the expression of Hexokinase I and II
  • Squamous cells carcinoma represents 30% of all cases of non-small cell lung cancer (NSCLC) and it is associated with smoking.
  • NSCLC non-small cell lung cancer
  • the inventors tested 3555 bioactive small molecules from four small molecule libraries against three squamous cell lung carcinoma cell lines (H520, SK-MES-1, and H596 cells) to identify compounds that decrease glucose consumption per cell.
  • This screen yielded a list of novel inhibitors of glucose consumption in squamous cell lung carcinoma cell lines that includes clinically relevant kinase inhibitors (z.e., pacritinib and dovitinib) and topoisomerase inhibitors (z.e., camptothecin and idarubicin). From this list, the inventors prioritized compounds (1) that decreased glucose consumption in all three cell lines with an EC50 value ⁇ 1 pM; (2) that had been used in mice and humans; (3) that had not been previously linked to glucose consumption; and (4) that did not dramatically decrease cell growth at the time point measured (24 hours after treatment). This led the inventors to further investigate pacritinib, a Janus kinase 2 (JAK2) and Fms Related Receptor Tyrosine Kinase 3 (FLT3) inhibitor used for the treatment of myelofibrosis.
  • pacritinib a Janus kinase 2 (JAK2) and Fms Related Receptor Tyrosine Kina
  • FIGs. 27A and 27B pacritinib reduced glucose consumption (FIG. 27A) in all three SqCLC cell with an EC 50 of -1.2 pM and without significantly altering cell growth at 24h post-treatment (FIG. 27B).
  • This effect required at least a 16h incubation time, suggesting that the decrease in glucose consumption is likely due to alterations in transcription or translation and not due to the direct inhibition of proteins involved in glucose consumption (/'. ⁇ ?., glucose transporters GLUT1 and GLUT3, hexokinases HKI and HKII).
  • FIGs. 28A and 28B Western blot and Q-PCR analyses show that pacritinib reduced the mRNA (FIG. 28A) and protein (FIG. 28B) levels of HKI and HKII without affecting expression levels of GLUT1 and GLUT3 in all three SqCLC cell lines, suggesting that pacritinib blocks glucose consumption by decreasing HKI and HKII levels.
  • Pacritinib is a well-known inhibitor of JAK2 and FLT3, so the inventors investigated whether the inhibition of one of the known targets results in a decrease of HKI and HKII levels and therefore a decrease in glucose consumption.
  • the inventors overexpressed JAK2 and FLT3 in SK-MES-1, H520 and H596 and measured the effect of 24h incubation with pacritinib.
  • the rationale for this experiment was that only the overexpression of the target protein should reduce the ability of pacritinib to affect glucose consumption.
  • Overexpression of FLT3 dramatically decreased the ability of pacritinib to alter glucose consumption (FIG. 29), whereas overexpression of JAK2 did not have any effect (data not shown). Consistent with the role of FLT3 in regulating Hexokinase I and II expression, overexpression of FLT3 resulted in an increase in the protein levels of the hexokinases and a consequent increase in glucose consumption.
  • pacritinib regulates HKI and HK2 via FLT3
  • the inventors tested the effect of other inhibitors that have a Ki for FLT3 similar to or lower than pacritinib: Quizartinib, Dovitinib, Cabozantinib, and FLT3 inhibitor.
  • FIGs. 30A-30C all inhibitors tested reduced glucose consumption in SK-MES-1, H20 and H596 similar to pacritinib, suggesting that glucose consumption is regulated via FLT3.

Abstract

Aspects of the present disclosure are directed to methods for treating a subject having cancer. Certain aspects relate to administration of a cancer therapy after observing a change in glucose consumption due to administration of the same or a different cancer therapy. Further aspects relate to methods for evaluating the efficacy of a cancer therapy, such as a kinase inhibitor, by administering to a subject a therapeutically effective amount of the cancer therapy and measuring resulting changes in glucose consumption.

Description

METHODS AND SYSTEMS FOR ANALYSIS OF DRUG TARGET ENGAGEMENT
AND TREATMENT OF CANCER
[0001] This application claims benefit of priority of U.S. Provisional Patent Application No. 63/137,360, filed January 14, 2021, which is hereby incorporated by reference in its entirety.
BACKGROUND
I. Field of the Disclosure
[0002] Aspects of this disclosure relate, generally, to at least the fields of cancer biology and medicine and, more specifically, to methods of measuring biomarkers of drug target engagement in cancer.
II. Background
[0003] Metabolic reprogramming is an emerging hallmark of cancer, and most aggressive human cancers present with elevated glucose consumption relative to surrounding tissue. 18F- FDG PET measures this elevated glucose consumption and has become an important tool in the care of cancer patients.
[0004] There exists a need in the art for development of noninvasive methods of determining drug target engagement in tumors (e.g., 18F-FDG PET) to measure pharmacodynamic biomarkers in cancer (e.g., glucose consumption) to allow for stratification and treatment of cancer patients having increased sensitivity to drugs.
SUMMARY
[0005] Aspects of the present disclosure address needs in the art by providing methods for treating subjects with cancer (e.g., lung cancer) and methods for evaluating the efficacy of a kinase inhibitor by comparing glucose consumption levels in a tumor of the subject before and after treatment with the cancer therapy. Accordingly, provided herein, in some aspects, are methods for treating a subject with cancer comprising measuring a first glucose consumption level in a tumor of the subject before administration of a dose of a kinase inhibitor; administering the kinase inhibitor dose to the subject; measuring a second glucose consumption level in the tumor after administration of the kinase inhibitor dose; and continuing or modifying the therapy based on the second glucose consumption level relative to the first glucose consumption level. In some embodiments, the second glucose consumption level is reduced relative to the first glucose consumption level, and administration of the kinase inhibitor is continued. In some embodiments, the second glucose level is unchanged or is not reduced relative to the first glucose consumption level, and administration of the therapy is modified. In some embodiments, modification of the therapy comprises an increase in the dose of the cancer therapy. In some embodiments, modification of the therapy comprises administration of an alternative cancer therapy different from the kinase inhibitor previously administered. In some embodiments, the kinase inhibitor is a Fms Related Receptor Tyrosine Kinase 3 (FLT3) inhibitor. In some embodiments, the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
[0006] Embodiments of the disclosure include methods for treating a subject having cancer, methods for evaluating the efficacy of a cancer therapy used to treat a subject having cancer, methods for predicting a subject ’s response to a cancer therapy, methods for identifying a subject with cancer as a candidate for a cancer therapy, and methods and compositions for treating a subject having lung cancer. Methods of the disclosure can include 1, 2, 3, 4, 5, 6, or more of the following steps: measuring a first glucose consumption level in a subject, providing a first dose of a cancer therapy to a subject, measuring a second glucose consumption level in a subject, comparing a first glucose consumption level measured in a subject to a second glucose consumption level measured in the subject, providing a second dose of a cancer therapy to a subject, providing three or more doses of a cancer therapy a subject, providing a combination cancer therapy to a subject, providing an alternative therapy to a subject, determining a subject to have cancer, providing two or more types of cancer therapy to a subject, identifying one or more cancer therapy as being in need of evaluation of efficacy, evaluating the efficacy of a cancer therapy, identifying a subject as being a candidate for a cancer therapy, and predicting a subject’s response to a cancer therapy. Certain embodiments of the disclosure may exclude one or more of the preceding elements and/or steps.
[0007] Disclosed herein, in some aspects, is a method of treating a subject for cancer comprising administering a dose of a kinase inhibitor to the subject, wherein a first glucose consumption level and a second glucose consumption level were measured in a tumor of the subject, wherein a previous dose of the kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was reduced relative to the first glucose consumption level.
[0008] Disclosed herein, in some aspects, is a method of treating a subject for cancer comprising administering a cancer therapy to the subject, wherein a first glucose consumption level and a second glucose consumption level were measured in a tumor of the subject, wherein a dose of a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, wherein the second glucose consumption level was not reduced relative to the first glucose consumption level, and wherein the cancer therapy does not comprise the kinase inhibitor.
[0009] Disclosed herein, in some aspects, is a method for evaluating efficacy of a kinase inhibitor in a subject having cancer, the method comprising: (a) measuring a first glucose consumption level in a tumor of the subject; (b) administering a dose of the kinase inhibitor to the subject; and (c) measuring a second glucose consumption level in the tumor at most 48 hours after administering the kinase inhibitor to the subject. In some embodiments, the method further comprises identifying the kinase inhibitor as effective, wherein the second glucose consumption level is reduced compared to the first glucose consumption level. In some embodiments, the method further comprises (d) administering an additional dose of the kinase inhibitor to the subject, wherein the second glucose consumption level is reduced compared to the first glucose consumption level. In some embodiments, the method further comprises identifying the kinase inhibitor as ineffective, wherein the second glucose consumption level is not reduced compared to the first glucose consumption level. In some embodiments, the method further comprises (d) administering an alternative cancer therapy, wherein the second glucose consumption level is not reduced compared to the first glucose consumption level.
[0010] Disclosed herein, in some aspects, is a method for evaluating efficacy of a kinase inhibitor in a subject having cancer, the method comprising: (a) administering the kinase inhibitor to the subject; and (b) measuring a glucose consumption level in a tumor of the subject at most 48 hours after administering the kinase inhibitor. In some embodiments, a reference glucose consumption level was measured in the tumor prior to administering the kinase inhibitor to the subject. In some embodiments, the method further comprises administering an additional dose of the kinase inhibitor to the subject, wherein the glucose consumption level is reduced relative to the reference glucose consumption level. In some embodiments, the method further comprises administering an alternative cancer therapy to the subject, wherein the glucose consumption level is not reduced relative to the reference glucose consumption level. [0011] Disclosed herein, in some aspects, is a method of treating a subject for cancer comprising: (a) selecting a subject for treatment, wherein a first glucose consumption level and a second glucose consumption level were measured in the subject, wherein a first dose of a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was reduced relative to the first glucose consumption level; and (b) administering to the subject a second dose of the kinase inhibitor.
[0012] Disclosed herein, in some aspects, is a method of treating a subject for cancer comprising: (a) selecting a subject for treatment, wherein a first glucose consumption level and a second glucose consumption level were measured in the subject, wherein a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was not reduced compared to the first glucose consumption level; and (b) administering an alternative cancer therapy to the subject, wherein the alternative cancer therapy does not comprise the kinase inhibitor.
[0013] Disclosed herein, in some aspects, is a method of treating a subject for cancer, the method comprising: (a) measuring a first glucose consumption level in a tumor of the subject; (b) administering to the subject a first dose of a kinase inhibitor; (c) subsequent to (b), measuring a second glucose consumption level in the tumor; and (d) administering to the subject a second dose of the kinase inhibitor, wherein the second glucose consumption level is reduced relative to the first glucose consumption level.
[0014] Disclosed herein, in some aspects, is a method of treating a subject for cancer, the method comprising: (a) measuring a first glucose consumption level in a tumor of the subject; (b) administering to the subject a kinase inhibitor; (c) subsequent to (b), measuring a second glucose consumption level in the tumor; and (d) administering to the subject an alternative cancer therapy, wherein the second glucose consumption level is not reduced relative to the first glucose consumption level, wherein the alternative cancer therapy does not comprise the kinase inhibitor.
[0015] In some embodiments of the methods disclosed herein, the second glucose consumption level was measured at most 48 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments of the methods disclosed herein, the second glucose consumption level was measured at most 36 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments, the second glucose consumption level was measured at most 24 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments, the second glucose consumption level was measured at most 18 hours after administering the previous dose, the dose, or the first dose of the kinase inhibitor. In some embodiments, the second glucose consumption level was measured at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours (or any range or value derivable therein) after administering the previous dose, the dose, or the first dose of the kinase inhibitor.
[0016] In some embodiments of the methods disclosed herein, measuring the glucose consumption level comprises positron emission tomography (PET). In some embodiments, the first glucose consumption level and the second glucose consumption level were measured using PET. In some embodiments, measuring the first glucose consumption level comprises PET. In some embodiments, measuring the second glucose consumption level comprises PET. In some embodiments, measuring the glucose consumption level comprises administering fluorodeoxyglucose to the subject. In some embodiments, measuring the first glucose consumption level and the second glucose consumption level comprises administering fluorodeoxyglucose to the subject. In some embodiments, measuring the first glucose consumption level comprises administering fluorodeoxyglucose to the subject. In some embodiments, measuring the second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
[0017] In some embodiments, the kinase inhibitor is a FLT3 inhibitor. In some embodiments, the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359. In some embodiments, the kinase inhibitor is afatinib. In some embodiments, the kinase inhibitor is buparlisib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is ceritinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the kinase inhibitor is dovitinib. In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is pacritinib. In some embodiments, the kinase inhibitor is ponatinib. In some embodiments, the kinase inhibitor is trametinib. In some embodiments, the kinase inhibitor is vemurafenib. In some embodiments, the kinase inhibitor is quizartinib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is TCS 359.
[0018] In some embodiments, when the second glucose consumption level is reduced compared to or relative to the first or reference glucose consumption level, the second or additional dose of the kinase inhibitor is the same amount as the previous or first dose of the kinase inhibitor. In some embodiments, the second or additional dose of the kinase inhibitor is a lower amount than the previous or first dose of the kinase inhibitor.
[0019] In some embodiments, when the second glucose consumption level is not reduced compared to or relative to the first or reference glucose consumption level, the cancer therapy or alternative cancer therapy is an alternative kinase inhibitor. In some embodiments, the cancer therapy or alternative cancer therapy is chemotherapy, radiotherapy, or immunotherapy.
[0020] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is squamous cell lung carcinoma.
[0021] “Individual, “subject,” and “patient” are used interchangeably and can refer to a human or non-human.
[0022] As used herein, “treat,” “treating,” or “treatment” or equivalent terminology refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development, or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (z.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. The results of treatment can be determined by methods known in the art, such as determination of reduction of pain as measured by reduction of requirement for administration of opiates or other pain medication, determination of reduction of tumor burden, determination of restoration of function, or other methods known in the art.
[0023] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.
[0024] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
[0025] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.
[0026] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[0027] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristics of the disclosure.
[0028] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.
[0029] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure. [0030] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0032] FIG. 1 illustrates some methods for studying glucose consumption by cells. [0033] FIG. 2 illustrates a luminescence-based method for measuring cellular glucose consumption.
[0034] FIG. 3 illustrates a high-throughput method for measuring cellular glucose consumption.
[0035] FIG. 4 provides an overview of lung cancer cell lines and small molecule libraries screened for new regulators of glucose consumption by cells using the high throughput method illustrated in FIG. 3.
[0036] FIGs. 5A-5C show glucose consumption in A549 (FIG. 5A), H460 (FIG. 5B) and HCC827 (FIG. 5C) lung cancer cells after treatment with various kinase inhibitors.
[0037] FIGs. 6A-6B show glucose consumption of H460 (FIG. 6A) and HCC827 (FIG. 6B) lung cancer cells after treatment with kinase inhibitors.
[0038] FIG. 7 provides the structure of milciclib and IC50 values for inhibition of CDK and TRK proteins by milciclib.
[0039] FIGs. 8A-8C show glucose consumption by H460 lung cancer cells in culture and in vivo after treatment with milciclib. FIG. 8A. Normalized glucose consumption by H460 cells as a function of milciclib concentration. FIG. 8B. Pre- and post-treatment 18F-FDG PET imaging of glucose consumption by H460 tumors in mice treated with either vehicle (top) or milciclib (bottom). FIG. 8C. Quantification of milciclib-induced changes in glucose consumption in H460 cells (left panel) and the brain, heart, liver, and muscle of mice (right panel) pre- and post-treatment with vehicle or milciclib.
[0040] FIGs. 9A-9B show GLUT1, GLUT3, Hexokinase 1, and Hexokinase 2 mRNA (FIG. 9A) and protein (FIG. 9B) levels in H460 lung cancer cells after milciclib treatment compared to vehicle treatment.
[0041] FIG. 10 illustrates a method of assessing glucose transport and hexokinase activity by H460 lung cancer cells using a fluorescent nanosensor.
[0042] FIGs. 11A-11B show glucose transport and hexokinase activity in H460 lung cancer cells as measured using the fluorescent nanosensor illustrated in FIG. 10. FIG. 11A. Change in fluorescence after vehicle treatment (left) or milciclib treatment (right). FIG. 11B. Quantification of the results in FIG. 11A. The graph on the left shows that milciclib treatment does not affect glucose phosphorylation, which is measured by the fluorescent nanosensor when the cells are treated with glucose and Cytochalasin B. The graph on the right shows that milciclib treatment decreases glucose transport, which is measured by the fluorescent nanosensor when the cells are treated with glucose only and when glucose phosphorylation activity is unaffected. [0043] FIGs. 12A-12B show glucose consumption by H460 lung cancer cells overexpressing CDK7 before and after treatment with milciclib. FIG. 12A. Glucose consumption in H460 cells overexpressing CDK7. FIG. 12B. Glucose consumption in H460 cells overexpressing CDK7 after treatment with milciclib.
[0044] FIG. 13A-13B show glucose consumption by lung cancer cell lines and following overexpression of CDK7 before and after treatment with CDK7 inhibitors THZ1 (FIG. 13A) and LDC4297 (FIG. 13B).
[0045] FIGs. 14A-14B show glucose consumption by H460 and HCC827 overexpressing PTEN or PIK3CA. FIG. 14A. Phosphorylated CDK7, CDK7, and GLUT1 protein levels in H460 and HCC827 overexpressing PTEN or PIK3CA was measured by Western blot. FIG. 14B. Glucose consumption by H460 and HCC827 overexpressing PTEN or PIK3CA after treatment with milciclib.
[0046] FIGs. 15A-15C show glucose consumption and cell growth data for H460 lung cancer cells overexpressing GLUT1 after treatment with milciclib. FIG. 15A. GLUT1 protein levels in H460 cells was measured by Western blot. FIG. 15B. Glucose consumption by H460 cells overexpressing GLUT1 up to 24 hours post- milciclib treatment. FIG. 15C. Growth of H460 cells overexpressing GLUT1 up to 48 hours post-milciclib treatment.
[0047] FIG. 16 provides an overview of lung cancer cell lines that model the genetics of human lung cancer and small molecule inhibitors that can target many of the oncogenic drivers of lung cancer.
[0048] FIG. 17 provides an overview of the experimentally-tested lung cancer cell lines and small molecule inhibitors for which glucose consumption was measured 24 hours posttreatment and cell growth was measured 72 hours post-treatment.
[0049] FIG. 18 shows glucose consumption by nine different lung cancer cell lines 24 hours after treatment with afatinib. W: wild-type; M: mutant.
[0050] FIG. 19 shows glucose consumption by nine different lung cancer cell lines 24 hours after treatment with erlotinib. W: wild-type; M: mutant.
[0051] FIGs. 20A-20B. FIG. 20A shows glucose consumption by nine different lung cancer cell lines 24 hours after treatment with buparlisib. FIG. 20B shows the levels of phosphorylated AKT and total AKT in A549 lung cancer cells (top) and the ratio of phosphorylated AKT to total AKT in six different lung cancer cell lines after treatment with buparlisib (bottom).
[0052] FIG. 21 shows growth of nine different cancer cell lines 72 hours after treatment with afatinib. [0053] FIG. 22 shows growth of nine different cancer cell lines 72 hours after treatment with erlotinib.
[0054] FIG. 23 shows a receiver operating characteristic (ROC) curve plotting the sensitivity and specificity of using early inhibition of glucose consumption as a predictor of the inhibition of cell growth (defined as >40% growth inhibition) as a function of decrease in glucose consumption. The results show that early inhibition of glucose consumption is a strong predictor of drug therapeutic efficacy.
[0055] FIG. 24 shows glucose consumption by and growth of H460 (left), A549 (middle), and H2229 (right) cancer cells after treatment with dovitinib.
[0056] FIGs. 25A-25C show in vivo glucose consumption by and growth of PC9 lung cancer cells treated with dovitinib or erlotinib. FIG. 25A. Pre- and post-treatment 18F-FDG PET imaging of glucose consumption by PC9 tumors in mice treated with vehicle (top), dovitinib (middle), or erlotinib (bottom). FIG. 25B. Quantification of drug-induced changes in glucose consumption by PC9 tumors in mice. FIG. 25C. Quantification of drug-induced changes in PC9 tumor size for up to 12 days of treatment with kinase inhibitors.
[0057] FIGs. 26A-26D show glucose consumption and cell growth data for lung cancer cell lines overexpressing GLUT1 after treatment with kinase inhibitors. H460 lung cancer cells were treated with dovitinib (FIG. 26A) or buparlisib (FIG. 26B) and HCC827 lung cancer cells were treated with buparlisib (FIB. 26C) or trametinib (FIG. 26D).
[0058] FIGs. 27A-27B show glucose consumption (FIG. 27A) and cell growth (FIG. 27B) measured 24h after incubation with pacritinib in SK-MES-1, H520, and H596 squamous lung carcinoma cells. Data points are mean ± SEM, n=6.
[0059] FIGs. 28A-28B show normalized mRNA levels (FIG. 28A) and protein levels (FIG. 28B) of Hexokinase 1 (HK1), Hexokinase 2 (HK2), GLUT1, and GLUT3 after incubation of SK-MES-1, H520, and H596 squamous lung carcinoma cells with 10 pM pacritinib for 24h. Data points are mean ± SEM, n=2.
[0060] FIGs. 29A-29C show the effect of FLT3 overexpression on glucose consumption in SK-MES-1 (FIG. 29A), H520 (FIG. 29B), and H596 (FIG. 29C) squamous lung carcinoma cells. Data points are mean ± SEM, n=2.
[0061] FIGs. 30A-30C show the effect of different FLT3 inhibitors on glucose consumption in SK-MES-1 (FIG. 30A), H520 (FIG. 30B), and H596 (FIG. 30C) squamous lung carcinoma cells. Data points are mean ± SEM, n=4.
[0062] FIGs. 31A-31G show glucose consumption by nine different lung cancer cell lines 24 hours after treatment with cabozatinib (FIG. 31A), ceritinib (FIG. 31B), crizotinib (FIG. 31C), dovitinib (FIG. 31D), ponatinib (FIG. 31E), trametinib (FIG. 31F), and vemurafenib (FIG. 31G).
DETAILED DESCRIPTION
[0063] The present disclosure is based, at least in part, on the surprising discovery that certain kinase inhibitors, including afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, and vemurafenib, decrease glucose consumption within 24 hours of drug treatment specifically in cancer cells for which the drug has a longterm therapeutic effect. Thus, a decrease in glucose consumption in a tumor indicates that that the kinase inhibitor has pharmacodynamically engaged its target in the tumor and is having a biological effect on the tumor, which suggests that the treatment regimen with the kinase inhibitor should be continued. Conversely, no change or an increase in glucose consumption in a tumor indicates that the kinase inhibitor has not pharmacodynamically engaged its target or the target itself is not important for the tumor, which suggests that the treatment regimen should be altered by administering more of the drug and/or a different drug.
[0064] Accordingly, in some embodiments, disclosed are methods for treating a subject for cancer comprising measuring a first glucose consumption level in a tumor of the subject before administration of a dose of a kinase inhibitor; administering the kinase inhibitor dose to the subject; measuring a second glucose consumption level in the tumor after administration of the kinase inhibitor dose; and continuing or modifying administration of the therapy based on the second glucose consumption level relative to the first glucose consumption level.
[0065] In some embodiments, the second glucose consumption level is reduced relative to the first glucose consumption level, and administration of the kinase inhibitor is continued. In some embodiments, the second glucose level is unchanged or is not reduced relative to the first glucose consumption level, and administration of the therapy is modified. In some embodiments, modification of the therapy comprises an increase in the dose of the kinase inhibitor. In some embodiments, modification of the cancer therapy comprises administration of an alternative cancer therapy different from the kinase inhibitor previously administered.
I. Glucose Consumption Measurement Methods
[0066] Certain aspects of the present disclosure are related to measurement of glucose consumption by cells using 18F-FDG PET. Cancer treatment and outcome depend largely on the accurate diagnosis and staging of disease. There is extensive data in the literature indicating the importance of 18F-FDG PET imaging in accurately characterizing disease, as well as determining stage and sites of recurrent disease in many cancer types. For these indications, functional imaging with PET provides unique information which is not available from standard medical imaging modalities such as ultrasound, X-ray, computerized tomography (CT) or magnetic resonance imaging (MRI).
[0067] In some embodiments of the methods disclosed herein, measuring glucose consumption levels comprises positron emission tomography (PET). In some embodiments, a first glucose consumption level and a second glucose consumption level are measured using PET. In some embodiments, measuring the first glucose consumption level comprises PET. In some embodiments, measuring the second glucose consumption level comprises PET. In some embodiments, a glucose consumption level, as measured herein, is a signal level from a PET measurement. In some embodiments, measuring glucose consumption levels comprises administering fluorodeoxyglucose to the subject. In some embodiments, measuring a first glucose consumption level and a second glucose consumption level comprises administering fluorodeoxyglucose to the subject. In some embodiments, measuring the first glucose consumption level comprises administering fluorodeoxyglucose to the subject. In some embodiments, measuring the second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
[0068] Positron emission tomography (PET) is a nuclear medicine procedure based on the measurement of positron emission from radiolabelled tracer molecules. These radiotracers allow biologic processes to be measured and whole body images to be obtained, which demonstrates sites of radiotracer accumulation. A common radiotracer in use today is fluorodeoxyglucose, also known as 18F, fluorodeoxyglucose Fl 8, 18F-fluorodeoxyglucose, [18F]FDG, 18F-FDG, or FDG), a radiolabelled sugar (glucose) molecule. Chemically, 18F-FDG is 2-deoxy-2-[18F]fluoro-D-glucose. 18F-FDG is a glucose analog and is formed by replacing one of the 2-hydrogens of 2-deoxy-D-glucose (2-DG) with the positron-emitting isotope fluorine- 18, which emits paired gamma rays, allowing distribution of the tracer to be imaged by external gamma camera(s). More specifically, the positron-emitting radionuclide fluorine- 18 is substituted for the normal hydroxyl group at the C-2 position of a glucose molecule.
[0069] 18F-FDG, as a glucose analog, is taken up by high-glucose-using cells such as brain, brown adipocytes, kidney, and cancer cells, where phosphorylation prevents the glucose from being released again from the cell once it has been taken up by the cell. The 2-hydroxyl group (-OH) in normal glucose is needed for further glycolysis, or glucose metabolism, but 18F-FDG is missing this 2-hydroxyl. Thus, like 2-deoxy-D-glucose (2-DG), 18F-FDG cannot be further metabolized in cells. The [18F]FDG-6-phosphate formed when 18F-FDG enters the cell thus cannot move out of the cell before radioactive decay. As a result, the distribution of 18F-FDG is a good reflection of the distribution of glucose uptake and metabolism by cells in the body.
[0070] After 18F-FDG decays radioactively, however, its 2-fluorine is converted to 18O“. The molecule then picks up a proton H+ from a hydronium ion in its aqueous environment and becomes glucose-6-phosphate labeled with harmless nonradioactive heavy oxygen in the hydroxyl at the C-2 position. The presence of the newly-formed 2-hydroxyl now allows the molecule to be metabolized normally in the same way as ordinary glucose, producing nonradioactive end-products.
[0071] Although in theory all 18F-FDG is metabolized as above with a radioactivity elimination half-life of 110 minutes (the same as that of fluorine- 18), clinical studies have shown that the radioactivity of 18F-FDG partitions into two major fractions. About 75% of the fluorine- 18 activity remains in tissues and is eliminated with a half-life of 110 minutes, presumably by decaying in place to 0-18 to form [18O]O-glucose-6-phosphate, which is nonradioactive. This molecule can soon be metabolized to carbon dioxide and water, after transmutation of the fluorine to oxygen ceases to prevent metabolism). Another fraction of 18F-FDG, representing about 20% of the total fluorine- 18 activity of an injection, is eliminated renally by two hours after a dose of 18F-FDG, with a rapid half-life of about 16 minutes. This portion makes the renal-collecting system and bladder prominent in a normal PET scan and indicates that the 20% portion of the total fluorine- 18 tracer activity is eliminated pharmacokinetic ally much more quickly than the isotope itself can decay. All radioactivity of 18F-FDG, both the 20% portion which is rapidly excreted in urine in the first several hours after administration of the 18F-FDG and the 80% portion which remains in the patient, decays with a half-life of 110 minutes. Thus, within 24 hours (13 half-lives after the injection), the radioactivity in the patient and in any initially voided urine which may have contaminated bedding or objects after the PET exam will have decayed to 2 13 = 1/8192 of the initial radioactivity of the dose.
[0072] The uptake of 18F-FDG by tissues is a marker for the tissue uptake of glucose, which in turn is closely correlated with certain types of tissue metabolism. Specifically, 18F-FDG is taken up by cells, phosphorylated by hexokinase, and retained by tissues with high metabolic activity, such as most types of malignant tumors. Thus, imaging with 18F-FDG PET is used to determine sites of abnormal glucose metabolism and can be used to characterize and localize many types of tumors and/or to diagnose, stage, and monitor treatment of cancers. [0073] In body-scanning applications in searching for tumor or metastatic disease, a dose of 18F-FDG in solution (for example, 5 to 10 millicuries or 200 to 400 MBq) is injected rapidly into a saline drip running into a vein of a subject who has been fasting for at least six hours and who has a suitably low blood sugar. The subject then waits about an hour for the sugar to distribute and be taken up into organs which use glucose. During this time, physical activity should be kept to a minimum in order to minimize uptake of the radioactive sugar into muscles, which can cause unwanted artifacts in the scan. Then, the patient is placed in the PET scanner for a series of one or more static two-dimensional or three-dimensional images of the distribution of 18F-FDG within the body. Tumor 18F-FDG uptake is analyzed in terms of Standardized Uptake Value (SUV).
II. Kinase Inhibitors
[0074] Certain aspects of the disclosure are related to administration of one or more kinase inhibitors to a subject in need thereof before and after measuring glucose consumption levels in a tumor of the subject. A protein kinase inhibitor (also “kinase inhibitor”) is a type of enzyme inhibitor that blocks the action of one or more protein kinases. Protein kinases are enzymes that chemically add the terminal y-phosphate group of adenosine triphosphate to a protein, in some cases modulating function of the protein and/or regulating biological processes involving the protein. More specifically, phosphorylation results in a functional change of the target protein (substrate) by regulating signaling pathways by amplification or cellular location, or by interactions with regulatory proteins. Human cells have many different kinases, and they help control important functions, such as cell signaling, metabolism, division, and survival.
[0075] The phosphate groups added by kinases are usually added to serine, threonine, or tyrosine amino acids on the protein. Most kinases act on both serine and threonine, while tyrosine kinases act on tyrosine, and a number of kinases act on all three. There are also protein kinases that phosphorylate other amino acids, including histidine kinases that phosphorylate histidine residues.
[0076] In some embodiments, the kinase inhibitor is imatinib, buparlisib, dovitinib, pacritinib, nilotinib, dasatinib, bosutinib, ponatinib, gefitinib, gilteritinib, quizartinib, crenolanib, erlotinib, afatinib, osimertinib, lapatinib, neratinib, sorafenib, sunitinib, pazopanib, axitinib, lenvatinib, cabozatinib, vandetanib, regorafenib, vemurafenib, dabrafenib, trametinib, cobimetinib, crizotinib, certinib, alectinib, brigatinib, lorlatinib, ibrutinib, acalibrutinib, midostaurin, ruxolitinib, idelalisib, copanlisib, palbociclib, ribociclib, or abemaciclib. These inhibitors are described in Kannaiyan R, Mahadevan D, “A comprehensive review of protein kinase inhibitors for cancer therapy,” Expert Rev Anticancer Ther, 2018;18(12):1249-1270, for example, incorporated herein by reference in its entirety. These kinase inhibitors inhibit Bcr- Abl tyrosine kinases, Epidermal Growth Factor receptor tyrosine kinases, Vascular Endothelial Growth Factor Receptor (VEGFR) tyrosine kinases, BRAF kinases, mitogen-activated protein kinases, anaplastic lymphoma kinase, Src family kinases like Bruton tyrosine kinase, Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FET3), Janus family kinases like JAK 2, lipid kinases like phosphoinositide 3-kinase, and/or cyclin dependent kinases, for example. In some embodiments, the kinase inhibitor is a FET3 inhibitor. In some embodiments, the FET3 inhibitor is midostaurin, gilteritinib, quizartinib (AC220), crenolanib, sorafenib, sunitinib, cabozatinib (XE184), or TCS 359. FET3 and various examples of FET3 inhibitors are discussed in, for example, Gebru MT, Wang HG. “Therapeutic targeting of FET3 and associated drug resistance in acute myeloid leukemia,” J Hematol Oncol. 2020 Nov 19; 13(1): 155., incorporated by reference herein in its entirety.
[0077] In some embodiments, the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359. In some embodiments, the kinase inhibitor is afatinib. In some embodiments, the kinase inhibitor is buparlisib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is ceritinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the kinase inhibitor is dovitinib. In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is pacritinib. In some embodiments, the kinase inhibitor is ponatinib. In some embodiments, the kinase inhibitor is trametinib. In some embodiments, the kinase inhibitor is vemurafenib. In some embodiments, the kinase inhibitor is quizartinib. In some embodiments, the kinase inhibitor is cabozatinib. In some embodiments, the kinase inhibitor is TCS 359.
[0078] As disclosed herein, kinase inhibitors can decrease glucose consumption within 24 hours of treatment with the inhibitors specifically in cancer cells for which the inhibitors have a long-term therapeutic effect. A decrease in glucose consumption in a tumor may indicate that that the kinase inhibitor has pharmacodynamically engaged its target in the tumor and is having a biological effect on the tumor. This suggests that the current treatment regimen with the kinase inhibitor should be continued. Conversely, no change or an increase in glucose consumption in a tumor indicates that the kinase inhibitor has not pharmacodynamically engaged its target or the target itself is not important for the tumor. This suggests that the kinase inhibitor treatment regimen should be altered by administering more of the inhibitor and/or an alternative cancer therapy, for example, a different kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy.
[0079] In some embodiments of the methods disclosed herein, a dose of the kinase inhibitor is administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level. In some embodiments, the second glucose consumption level is measured at most 18 to 48 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 48 hours, or any range or value derivable therein, after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 48 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 36 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 24 hours after administering the dose of the kinase inhibitor to the subject. In some embodiments, the second glucose consumption level is measured at most 18 hours after administering the dose of the kinase inhibitor to the subject.
[0080] Administration of the kinase inhibitor may comprise administration of at least 1, 2, 3, 4, 5, or more kinase inhibitors. Any of these therapies may also be excluded. Combinations of these therapies may also be administered.
III. Therapeutic Methods
[0081] Aspects of the disclosure are directed to compositions and methods of administering therapeutically effective amounts one or more cancer therapies to a subject or patient in need thereof before and after measuring glucose consumption levels in a tumor of the subject. The compositions of the disclosure may be used for in vivo, in vitro, or ex vivo administration. The route of administration of the composition may be, for example, intratumoral, intravenous, intramuscular, intraperitoneal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, through inhalation, or through a combination of two or more routes of administration. The cancer therapies may be administered via the same or different routes of administration.
[0082] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain embodiments, the cancer may originate in the blood, bladder, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.
[0083] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing ’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
[0084] In some embodiments, disclosed are methods for treating cancers comprising lung cancers. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is squamous cell lung carcinoma.
[0085] In some embodiments, the cancer therapy comprises a local cancer therapy. In some embodiments, the cancer therapy comprises a systemic cancer therapy. In some embodiments, the cancer therapy excludes a systemic cancer therapy. In some embodiments, the cancer therapy excludes a local cancer therapy.
A. Radiotherapy
[0086] In some embodiments, a radiotherapy, such as ionizing radiation, is administered to a subject. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A preferred non-limiting example of ionizing radiation is an x-radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.
[0087] In some embodiments, the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some embodiments, the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some embodiments, the internal radiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. In some embodiments, the radiotherapy is administered to a primary tumor.
[0088] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the does may be about 1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.
[0089] In some embodiments, the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of radiation is at least, at most, or about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range therein). In some embodiments, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.
B. Cancer Immunotherapy
[0090] In some embodiments, the cancer therapy comprises a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor- associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines. Various immunotherapies are known in the art, and examples are described below.
1. Checkpoint Inhibitors and Combination Treatment
[0091] In some embodiments, the cancer immunotherapy comprises immune checkpoint inhibitors, examples of which are further described below. As disclosed herein, “checkpoint inhibitor therapy” (also “immune checkpoint blockade therapy”, “immune checkpoint therapy”, “ICT,” “checkpoint blockade immunotherapy,” or “CBI”), refers to cancer therapy comprising providing one or more immune checkpoint inhibitors to a subject suffering from or suspected of having cancer. a. PD-1, PDL1, and PDL2 inhibitors
[0092] PD-1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD- 1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and/or PDL1 activity.
[0093] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.
[0094] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014/0294898, US 2014/022021, and US2011/0008369, all incorporated herein by reference.
[0095] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD- 1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g. , an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX- 1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.
[0096] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.
[0097] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. b. CTLA-4, B7-1, and B7-2
[0098] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co- stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and/or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.
[0099] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. [0100] Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. WO200 1/014424, W02000/037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.
[0101] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and Yervoy®) or antigen binding fragments and variants thereof (see, e.g., WO 01/14424).
[0102] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on PD-1, B7-1, or B7-2 as the above- mentioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. c. LAG3
[0103] Another immune checkpoint that can be targeted in the methods provided herein is the lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3. The complete mRNA sequence of human LAG3 has the Genbank accession number NM_002286. LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG3 ’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+ T cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors of the disclosure may block one or more functions of LAG3 activity.
[0104] In some embodiments, the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0105] Anti-human-LAG3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-LAG3 antibodies can be used. For example, the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. The anti-LAG3 antibodies disclosed in: US 9,505,839 (BMS-986016, also known as relatlimab); US 10,711,060 (IMP-701, also known as LAG525); US 9,244,059 (IMP731, also known as H5L7BW); US 10,344,089 (25F7, also known as LAG3.1); WO 2016/028672 (MK-4280, also known as 28G-10); WO 2017/019894 (BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11:6 (LBL-007) can be used in the methods disclosed herein. These and other anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: WO 2016/028672, WO 2017/106129, WO 2017062888, WO 2009/044273, WO 2018/069500, WO 2016/126858, WO 2014/179664, WO 2016/200782, WO 2015/200119, WO 2017/019846, WO 2017/198741, WO 2017/220555, WO 2017/220569, WO 2018/071500, WO
2017/015560; WO 2017/025498, WO 2017/087589 , WO 2017/087901, WO 2018/083087, WO 2017/149143, WO 2017/219995, US 2017/0260271, WO 2017/086367, WO
2017/086419, WO 2018/034227, and WO 2014/140180. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.
[0106] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. d. TIM-3 [0107] Another immune checkpoint that can be targeted in the methods provided herein is the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has the Genbank accession number NM_032782. TIM-3 is found on the surface IFNy- producing CD4+ Thl and CD8+ Tel cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates the T-cell exhaustion. TIM-3 has also been shown as a CD4+ Thl -specific cell surface protein that regulates macrophage activation. Inhibitors of the disclosure may block one or more functions of TIM- 3 activity.
[0108] In some embodiments, the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
[0109] Anti-human-TIM-3 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIM-3 antibodies can be used. For example, anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the claimed invention can be found in, for example: US 9,605,070, US 8,841,418, US2015/0218274, and US 2016/0200815. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.
[0110] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. 2. Activation of co-stimulatory molecules
[0111] In some embodiments, the cancer immunotherapy comprises an activator of a costimulatory molecule. In some embodiments, the activator comprises an agonist of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Activators include activating antibodies, polypeptides, compounds, and nucleic acids.
3. Dendritic cell therapy
[0112] In some embodiments, the cancer immunotherapy comprises dendritic cell therapy. Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.
[0113] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Other adjuvants include proteins or other chemicals that attract and/or activate dendritic cells, such as granulocyte macrophage colony- stimulating factor (GM-CSF).
[0114] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.
[0115] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor- specific peptide/protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.
[0116] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets. 4. CAR-T cell therapy
[0117] In some embodiments, the cancer immunotherapy comprises chimeric immunoreceptors. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.
[0118] The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a “living drug”. CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signaling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.
[0119] Example CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).
5. Cytokine therapy
[0120] In some embodiments, the cancer immunotherapy comprises cytokine therapy. Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.
[0121] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNk).
[0122] Interleukins have an array of immune system effects. IE-2 is an example interleukin cytokine therapy. 6. Adoptive T-cell therapy
[0123] In some embodiments, the cancer immunotherapy comprises adoptive T-cell therapy. Adoptive T cell therapy is a form of passive immunization by the transfusion of T- cells (adoptive cell transfer). They are found in blood and tissue and usually activate when they find foreign pathogens. Specifically they activate when the T-cell’s surface receptors encounter cells that display parts of foreign proteins on their surface antigens. These can be either infected cells, or antigen presenting cells (APCs). They are found in normal tissue and in tumor tissue, where they are known as tumor infiltrating lymphocytes (TILs). They are activated by the presence of APCs such as dendritic cells that present tumor antigens. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.
[0124] Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.
[0125] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, embodiments of the disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some embodiments, the patient is one that has been determined to be resistant to a therapy described herein. In some embodiments, the patient is one that has been determined to be sensitive to a therapy described herein.
C. Oncolytic virus
[0126] In some embodiments, the cancer therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy D. Polysaccharides
[0127] In some embodiments, the cancer therapy comprises polysaccharides. Certain compounds found in mushrooms, primarily polysaccharides, can up-regulate the immune system and may have anti-cancer properties. For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophage, NK cells, T cells and immune system cytokines and have been investigated in clinical trials as immunologic adjuvants.
E. Neoantigens
[0128] In some embodiments, the cancer therapy comprises neoantigen administration. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, as identified using RNA sequencing data, is higher in tumors with a high mutational burden. The level of transcripts associated with cytolytic activity of natural killer cells and T cells positively correlates with mutational load in many human tumors.
F. Chemotherapies
[0129] In some embodiments, the cancer therapy comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon- a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydiazine derivatives (e.g., procarbazine), and adreocortical suppressants (e.g., taxol and mitotane). In some embodiments, cisplatin is a particularly suitable chemotherapeutic agent.
[0130] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications including about 15 mg/m2 to about 20 mg/m2 for 5 days every three weeks for a total of three courses being contemplated in certain embodiments. In some embodiments, the amount of cisplatin delivered to the cell and/or subject in conjunction with the construct comprising an Egr-1 promoter operatively linked to a polynucleotide encoding the therapeutic polypeptide is less than the amount that would be delivered when using cisplatin alone.
[0131] Other suitable chemotherapeutic agents include antimicro tubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter/TNFa construct delivered via an adenoviral vector and doxorubicin was determined to be effective in overcoming resistance to chemotherapy and/or TNF-a, which suggests that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-a.
[0132] Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for an adult include about 60 mg/m2 to about 75 mg/m2 at about 21 -day intervals or about 25 mg/m2 to about 30 mg/m2 on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg/m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone- marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
[0133] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and/or ifosfamide, melphalan (E-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR® is available from Adria), is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg/kg/day to about 5 mg/kg/day, intravenous doses include, for example, initially about 40 mg/kg to about 50 mg/kg in divided doses over a period of about 2 days to about 5 days or about 10 mg/kg to about 15 mg/kg about every 7 days to about 10 days or about 3 mg/kg to about 5 mg/kg twice a week or about 1.5 mg/kg/day to about 3 mg/kg/day. Because of adverse gastrointestinal effects, the intravenous route is preferred. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities. [0134] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluouracil; 5-FU) and floxuridine (fluorode- oxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg/m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.
[0135] Gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., “gemcitabine”), another suitable chemotherapeutic agent, is recommended for treatment of advanced and metastatic pancreatic cancer, and will therefore be useful in the present disclosure for these cancers as well.
[0136] The amount of the chemotherapeutic agent delivered to the patient may be variable. In one suitable embodiment, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutic s of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.
G. Surgery
[0137] In some embodiments, the cancer therapy comprises surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs ’ surgery).
[0138] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
H. Other Agents
[0139] It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment, for example. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti- hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
IV. Cancer Treatment
[0140] Aspects of the present disclosure are directed to methods comprising treatment of a subject suffering from, or suspected of having, cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is squamous cell lung carcinoma.
[0141] In some embodiments, the disclosed methods comprise treating a subject suffering from a cancer with a kinase inhibitor. In some embodiments, the kinase inhibitor is a FLT3 inhibitor. In some embodiments, the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359. In some embodiments, the kinase inhibitor is afatinib. In some embodiments, the kinase inhibitor is buparlisib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is ceritinib. In some embodiments, the kinase inhibitor is crizotinib. In some embodiments, the kinase inhibitor is dovitinib. In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is pacritinib. In some embodiments, the kinase inhibitor is ponatinib. In some embodiments, the kinase inhibitor is trametinib. In some embodiments, the kinase inhibitor is vemurafenib. In some embodiments, the kinase inhibitor is quizartinib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is TCS 359.
[0142] In some embodiments, the kinase inhibitor is administered subsequent to measurement of a first or reference glucose consumption level in a tumor of the subject, and a second glucose consumption level is measured subsequent to administration of the kinase inhibitor. As disclosed herein, changes in glucose consumption following administration of a cancer therapy, for example, a kinase inhibitor, can be used to predict the long-term therapeutic effect of the therapy, with decreases in glucose consumption in a tumor indicating that the therapy has pharmacodynamically engaged its target in the tumor and is having a biological effect on the tumor, and no changes or increases in glucose consumption in a tumor indicating that the therapy has not pharmacodynamically engaged its target or the target itself is not important for the tumor. Pharmacodynamic engagement of a kinase inhibitor target by the kinase inhibitor, for example, suggests that the treatment regimen with the kinase inhibitor should be continued, whereas lack of pharmacodynamic engagement of the kinase inhibitor target by the kinase inhibitor suggests that the treatment regimen should be altered by administering more of the kinase inhibitor and/or a different cancer therapy.
[0143] Accordingly, in some embodiments, disclosed are methods for treating a subject for cancer comprising measuring a first glucose consumption level in a tumor of the subject before administration of a dose of a cancer therapy; administering the cancer therapy dose to the subject; measuring a second glucose consumption level in the tumor after administration of the cancer therapy dose; and continuing or modifying administration of the cancer therapy based on the second glucose consumption level relative to the first glucose consumption level.
[0144] In some embodiments, the second glucose consumption level is reduced relative to the first glucose consumption level, and administration of the cancer therapy is continued. In some embodiments, the second glucose level is unchanged or is not reduced relative to the first glucose consumption level, and administration of the cancer therapy is modified. In some embodiments, modification of the cancer therapy comprises an increase in the dose of the cancer therapy. In some embodiments, modification of the cancer therapy comprises administration of an alternative cancer therapy different from the cancer therapy previously administered.
[0145] In some embodiments, the cancer therapy comprises a kinase inhibitor. Thus, in some embodiments, the method comprises administering a dose of a kinase inhibitor to the subject. In some embodiments, the cancer therapy comprises a cancer therapy other than the kinase inhibitor, or an alternative cancer therapy. Thus, in some embodiments, the method comprises administering a cancer therapy other than the kinase inhibitor, or an alternative cancer therapy, to the subject.
[0146] In some embodiments of the method, a first glucose consumption level and a second glucose consumption level are measured in a tumor of the subject. In some embodiments, the dose of the kinase inhibitor is administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level. If the second glucose consumption level is reduced relative to the first glucose consumption level, then a second dose of the kinase inhibitor is administered to the subject. The second dose of the kinase inhibitor can be the same as or a lower amount than the previous or first dose of the kinase inhibitor. If the second glucose consumption level is not reduced relative to the first glucose consumption level, then a cancer therapy other than the kinase inhibitor is administered to the subject. The cancer therapy other than the kinase inhibitor, or alternative cancer therapy, can be an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy.
[0147] In further embodiments, disclosed is a method for evaluating the efficacy of a kinase inhibitor in a subject having cancer comprising administering a dose of the kinase inhibitor to the subject and measuring a second glucose consumption level in the tumor at most 48 hours after administering the kinase inhibitor to the subject. In some embodiments, the method further comprises measuring a first or reference glucose consumption level in a tumor of the subject prior to administration of the dose of the kinase inhibitor to the subject. In some embodiments, the kinase inhibitor is identified as effective when the second glucose consumption level is reduced compared to the first or reference glucose consumption level, and the method further comprises administering an additional dose of the kinase inhibitor to the subject. The additional dose of the kinase inhibitor can be the same as or a lower amount that the first or previous dose of the kinase inhibitor. In some embodiments, the kinase inhibitor is identified as ineffective when the second glucose consumption level is unchanged or not reduced compared to the first or reference glucose consumption level, and an alternative cancer therapy comprising an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy can be administered to the subject.
[0148] In still further embodiments, the disclosed methods comprise identifying one or more subjects as being candidates for treatment with a kinase inhibitor or an alternative cancer therapy other than the kinase inhibitor and/or identifying one or more subjects as subjects as having increased sensitivity to a kinase inhibitor or an alternative cancer therapy other than the kinase inhibitor. For example, in some embodiments, disclosed is a method of treating a subject for cancer comprising selecting a subject for treatment with a second dose of a kinase inhibitor when a second glucose consumption level measured in the subject after administration of a first dose of the kinase inhibitor is reduced relative to a first glucose consumption level measured in the subject before administration of a first dose of the kinase inhibitor. The second dose of the kinase inhibitor can be the same as or a lower amount that the first dose of the kinase inhibitor. Also disclosed is a method of treating a subject for cancer comprising selecting a subject for treatment with an alternative cancer therapy when a second glucose consumption level measured in the subject after administration of a dose of a kinase inhibitor is unchanged or not reduced relative to a first glucose consumption level measured in the subject before administration of a dose of the kinase inhibitor. The alternative cancer therapy can comprise an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy. Thus, drug target engagement in tumors can be used as a pharmacodynamic biomarker to allow for stratification and treatment of cancer patients having increased sensitivity to drugs.
V. Administration of Therapeutic Compositions
[0149] Embodiments of the disclosure relate to compositions and methods comprising therapeutic compositions. The one or more cancer therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. In some embodiments, the one or more cancer therapies are administered in a separate composition. In some embodiments, the one or more cancer therapies are in the same composition. Various combinations of the agents may be employed.
[0150] Compositions according to the present invention can be prepared according to standard techniques and may comprise water, buffered water, saline, glycine, dextrose, iso- osmotic sucrose solutions and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and the like. The preparation of compositions that contains the cancer therapies will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
[0151] The cancer therapies of the disclosure may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intraarterially, intravenously, intraperitoneally, subcutaneously, intramuscularly, intratumorally, topically, orally, transdermally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual ’s clinical history and response to the treatment, and the discretion of the attending physician.
[0152] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.
[0153] The therapy provided herein comprises administration of one or more cancer therapies. In some embodiments, the one or more cancer therapies comprise a kinase inhibitor. In some embodiments, the one or more therapies comprise an alternative cancer therapy to the kinase inhibitor, and the alternative cancer therapy can comprise an alternative kinase inhibitor or chemotherapy, radiotherapy, or immunotherapy. In some embodiments, the one or more cancer therapies are administered sequentially, with the kinase inhibitor administered before the alternative cancer therapy. [0154] In some embodiments, the one or more cancer therapies are administered within 1 week, within 2 weeks, within 3 weeks, or within 1 month after administration of a first dose of a kinase inhibitor. In some embodiments, the one or more cancer therapies are administered within 1 week after administration of a first dose of a kinase inhibitor. In some embodiments, the one or more cancer therapies are administered within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, or within 6 days after administration of a first dose of a kinase inhibitor. In some embodiments, the one or more cancer therapies are administered within 48 hours, 47 hours, 46 hours, 45 hours, 44 hours, 43 hours, 42 hours, 41 hours, 40 hours, 39 hours, 38 hours, 37 hours, 36 hours, 35 hours, 34 hours, 33 hours, 32 hours, 31 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour after administration of a first dose of a kinase inhibitor.
[0155] In some embodiments of the methods disclosed herein, a single dose of the one or more cancer therapies are administered. In some embodiments of the methods disclosed herein, multiple doses of the one or more second cancer therapies are administered. In some embodiments, the method comprises administering multiple doses of the one or more cancer therapies, and the multiple doses are administered on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive days. In some embodiments, the method comprises administering multiple doses of the one or more cancer therapies, and the multiple doses are administered on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 non-consecutive days. Administration of the multiple doses on consecutive or non-consecutive days can comprise a cycle, and the cycle may be repeated once a month for one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve consecutive or non-consecutive months, or once a year for one, two, three, four, or five consecutive or non-consecutive years.
[0156] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect. In the practice in certain embodiments, it is contemplated that doses in the range from 10 mg/kg to 200 mg/kg can affect the protective capability of these agents. Thus, it is contemplated that doses include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 pg/kg, mg/kg, pg/day, or mg/day or any range derivable therein. Furthermore, such doses can be administered at multiple times during a day, and/or on multiple days, weeks, or months.
[0157] In certain embodiments, the effective dose of the pharmaceutical composition is one which can provide a blood level of about 1 pM to 150 pM. In another embodiment, the effective dose provides a blood level of about 4 pM to 100 pM.; or about 1 pM to 100 pM; or about 1 pM to 50 pM; or about 1 pM to 40 pM; or about 1 pM to 30 pM; or about 1 pM to 20 pM; or about 1 pM to 10 pM; or about 10 pM to 150 pM; or about 10 pM to 100 pM; or about 10 pM to 50 pM; or about 25 pM to 150 pM; or about 25 pM to 100 pM; or about 25 pM to 50 pM; or about 50 pM to 150 pM; or about 50 pM to 100 pM (or any range derivable therein). In other embodiments, the dose can provide the following blood level of the agent that results from a therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 pM or any range derivable therein. In certain embodiments, the therapeutic agent that is administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case the blood levels may refer to the amount of that agent. Alternatively, to the extent the therapeutic agent is not metabolized by a subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.
[0158] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.
[0159] It will be understood by those skilled in the art and made aware that dosage units of pg/kg or mg/kg of body weight can be converted and expressed in comparable concentration units of pg/ml or mM (blood levels), such as 4 pM to 100 pM. It is also understood that uptake is species and organ/tissue dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein. Examples
[0160] The following examples are included to demonstrate embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
1. Discovery of regulators of 18F-FDG consumption in cancer cells
[0161] Methods for studying glucose consumption such as those illustrated by FIG. 1 are slow, labor intensive, expensive, and difficult to scale up. To overcome these challenges, luminescence-based methods were used for measuring cellular glucose consumption, illustrated by FIG. 2. As shown in FIG. 3, these luminescence-based methods were employed by the inventors in a high-throughput method for measuring cellular glucose consumption by cells to quickly, easily, and cheaply identify potential regulators of glucose consumption by the cells. The lung cancer cell lines and small molecule libraries screened using this high- throughput method are illustrated by FIG. 4.
[0162] Glucose consumption by the cells was measured by the inventors 24 hours postsmall molecule treatment. As shown in FIG. 5, several new regulators of glucose consumption were identified by the high-throughput screen of small molecule libraries in A549 (FIG. 5A), H460 (FIG. 5B), and HCC827 (FIG. 5C) lung cancer cell lines. The inventors also measured glucose consumption as a function of inhibitor concentration, as shown in FIG. 6, and found that the drugs that inhibit glucose consumption in H460 (FIG. 6A) and HCC827 (FIG. 6B) lung cancer cells do so across a range of concentrations.
[0163] A new regulator of glucose consumption, milciclib, was selected for further experimentation. Milciclib inhibits CDK2, CDK4, CDK7, and TRKA. It is also known to limit the growth of a wide range of xenograft models, including ovarian, colon, pancreatic, and prostate cancer. FIG. 7 provides the structure of milciclib and IC50 values for inhibition of several CDK and TRK proteins by milciclib.
[0164] The inventors first investigated the ability of milciclib to inhibit glucose consumption by the NSCLC line H460. FIG. 8 shows that milciclib selectively inhibited glucose consumption by H460 lung cancer cells in culture and in vivo. Specifically, as the concentration of milciclib increased, glucose consumption by H460 cells decreased, as shown in FIG. 8A. The inventors also showed that milciclib treatment decreased glucose consumption in H460 tumors in mice. Pre- and post-treatment 18F-FDG PET imaging of glucose consumption by H460 tumors in mice treated with either vehicle or milciclib was performed and quantified, as shown in FIG. 8B and FIG. 8C, respectively. Milciclib treatment was found to significantly decrease glucose consumption in H460 tumors (p = 0.0007), while no significant changes in glucose consumption were observed by the brain, heart, liver, and muscle of mice bearing H460 tumors. The inventors also determined changes in GLUT1 protein levels as a result of milciclib treatment. As shown in FIG. 9, milciclib treatment decreased both GLUT1 mRNA (FIG. 9A) and protein (FIG. 9B) levels in H460 lung cancer cells compared to vehicle treatment.
[0165] Next, using the fluorescent nanosensor illustrated by FIG. 10, the inventors demonstrated that milciclib decreases glucose transport in H460 lung cancer cells. As shown in FIG. 11A, milciclib treatment resulted in a slower loss of fluorescence in the absence of Cytochalasin B compared to vehicle treatment, indicating diminished glucose transport into H460 cells in the presence of milciclib. FIG. 11B shows a quantification of the results in FIG. 11A. Milciclib has no effect on the rate of change of the fluorescent signal compared to vehicle in the cells treated with glucose and Cytochalasin B (left), demonstrating that Milciclib has no effect on glucose phosphorylation. Milciclib decreases the rate of change of the fluorescent signal compared to vehicle in the cells treated with glucose alone (right). Combined with the results from the first graph on the left, this data demonstrates that Milciclib blocks glucose transport.
[0166] Further, as shown in FIGs. 12A and 12B, milciclib inhibited CDK7 to inhibit glucose consumption in H460 lung cancer cells. CDK7 is a CDK- activating kinase and part of the TFIIH transcription complex. CDK7 overexpression increased GLUT1 protein levels and increased glucose consumption in H460 cells (FIG. 12A), and treatment of H460 cells overexpressing CDK7 with milciclib resulted in a smaller decrease in glucose consumption by the cells (FIG. 12B). Similarly, CDK7 inhibitors THZ1 (FIG. 13A) and LDC4297 (FIG. 13B) blocked glucose consumption by H460 lung cancer cells, and overexpressing CDK7 blocked this effect, demonstrating that these inhibitors block glucose consumption by inhibiting CDK7. [0167] The effects of PTEN and PIK3CA overexpression on glucose consumption by lung cancer cells were also examined. FIG. 14 shows glucose consumption by H460 and HCC827 overexpressing PTEN or PIK3CA. Phosphorylated CDK7, CDK7, and GLUT1 protein levels in H460 and HCC827 overexpressing PTEN or PIK3CA were measured by Western blot (FIG. 14A), and treatment of H460 and HCC827 overexpressing PTEN or PIK3CA with milciclib resulted in a decrease in glucose consumption by the cells (FIG. 14B). These results indicate that CDK7 drives glucose consumption downstream of mutant PIK3CA signaling.
[0168] The inventors confirmed that all of the results related to CDK7 inhibitors and glucose consumption were reproducible in H1975 cells, a NSCLC line having a PIK3CA mutation. The inventors also measured glucose consumption and cell growth data for H460 lung cancer cells overexpressing GLUT1 before and after treatment with milciclib, as shown in FIG. 15. GLUT1 overexpression in H460 cells was confirmed by Western blot (FIG. 15A), and GLUT1 overexpression increased glucose consumption by H460 cells up to 24 hours post- milciclib treatment (FIG. 15B). Further, GLUT1 overexpression increased growth of H460cells up to 48 hours post-milciclib treatment (FIG. 15C). These results indicate that inhibiting glucose consumption may be a key mechanism by which milciclib inhibits cell growth in PIK3CA mutant cancer cells.
[0169] In summary, through these experiments, the inventors identified: (1) a targetable protein that selectively activates glucose consumption in NSCLC; (2) the genetic driver of this effect; (3) an inhibitor of this protein that blocks glucose consumption sufficiently to limit cancer cell growth; and (4) a noninvasive clinical assay (18F-FDG PET) to measure drug pharmacodynamics.
2. Identification of 18F-FDG PET as a predictive biomarker of drug efficacy of clinically relevant inhibitors
[0170] For the following experiments, the inventors sought to use 18F-FDG PET as predictive biomarker of the efficacy of a broad range of kinase inhibitors. Further, the inventors aimed to probe the link between inhibition of glucose consumption and inhibition of cell growth to determine whether the drug concentration necessary to block glucose consumption is also sufficient to block cancer cell growth. Finally, the inventors investigated the functional relevancy of glucose consumption inhibition and its contribution to cancer cell growth inhibition.
[0171] The inventors selected lung cancer as a model system based due in part to the number lung cancer cell lines that model the genetics of human lung cancer and the number of readily-available small molecule inhibitors that can target many of the oncogenic drivers of lung cancer. These lung cancer cell lines and small molecule inhibitors are described in FIG. 16, while FIG. 17 provides an overview of the experimentally-tested lung cancer cell lines and small molecule inhibitors. Glucose consumption was measured 24 hours post-treatment of the cell lines, and cell growth was measured 72 hours post-treatment of the cell lines.
[0172] In a first set of experiments, the inventors measured glucose consumption by nine lung cancer cell lines after treatment with three different kinase inhibitors. In the studies highlighted in FIG. 18, the shown lung cancer cell lines were treated with increasing concentrations of afatinib, and glucose consumption by the cell lines was measured 24 hours after treatment. Glucose consumption was decreased in H1229, H1734, H1993, H3122, HCC827, and PC9 cells. In the studies highlighted in FIG. 19, the shown lung cancer cell lines were treated with increasing concentrations of erlotinib, and glucose consumption by the cell lines was measured 24 hours after treatment. Glucose consumption was decreased in H1734, HCC827, and PC9 cells. In the studies highlighted in FIG. 20A, the shown lung cancer cell lines were treated with increasing concentrations of buparlisib, and glucose consumption by the cell lines was measured 24 hours after treatment. Glucose consumption was decreased in H1734, H1993, H3122, H460, HCC827, and PC9 cells. In addition, FIG. 20B shows that treatment with increasing concentrations of buparlisib resulted in decreased phosphorylation of AKT in six different lung cancer cell lines. In the studies highlighted in FIGs. 31A-31G, the shown lung cancer cell lines were treated with increasing concentrations of cabozantinib (FIG. 31A), ceritinib (FIG. 31B), crizotinib (FIG. 31C), dovitinib (FIG. 31D), ponatinib (FIG. 31E), trametinib (FIG. 31F), and vemurafenib (FIG. 31G). Glucose consumption was decreased in H1734, H1993, and H3122 cells in response to cabozantinib (FIG. 31A). Glucose consumption was decreased in H1229, H1993, H2228, H3122, HCC827, and PC9 cells in response to ceritinib (FIG. 31B). Glucose consumption was decreased in H1229, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells in response to crizotinib (FIG. 31C). Glucose consumption was decreased in H1229, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells in response to dovitinib (FIG. 31D). Glucose consumption was decreased in A549, H1229, H1734, H1993, H2228, H3122, H460, HCC827, and PC9 cells in response to ponatinib (FIG. 31E). Glucose consumption was decreased in H1229, H1734, H1993, H3122, H460, HCC827, and PC9 cells in response to trametinib (FIG. 31F). Glucose consumption was decreased in A549, H1734, H3122, H460, HCC827, and PC9 cells in response to vemurafenib (FIG. 31G). These experiments demonstrate that kinases are strong drivers of glucose consumption.
[0173] For a second set of experiments, the inventors measured the growth of the nine lung cancer cell lines after treatment with kinase inhibitors. In FIG. 21, the lung cancer cell lines were treated with increasing concentrations of afatinib, and cell growth was measured 72 hours after treatment. Cell growth was decreased for A549, H1229, H1734, HCC827, and PC9 cells. In FIG. 22, the lung cancer cell lines were treated with increasing concentrations of erlotinib, and cell growth was measured 48 hours after treatment. Cell growth was decreased for HCC1745, HCC827 and PC9 cells. These experiments demonstrate that kinases are strong drivers of cancer cell growth.
[0174] When investigating the sensitivity and specificity of using early changes in glucose consumption in response to a kinase inhibitor as a predictive biomarker of drug efficacy, the inventors found that using cut-offs of a >40% inhibition of cell growth and a 21% decrease in glucose consumption, the approach had a sensitivity of 77% and a specificity of 79%. A >20% change in 18F-FDG is unlikely to be due to random fluctuations or measurement error (Weber et al., E r J Nucl Med Mol Imaging, 2006), and a >40% change in tumor volume is similar to the RECIST criteria of a 30% decrease in the largest tumor diameters. This indicates that early inhibition of glucose consumption is a strong predictor of drug therapeutic efficacy.
[0175] Further supporting this notion, FIG. 24 shows that glucose consumption by and growth of H460 cancer cells are decreased after treatment with dovitinib. Similarly, FIGs. 25A-25C show that in vivo glucose consumption by and growth of PC9 lung cancer cells are decreased after treatment with dovitinib or erlotinib. Specifically, pre- and post-treatment 18F- FDG PET imaging of glucose consumption by PC9 tumors in mice treated with vehicle (top), dovitinib (middle), or erlotinib (bottom) (FIG. 25A) showed significant drug-induced decreases in glucose consumption by PC9 tumors in mice (FIG. 25B). Additionally, as shown in FIG. 25C, quantification of drug-induced changes in PC9 tumor size after up to 12 days of treatment with kinase inhibitors revealed a decrease in PC9 tumor size.
[0176] Finally, in addition to demonstrating that early inhibition of glucose consumption is a strong predictor of drug therapeutic efficacy, the inventors have also demonstrated that early inhibition of glucose consumption is necessary for drug therapeutic efficacy. As shown in FIGs. 26A-26D, when GLUT1 is overexpressed in H460 (FIG. 26A and FIG. 26B) or HCC827 (FIG. 26C and FIG 26D) lung cancer cells, treatment with kinase inhibitors fails to inhibit glucose consumption and cell growth.
[0177] In summary, the inventors established that early decreases in cancer cell glucose consumption in response to a kinase inhibitor are a strong predictor of eventual changes in cell growth across kinase inhibitors, drug concentrations, and lung cancer genetics in culture and in vivo. Further, early decreases in cancer cell glucose consumption in response to a kinase inhibitor is necessary for the inhibitor to inhibit cancer cell growth. These studies provide a strong rationale for use of early 18F-FDG PET scans to predict therapeutic efficacy. [0178] Understanding the signaling pathways that drive 18F-FDG consumption in different cancers can lead to: better patient stratification based on knowledge of how 18F-FDG PET signal relates to underlying tumor biology; identification of new therapeutic targets for which 18F-FDG PET could serve as a pharmacodynamics biomarker; and development of a rational approach for using 18F-FDG PET as a pharmacodynamics biomarker of current therapies. Early predictive 18F-FDG PET-based biomarkers of drug efficacy could improve the care of cancer patients by: increasing the percent of patients treated with an effective therapy; reducing unnecessary side effects in patients who will not benefit from the drug; and allowing patients who will not benefit from the drug to seek additional treatments sooner.
3. Pacritinib reduces glucose consumption in a subset of lung squamous cell carcinoma by inhibiting the expression of Hexokinase I and II
[0179] Squamous cells carcinoma (SqCLC) represents 30% of all cases of non-small cell lung cancer (NSCLC) and it is associated with smoking. Currently there are few molecular- targeted therapies for this subset of patients pointing out to the need to identify new targetable pathways.
[0180] Using the inventors ’ previously validated high throughput assay, the inventors tested 3555 bioactive small molecules from four small molecule libraries against three squamous cell lung carcinoma cell lines (H520, SK-MES-1, and H596 cells) to identify compounds that decrease glucose consumption per cell.
[0181] This screen yielded a list of novel inhibitors of glucose consumption in squamous cell lung carcinoma cell lines that includes clinically relevant kinase inhibitors (z.e., pacritinib and dovitinib) and topoisomerase inhibitors (z.e., camptothecin and idarubicin). From this list, the inventors prioritized compounds (1) that decreased glucose consumption in all three cell lines with an EC50 value <~1 pM; (2) that had been used in mice and humans; (3) that had not been previously linked to glucose consumption; and (4) that did not dramatically decrease cell growth at the time point measured (24 hours after treatment). This led the inventors to further investigate pacritinib, a Janus kinase 2 (JAK2) and Fms Related Receptor Tyrosine Kinase 3 (FLT3) inhibitor used for the treatment of myelofibrosis.
[0182] As shown in FIGs. 27A and 27B, pacritinib reduced glucose consumption (FIG. 27A) in all three SqCLC cell with an EC 50 of -1.2 pM and without significantly altering cell growth at 24h post-treatment (FIG. 27B). This effect required at least a 16h incubation time, suggesting that the decrease in glucose consumption is likely due to alterations in transcription or translation and not due to the direct inhibition of proteins involved in glucose consumption (/'.<?., glucose transporters GLUT1 and GLUT3, hexokinases HKI and HKII).
[0183] As shown in FIGs. 28A and 28B, Western blot and Q-PCR analyses show that pacritinib reduced the mRNA (FIG. 28A) and protein (FIG. 28B) levels of HKI and HKII without affecting expression levels of GLUT1 and GLUT3 in all three SqCLC cell lines, suggesting that pacritinib blocks glucose consumption by decreasing HKI and HKII levels. [0184] Pacritinib is a well-known inhibitor of JAK2 and FLT3, so the inventors investigated whether the inhibition of one of the known targets results in a decrease of HKI and HKII levels and therefore a decrease in glucose consumption. The inventors overexpressed JAK2 and FLT3 in SK-MES-1, H520 and H596 and measured the effect of 24h incubation with pacritinib. The rationale for this experiment was that only the overexpression of the target protein should reduce the ability of pacritinib to affect glucose consumption. Overexpression of FLT3 dramatically decreased the ability of pacritinib to alter glucose consumption (FIG. 29), whereas overexpression of JAK2 did not have any effect (data not shown). Consistent with the role of FLT3 in regulating Hexokinase I and II expression, overexpression of FLT3 resulted in an increase in the protein levels of the hexokinases and a consequent increase in glucose consumption.
[0185] To further confirm that pacritinib regulates HKI and HK2 via FLT3, the inventors tested the effect of other inhibitors that have a Ki for FLT3 similar to or lower than pacritinib: Quizartinib, Dovitinib, Cabozantinib, and FLT3 inhibitor. As shown in FIGs. 30A-30C, all inhibitors tested reduced glucose consumption in SK-MES-1, H20 and H596 similar to pacritinib, suggesting that glucose consumption is regulated via FLT3.
* * *
[0186] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

WHAT IS CLAIMED:
1. A method of treating a subject for cancer comprising administering a dose of a kinase inhibitor to the subject, wherein a first glucose consumption level and a second glucose consumption level were measured in a tumor of the subject, wherein a previous dose of the kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, wherein the second glucose consumption level was reduced relative to the first glucose consumption level, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
2. The method of claim 1, wherein the second glucose consumption level was measured at most 48 hours after administering the previous dose of the kinase inhibitor.
3. The method of claim 2, wherein the second glucose consumption level was measured at most 36 hours after administering the previous dose of the kinase inhibitor.
4. The method of claim 3, wherein the second glucose consumption level was measured at most 24 hours after administering the previous dose of the kinase inhibitor.
5. The method of claim 4, wherein the second glucose consumption level was measured at most 18 hours after administering the previous dose of the kinase inhibitor.
6. The method of any of claims 1-5, wherein the dose of the kinase inhibitor is the same amount as the previous dose of the kinase inhibitor.
7. The method of any of claims 1-5, wherein the dose of the kinase inhibitor is a lower amount than the previous dose of the kinase inhibitor.
8. The method of any of claims 1-7, wherein the first glucose consumption level and the second glucose consumption level were measured using positron emission tomography (PET).
9. The method of any of claims 1-8, wherein the kinase inhibitor is a Fms Related Receptor Tyrosine Kinase 3 (FLT3) inhibitor.
10. The method of any of claims 1-8, wherein the kinase inhibitor is afatinib.
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11. The method of any of claims 1-8, wherein the kinase inhibitor is buparlisib.
12. The method of any of claims 1-8, wherein the kinase inhibitor is cabozantinib.
13. The method of any of claims 1-8, wherein the kinase inhibitor is ceritinib.
14. The method of any of claims 1-8, wherein the kinase inhibitor is crizotinib.
15. The method of any of claims 1-8, wherein the kinase inhibitor is dovitinib.
16. The method of any of claims 1-8, wherein the kinase inhibitor is pacritinib.
17. The method of any of claims 1-8, wherein the kinase inhibitor is ponatinib.
18. The method of any of claims 1-8, wherein the kinase inhibitor is trametinib.
19. The method of any of claims 1-8, wherein the kinase inhibitor is vemurafenib.
20. The method of any of claims 1-8, wherein the kinase inhibitor is quizartinib.
21. The method of any of claims 1-8, wherein the kinase inhibitor is cabozantinib.
22. The method of any of claims 1-8, wherein the kinase inhibitor is TCS 359.
23. The method of any of claims 1-22, wherein the cancer is lung cancer.
24. The method of claim 23, wherein the cancer is non- small cell lung cancer.
25. The method of claim 24, wherein the cancer is squamous cell lung carcinoma.
26. A method of treating a subject for cancer comprising administering a cancer therapy to the subject, wherein a first glucose consumption level and a second glucose consumption level were measured in a tumor of the subject, wherein a dose of a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, wherein the second glucose consumption level was not reduced relative to the first glucose consumption level, and wherein the cancer therapy does not comprise the kinase inhibitor, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
48
27. The method of claim 26, wherein the second glucose consumption level was measured at most 48 hours after administering the dose of the kinase inhibitor.
28. The method of claim 27, wherein the second glucose consumption level was measured at most 36 hours after administering the dose of the kinase inhibitor.
29. The method of claim 28, wherein the second glucose consumption level was measured at most 24 hours after administering the dose of the kinase inhibitor.
30. The method of claim 29, wherein the second glucose consumption level was measured at most 18 hours after administering the dose of the kinase inhibitor.
31. The method of any of claims 26-30, wherein the first glucose consumption level and the second glucose consumption level were measured using PET.
32. The method of any of claims 26-31, wherein the kinase inhibitor is a FLT3 inhibitor.
33. The method of any of claims 26-31, wherein the kinase inhibitor is afatinib,.
34. The method of any of claims 26-31, wherein the kinase inhibitor is buparlisib.
35. The method of any of claims 26-31, wherein the kinase inhibitor is cabozantinib.
36. The method of any of claims 26-31, wherein the kinase inhibitor is ceritinib.
37. The method of any of claims 26-31, wherein the kinase inhibitor is crizotinib.
38. The method of any of claims 26-31, wherein the kinase inhibitor is dovitinib.
39. The method of any of claims 26-31, wherein the kinase inhibitor is pacritinib.
40. The method of any of claims 26-31, wherein the kinase inhibitor is ponatinib.
41. The method of any of claims 26-31, wherein the kinase inhibitor is trametinib.
42. The method of any of claims 26-31, wherein the kinase inhibitor is vemurafenib.
43. The method of any of claims 26-31, wherein the kinase inhibitor is quizartinib.
44. The method of any of claims 26-31, wherein the kinase inhibitor is cabozantinib.
49
45. The method of any of claims 26-31, wherein the kinase inhibitor is TCS 359.33
46. The method of any of claims 26-45, wherein the cancer therapy is an alternative kinase inhibitor.
47. The method of any of claims 26-45, wherein the cancer therapy is chemotherapy, radiotherapy, or immunotherapy.
48. The method of any of claims 26-47, wherein the cancer is lung cancer.
49. The method of claim 48, wherein the cancer is non-small cell lung cancer.
50. The method of claim 48, wherein the cancer is squamous cell lung carcinoma.
51. A method for evaluating efficacy of a kinase inhibitor in a subject having cancer, the method comprising:
(a) measuring a first glucose consumption level in a tumor of the subject;
(b) administering a dose of the kinase inhibitor to the subject; and
(c) measuring a second glucose consumption level in the tumor at most 48 hours after administering the kinase inhibitor to the subject, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
52. The method of claim 51, wherein the second glucose consumption level is measured at most 36 hours after administering the dose of the kinase inhibitor.
53. The method of claim 52, wherein the second glucose consumption level is measured at most 24 hours after administering the dose of the kinase inhibitor.
54. The method of claim 53, wherein the second glucose consumption level is measured at most 18 hours after administering the dose of the kinase inhibitor.
55. The method of any of claims 51-54, wherein measuring the first glucose consumption level comprises administering fluorodeoxyglucose to the subject.
50
56. The method of any of claims 51-55, wherein measuring the first glucose consumption level comprises PET.
57. The method of any of claims 51-56, wherein measuring the second glucose consumption level comprises PET.
58. The method of any of claims 51-57, wherein measuring the second glucose consumption level comprises administering fluorodeoxyglucose to the subject.
59. The method of any of claims 51-58, wherein the kinase inhibitor is a FLT3 inhibitor.
60. The method of any of claims 51-58, wherein the kinase inhibitor is afatinib.
61. The method of any of claims 51-58, wherein the kinase inhibitor is buparlisib.
62. The method of any of claims 51-58, wherein the kinase inhibitor is cabozantinib.
63. The method of any of claims 51-58, wherein the kinase inhibitor is ceritinib.
64. The method of any of claims 51-58, wherein the kinase inhibitor is crizotinib.
65. The method of any of claims 51-58, wherein the kinase inhibitor is dovitinib.
66. The method of any of claims 51-58, wherein the kinase inhibitor is pacritinib.
67. The method of any of claims 51-58, wherein the kinase inhibitor is ponatinib.
68. The method of any of claims 51-58, wherein the kinase inhibitor is trametinib.
69. The method of any of claims 51-58, wherein the kinase inhibitor is vemurafenib.
70. The method of any of claims 51-58, wherein the kinase inhibitor is quizartinib.
71. The method of any of claims 51-58, wherein the kinase inhibitor is cabozantinib.
72. The method of any of claims 51-58, wherein the kinase inhibitor is TCS 359.60
73. The method of any of claims 51-72, further comprising identifying the kinase inhibitor as effective, wherein the second glucose consumption level is reduced compared to the first glucose consumption level.
74. The method of any of claims 51-72, further comprising (d) administering an additional dose of the kinase inhibitor to the subject, wherein the second glucose consumption level is reduced compared to the first glucose consumption level.
75. The method of claim 74, wherein the additional dose of the kinase inhibitor is the same amount as the dose of the kinase inhibitor.
76. The method of claim 74, wherein the additional dose of the kinase inhibitor is a lower amount than the dose of the kinase inhibitor.
77. The method of any of claims 51-72, further comprising identifying the kinase inhibitor as ineffective, wherein the second glucose consumption level is not reduced compared to the first glucose consumption level.
78. The method of any of claims 51-72, further comprising (d) administering an alternative cancer therapy, wherein the second glucose consumption level is not reduced compared to the first glucose consumption level.
79. The method of claim 78, wherein the alternative cancer therapy is an alternative kinase inhibitor.
80. The method of claim 78, wherein the alternative therapy is chemotherapy, radiotherapy, or immunotherapy.
81. The method of any of claims 51-80, wherein the cancer is lung cancer.
82. The method of claim 81, wherein the cancer is non-small cell lung cancer.
83. The method of claim 82, wherein the cancer is squamous cell lung carcinoma.
84. A method for evaluating efficacy of a kinase inhibitor in a subject having cancer, the method comprising:
(a) administering the kinase inhibitor to the subject; and
(b) measuring a glucose consumption level in a tumor of the subject at most 48 hours after administering the kinase inhibitor, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
85. The method of claim 84, wherein the glucose consumption level is measured at most 36 hours after administering the dose of the kinase inhibitor.
86. The method of claim 85, wherein the second glucose consumption level is measured at most 24 hours after administering the dose of the kinase inhibitor.
87. The method of claim 86, wherein the second glucose consumption level is measured at most 18 hours after administering the dose of the kinase inhibitor.
88. The method of any of claims 84-87, wherein measuring the glucose consumption level comprises administering fluorodeoxyglucose to the subject.
89. The method of any of claims 84-87, wherein measuring the glucose consumption level comprises PET.
90. The method of any of claims 84-89, wherein the kinase inhibitor is a FLT3 inhibitor.
91. The method of any of claims 84-89, wherein the kinase inhibitor is afatinib.
92. The method of any of claims 84-89, wherein the kinase inhibitor is buparlisib.
93. The method of any of claims 84-89, wherein the kinase inhibitor is cabozantinib.
94. The method of any of claims 84-89, wherein the kinase inhibitor is ceritinib.
95. The method of any of claims 84-89, wherein the kinase inhibitor is crizotinib.
96. The method of any of claims 84-89, wherein the kinase inhibitor is dovitinib.
97. The method of any of claims 84-89, wherein the kinase inhibitor is pacritinib.
98. The method of any of claims 84-89, wherein the kinase inhibitor is ponatinib.
99. The method of any of claims 84-89, wherein the kinase inhibitor is trametinib.
100. The method of any of claims 84-89, wherein the kinase inhibitor is vemurafenib.
53
101. The method of any of claims 84-89, wherein the kinase inhibitor is quizartinib.
102. The method of any of claims 84-89, wherein the kinase inhibitor is cabozantinib.
103. The method of any of claims 84-89, wherein the kinase inhibitor is TCS 359.91
104. The method of any of claims 84-103, wherein a reference glucose consumption level was measured in the tumor prior to administering the kinase inhibitor to the subject.
105. The method of claim 104, further comprising administering an additional dose of the kinase inhibitor to the subject, wherein the glucose consumption level is reduced relative to the reference glucose consumption level.
106. The method of claim 104, further comprising administering an alternative cancer therapy to the subject, wherein the glucose consumption level is not reduced relative to the reference glucose consumption level.
107. The method of claim 106, wherein the alternative cancer therapy is an alternative kinase inhibitor.
108. The method of claim 106, wherein the alternative therapy is chemotherapy, radiotherapy, or immunotherapy.
109. The method of any of claims 84-108, wherein the cancer is lung cancer.
110. The method of claim 109, wherein the cancer is non-small cell lung cancer.
111. The method of claim 110, wherein the cancer is squamous cell lung carcinoma.
112. A method of treating a subject for cancer comprising:
(a) selecting a subject for treatment, wherein a first glucose consumption level and a second glucose consumption level were measured in the subject, wherein a first dose of a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was reduced relative to the first glucose consumption level; and
54 (b) administering to the subject a second dose of the kinase inhibitor, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
113. The method of claim 112, wherein the second glucose consumption level was measured at most 48 hours after administering the first dose of the kinase inhibitor.
114. The method of claim 113, wherein the second glucose consumption level was measured at most 36 hours after administering the first dose of the kinase inhibitor.
115. The method of claim 114, wherein the second glucose consumption level was measured at most 24 hours after administering the first dose of the kinase inhibitor.
116. The method of claim 115, wherein the second glucose consumption level was measured at most 18 hours after administering the first dose of the kinase inhibitor.
117. The method of any of claims 112-116, wherein the first glucose consumption level and the second glucose consumption level were measured using PET.
118. The method of any of claims 112-117, wherein the second dose of the kinase inhibitor is the same amount as the first dose of the kinase inhibitor.
119. The method of any of claims 112-117, wherein the second dose of the kinase inhibitor is a lower amount than the first dose of the kinase inhibitor.
120. The method of any of claims 112-119, wherein the kinase inhibitor is a FLT3 inhibitor.
121. The method of any of claims 112-119, wherein the kinase inhibitor is afatinib.
122. The method of any of claims 112-119, wherein the kinase inhibitor is buparlisib.
123. The method of any of claims 112-119, wherein the kinase inhibitor is cabozantinib.
124. The method of any of claims 112-119, wherein the kinase inhibitor is ceritinib.
125. The method of any of claims 112-119, wherein the kinase inhibitor is crizotinib.
55
126. The method of any of claims 112-119, wherein the kinase inhibitor is dovitinib.
127. The method of any of claims 112-119, wherein the kinase inhibitor is pacritinib.
128. The method of any of claims 112-119, wherein the kinase inhibitor is ponatinib.
129. The method of any of claims 112-119, wherein the kinase inhibitor is trametinib.
130. The method of any of claims 112-119, wherein the kinase inhibitor is vemurafenib.
131. The method of any of claims 112-119, wherein the kinase inhibitor is quizartinib.
132. The method of any of claims 112-119, wherein the kinase inhibitor is cabozantinib.
133. The method of any of claims 112-119, wherein the kinase inhibitor is TCS 359.121
134. The method of any of claims 112-133, wherein the cancer is lung cancer.
135. The method of claim 134, wherein the cancer is non-small cell lung cancer.
136. The method of claim 135, wherein the cancer is squamous cell lung carcinoma.
137. A method of treating a subject for cancer comprising:
(a) selecting a subject for treatment, wherein a first glucose consumption level and a second glucose consumption level were measured in the subject, wherein a kinase inhibitor was administered to the subject after measuring the first glucose consumption level and before measuring the second glucose consumption level, and wherein the second glucose consumption level was not reduced compared to the first glucose consumption level; and
(b) administering an alternative cancer therapy to the subject, wherein the alternative cancer therapy does not comprise the kinase inhibitor, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
138. The method of claim 137, wherein the alternative cancer therapy is an alternative kinase inhibitor.
56
139. The method of claim 137, wherein the alternative therapy is chemotherapy, radiotherapy, or immunotherapy.
140. The method of any of claims 137-139, wherein the kinase inhibitor is afatinib.
141. The method of any of claims 137-139, wherein the kinase inhibitor is buparlisib.
142. The method of any of claims 137-139, wherein the kinase inhibitor is cabozantinib.
143. The method of any of claims 137-139, wherein the kinase inhibitor is ceritinib.
144. The method of any of claims 137-139, wherein the kinase inhibitor is crizotinib.
145. The method of any of claims 137-139, wherein the kinase inhibitor is dovitinib.
146. The method of any of claims 137-139, wherein the kinase inhibitor is pacritinib.
147. The method of any of claims 137-139, wherein the kinase inhibitor is ponatinib.
148. The method of any of claims 137-139, wherein the kinase inhibitor is trametinib.
149. The method of any of claims 137-139, wherein the kinase inhibitor is vemurafenib.
150. The method of any of claims 137-139, wherein the kinase inhibitor is quizartinib.
151. The method of any of claims 137-139, wherein the kinase inhibitor is cabozantinib.
152. The method of any of claims 137-139, wherein the kinase inhibitor is TCS 359.
153. The method of any of claims 137-139, wherein the cancer is lung cancer.
154. The method of claim 153, wherein the cancer is non-small cell lung cancer.
155. The method of claim 154, wherein the cancer is squamous cell lung carcinoma.
156. A method of treating a subject for cancer, the method comprising:
(a) measuring a first glucose consumption level in a tumor of the subject;
(b) administering to the subject a first dose of a kinase inhibitor;
57 (c) subsequent to (b), measuring a second glucose consumption level in the tumor; and
(d) administering to the subject a second dose of the kinase inhibitor, wherein the second glucose consumption level is reduced relative to the first glucose consumption level, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
157. The method of claim 156, wherein the second glucose consumption level was measured at most 48 hours after administering the first dose of the kinase inhibitor.
158. The method of claim 156 or 157, wherein the kinase inhibitor is a FLT3 inhibitor.
159. The method of claim 156 or 157, wherein the kinase inhibitor is afatinib.
160. The method of claim 156 or 157, wherein the kinase inhibitor is buparlisib.
161. The method of claim 156 or 157, wherein the kinase inhibitor is cabozantinib.
162. The method of claim 156 or 157, wherein the kinase inhibitor is ceritinib.
163. The method of claim 156 or 157, wherein the kinase inhibitor is crizotinib.
164. The method of claim 156 or 157, wherein the kinase inhibitor is dovitinib.
165. The method of claim 156 or 157, wherein the kinase inhibitor is pacritinib.
166. The method of claim 156 or 157, wherein the kinase inhibitor is ponatinib.
167. The method of claim 156 or 157, wherein the kinase inhibitor is trametinib.
168. The method of claim 156 or 157, wherein the kinase inhibitor is vemurafenib.
169. The method of claim 156 or 157, wherein the kinase inhibitor is quizartinib.
170. The method of claim 156 or 157, wherein the kinase inhibitor is cabozantinib.
171. The method of claim 156 or 157, wherein the kinase inhibitor is TCS 359.159
58
172. A method of treating a subject for cancer, the method comprising:
(a) measuring a first glucose consumption level in a tumor of the subject;
(b) administering to the subject a kinase inhibitor;
(c) subsequent to (b), measuring a second glucose consumption level in the tumor; and
(d) administering to the subject an alternative cancer therapy, wherein the second glucose consumption level is not reduced relative to the first glucose consumption level, wherein the alternative cancer therapy does not comprise the kinase inhibitor, wherein the kinase inhibitor is afatinib, buparlisib, cabozantinib, ceritinib, crizotinib, dovitinib, pacritinib, ponatinib, trametinib, vemurafenib, quizartinib, cabozantinib, or TCS 359.
173. The method of claim 172, wherein the second glucose consumption level was measured at most 48 hours after administering the first dose of the kinase inhibitor.
174. The method of claim 172 or 173, wherein the alternative cancer therapy is an alternative kinase inhibitor.
175. The method of claim 172 or 173, wherein the alternative therapy is chemotherapy, radiotherapy, or immunotherapy.
176. The method of any of claims 172-175, wherein the kinase inhibitor is a FLT3 inhibitor.
177. The method of any of claims 172-175, wherein the kinase inhibitor is afatinib.
178. The method of any of claims 172-175, wherein the kinase inhibitor is buparlisib.
179. The method of any of claims 172-175, wherein the kinase inhibitor is cabozantinib.
180. The method of any of claims 172-175, wherein the kinase inhibitor is ceritinib.
181. The method of any of claims 172-175, wherein the kinase inhibitor is crizotinib.
59
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