WO2017062426A1 - Methods and compositions for treatment of metastatic and refractory cancers and tumors - Google Patents
Methods and compositions for treatment of metastatic and refractory cancers and tumors Download PDFInfo
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- WO2017062426A1 WO2017062426A1 PCT/US2016/055458 US2016055458W WO2017062426A1 WO 2017062426 A1 WO2017062426 A1 WO 2017062426A1 US 2016055458 W US2016055458 W US 2016055458W WO 2017062426 A1 WO2017062426 A1 WO 2017062426A1
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Definitions
- T cells crucial for controlling the growth of immunogenic tumors 1 , rely upon many of the same signaling pathways targeted by pharmaceutical inhibitors. For instance, engagement of the T cell receptor (TCR) and co-stimulatory receptors activates the Ras-MAPK (mitogen-activated protein kinase) and PI3K-AKT signaling cascades, which are necessary for proliferation and effector function in T cells 2 . Additionally, the development of myeloid leukocytes also depends upon these pathways 3 .
- TCR T cell receptor
- Ras-MAPK mitogen-activated protein kinase
- PI3K-AKT signaling cascades which are necessary for proliferation and effector function in T cells 2 .
- myeloid leukocytes also depends upon these pathways 3 .
- a method of treating a mammalian subject with cancer comprises
- a composition or kit for treatment of a mammalian subject with a metastatic or refractory cancer or tumor comprises (a) an effective amount of a small molecule kinase inhibitor of a target enzyme in the MEK/MAPK pathway and (b) an effective amount of a molecule that induces T cell proliferation in the presence of said inhibitor.
- the components (a) and (b) are formulated with a pharmaceutically acceptable carrier or diluent.
- FIG. 1 A shows eight plots of targeted small molecule inhibitors that suppress T cell responses in vitro.
- A2780 cells were cultured for 3 days and human T cells were activated with Concanavalin A (ConA) for 7 days in the indicated compounds, BKM120, OSI127, Trametrinib, RAF, LBH589 and PD0332991 and ABT747.
- Plots show the normalized percent inhibition (NPI) vs. concentration in log ( ⁇ ).
- FIG. IB shows human PBMCs that were stained with CELLTRACE stain and activated with either aAPCs or ConA in the presence of inhibitors.
- Four plots show proliferation of CD8 cells after 7 days is shown.
- FIG. 1C shows quantifications of the division index normalized to vehicle for CD4 and CD8 T cells.
- Four box plots show mean, min, and max from three experiments with cells from a single donor, and red lines show values from single experiments with two additional donors.
- FIG. ID is a trace showing proliferation of CD8 cells after 7 days with trametinib.
- FIG. IE are two traces of mouse splenic T cells that, primed with tumor antigen, pulsed DCs in the presence of the inhibitors.
- FIG. IF is a trace showing proliferation of CD8 cells after 7 days is shown.
- FIG. 1G is a bar graph showing mouse splenic T cells that were primed with tumor antigen and pulsed DCs for 7 days.
- FIG 2A is a graph showing that IL-15 can rescue T cell functions from MEK inhibitors.
- FIG. 2B shows human PBMCs stained with CELLTRACE stain and activated with aAPCs +/- IL-15 (10 ng/ml) or ALT-803 (35.7 ng/ml) in the presence of trametinib.
- FIG. 2C is a blot showing FACS-sorted human CD8 T cells that were stimulated by anti-CD3/CD28 crosslinking in the presence of the indicated compounds as described in methods. After 10 min, cells were harvested for Western blotting.
- FIG. 2E shows three graphs of mouse T cells isolated from spleens
- FIG. 2F shows mouse splenic T cells that were primed with tumor antigen-pulsed DCs for 7 days and then recalled with fresh tumor antigen-pulsed DCs in the presence of trametinib +/- ALT-803 (35.7 ng/ml) in an IFN- ⁇ ELISpot assay.
- * P ⁇ 0.05
- FIG. 2G shows mice that were injected with ID8-OVA cells, treated with trametinib (1.0 mg/kg) or vehicle +/- ALT-803 (0.2 mg/kg), and injected with
- FIG. 2H shows quantification of the percent of OT-I T cells from FIG. 2G that divided.
- FIG 3 A illustrates that combination therapy with trametinib and ALT-803 can drive rejection of a murine KRas -mutated breast tumor cell line.
- Brpkpl 10 cells were cultured with vehicle, 8 nM, or 200 nM trametinib and analyzed by Western blot.
- FIG. 3B show mice that were injected with Brpkpl 10 cells subcutaneously (day 0) and treated once daily with trametinib (1.0 mg/kg) beginning on day 3 until day 13.
- ALT- 803 0.2 mg/kg was administered i.p. on days 3, 8, and 13.
- n 20 for trametinib and trametinib + ALT- 803 groups.
- FIG. 3C show four plots showing growth of tumors from individual mice from FIG. 3B.
- FIG. 3D show plots of percentage of mice with tumors ⁇ 100 mm 3 from FIG. 3B.
- Trametinib + ALT survival curve is significant from all other curves, P ⁇ 0.05 with log- rank test.
- FIG. 3E show mice that had rejected tumors from FIG. 3B were rechallenged about 25 days later with Brpkpl 10 cells contralaterally and tumor growth was compared to cells injected into naive mice.
- FIG 4A show that trametinib acts through mechanisms dependent on the microenvironment and CD8 T cells.
- Brpkpl 10 cells were cultured for 2 days with trametinib and proliferation was quantified by MTS assay.
- FIG. 4B show that mice with Brpkpl 10 subcutaneous tumors were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9. Tumors were excised on day 10, frozen in
- FIG. 4D shows LLC cells that were cultured with vehicle, 8 nM, or 200 nM trametinib and analyzed by Western blot.
- FIG. 4E illustrate that for LLC cells cultured for 2 days with trametinib, proliferation was quantified by MTS assay.
- FIG. 4F illustrate mice with intraperitoneal LLC tumors that were gavaged with trametinib once daily from day 4-14.
- FIG. 4G illustrate mice with subcutaneous LLC tumors that were gavaged with trametinib once daily from day 3-13. P ⁇ 0.05, Mann-Whitney.
- FIG. 4H illustrate that Brpkpl 10 tumor-bearing mice were treated as in FIG 4B, except that anti-CD 8a or control anti-LTF was also administered.
- FIG. 41 illustrate mice with Brpkpl 10 subcutaneous tumors that were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9 and harvested on day 10, stimulated with PMA/Ionomycin ex vivo for 5 hrs, and stained for intracellular IFN- ⁇ .
- FIG 5 A illustrates that trametinib reduces the accumulation of Ly6C+ MDSCs in tumors.
- Mice with Brpkpl 10 subcutaneous tumors were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9, harvested on day 10, and analyzed by flow cytometry. Percentages of cell populations found in dissociated tumors from 3 independent experiments.
- FIG. 5B illustrates that mice with Brpkpl 10 subcutaneous tumors were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9, harvested on day 10, and analyzed by flow cytometry. Percentages of cell populations found in dissociated tumors from 3 independent experiments.
- FIG. 5C illustrates that mice with Brpkpl 10 subcutaneous tumors were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9, harvested on day 10, and analyzed by flow cytometry. Percentages of cell populations found in dissociated tumors from 3 independent experiments.
- FIG. 5D illustrates the mice with Brpkpl 10 subcutaneous tumors that were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9, harvested on day 10, and analyzed by flow cytometry.
- This figure shows representative plots of gating for Ly6C hl and Ly6G + from CD1 lb + MHCII " cells in tumors.
- FIG. 5E shows that mice with LLC subcutaneous tumors were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7-9, harvested on day 10, and analyzed by flow cytometry. Percentages of Ly6C hi Ly6G " cells in LLC tumors were from 3 independent experiments.
- FIG. 5F illustrate in two bar graphs that CD1 lb + MHCH “ Ly6C hi , CD1 lb + MHCII "
- Ly6G + , or CDl lc + MHCII + dendritic cells were FACS sorted from advanced tumor bearing or naive mice and analyzed by qPCR. Expression normalized to TATA binding protein is shown.
- FIG. 5G shows in two charts the percentages or total numbers of cell populations from spleens of Brpkpl 10 tumor bearing mice of FIG. 5F from one of 3 independent experiments. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, unpaired t test.
- FIG. 5H shows in two charts the percentages or total numbers of cell populations from spleens of Brpkp 110 tumor bearing mice of FIG. 5F from one of 3 independent experiments. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, unpaired t test.
- FIG. 51 shows in two charts the percentages or total numbers of cell populations from spleens of Brpkp 110 tumor bearing mice of FIG. 5F from one of 3 independent experiments. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, unpaired t test.
- FIG 6A illustrates that trametinib selectively reduces the differentiation of Ly6C + MDSCs from bone marrow.
- MDSCs were differentiated from mouse bone marrow with IL-6 and GM-CSF in the presence of trametinib (200 nM) for 4 days. Shown are representative plots from 4 experiments. *p ⁇ 0.05, Mann-Whitney test.
- FIG. 6B shows the total number of cells from 4 experiments described as in FIG. 6A. *p ⁇ 0.05, Mann-Whitney test.
- FIG. 6C shows that MDSCs were differentiated from mouse bone marrow with Brpkpl 10 conditioned medium (50%) in the presence of trametinib (200 nM) or vehicle for 4 days by representative plots from 4 experiments. *p ⁇ 0.05, Mann-Whitney test.
- FIG. 6D shows the total number of cells from 4 experiments as described in FIG. 6C. *p ⁇ 0.05, Mann- Whitney test.
- FIG. 6E shows that MDSCs differentiated with Brpkpl 10 conditioned medium were added to mouse splenocytes activated with anti-CD3 and anti-CD28 and cultured for 3 days.
- T trametinib 200 nM.
- FIG 7A illustrates that osteopontin chemoattracts MDSCs and is reduced by trametinib treatment of tumor cells.
- LC-MS/MS data are shown from supernatants of
- Y axis MS count (abundance) in vehicle supernatants.
- FIG. 7B shows osteopontin concentration measured from supernatants of Brpkpl 10 cells cultured overnight in the indicated conditions.
- FIG. 7C shows osteopontin concentration from plasma samples collected from Brpkpl 10-bearing mice (or naive tumor-free mice) gavaged with trametinib on days 7-9, and harvested on day 10.
- FIG. 7D shows that GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G " populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis).
- FIG. 7E shows that GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G " populations and assayed for their ability to migrate in a transwell assay towards osteopontin (chemotaxis) or within the presence of osteopontin (chemokinesis).
- FIG. 8 illustrates in 16 plots that stimulation strength affects T cell sensitivity to PI3K and MEK inhibitors.
- Human PBMCs were stained with CELLTRACE and activated with either aAPCs loaded with anti-CD3 and anti-CD28 or anti-CD3 alone at various concentrations in the presence of inhibitors. Proliferation of CD8 and CD4 cells after 7 days is shown.
- FIG. 9A illustrates that OSI906 preserves T cell function at the concentrations required to inhibit MCF-7 cells.
- Plot shows NPI vs. concentration.
- FIG. 9B shows that human PBMCs were stained with CELLTRACE reagent and activated with aAPCs in the presence of OSI906. Proliferation of CD8 cells after 7 days is shown.
- FIG. 9C shows that human A2780 and MCF-7 cells and murine ID8 cells were blotted for IGF-1R expression.
- FIG. 9D shows the division indices of CD8 T cells from the experiment described in FIG. 9B are plotted relative to vehicle treatment alongside the proliferation of MCF-7 cells treated with OSI906 for 2 days, measured by percent of vehicle in an MTS assay. Representative results from one of two independent experiments.
- FIG. 9E shows the division indices of CD4 T cells from the experiment described in FIG. 9B are plotted relative to vehicle treatment alongside the proliferation of MCF-7 cells treated with OSI906 for 2 days, measured by percent of vehicle in an MTS assay. Representative results from one of two independent experiments.
- FIG. 10 is a table that illustrates that IL-15, IL-2, and IL-7 can rescue T cell proliferation from MEK inhibition.
- IL-2 20 U/ml
- IL-7 2 ng/ml
- IL-15 10 ng/ml
- IL-21 100 ng/ml
- IL-27 50 ng/ml
- FIGs. 11A, 11B and 11C show that trametinib does not significantly affect macrophage or Treg proportions in Brpkpl 10 mice.
- Mice with Brpkpl 10 subcutaneous tumors were gavaged with trametinib (1.0 mg/kg) or vehicle on days 7, 8, 9 and harvested on day 10 and analyzed by flow cytometry. Percentages of indicated cell populations found in dissociated tumors.
- FIG. 12 illustrates that isolation of Ly6G + and Ly6G " bone marrow-derived MDSCs.
- GM-CSF and IL-6 in vitro derived MDSCs were separated with Ly6G-MACS microbeads into Ly6G + and Ly6G " populations.
- Pre- and post-sort cell populations were analyzed for Ly6G and Ly6C expression by flow cytometry.
- T cell activation is necessary for control of cancer cell growth, spread and tumor reduction, such suppressive effects on T cell activation are not desirable in a cancer therapy or therapeutic.
- some signaling molecule inhibitors require the presence of CD8 T cells for tumor suppression, and are largely independent of direct inhibition of tumor cell proliferation.
- the MEK inhibitor, trametinib impairs the
- MDSCs Myeloid-Derived Suppressor Cells
- the inventors have shown that the combined anti-inflammatory activity of trametinib on multiple cell types, irrespective of the tumor cell cycle, could be responsible for its anti-tumor effects.
- the inventors have identified an explanation for the effect of such inhibitors, e.g., MEK inhibitors, on MDSCs and thus provide methods and compositions to overcome the suppressive effects of inhibitors of signaling molecule pathways on T cells in the treatment of cancers, including refractory cancers.
- a method of treating a mammalian subject with cancer comprises, in one aspect, administering to the subject having a cancer, including a metastatic or refractory cancer or tumor, a combination therapy.
- the combination therapy involves administering a small molecule kinase inhibitor of a target enzyme of the MEK/MAPK pathway and a molecule that induces T cell proliferation in the presence of the inhibitor.
- the combination of components (a) and (b) reduce the proliferation of the cancer and tumor cells in vivo.
- compositions or kits containing components (a) and (b) are further described herein for use in the treatment of cancer.
- a or “an” refers to one or more, for example, “an inhibitor” is understood to represent one or more such compounds, molecules, peptides or antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used
- the term "subject" as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research.
- the subject of these methods and compositions is a human.
- Still other suitable subjects include, without limitation, murine, rat, canine, feline, porcine, bovine, ovine, and others.
- neoplastic disease refers to any disease, condition, trait, genotype or phenotype characterized by unregulated or abnormal cell growth, proliferation or replication.
- a “cancer cell” is a cell that divides and reproduces abnormally with uncontrolled growth. This cell can break away from the site of its origin (e.g., a tumor) and travel to other parts of the body and set up another site (e.g., another tumor), in a process referred to as metastasis.
- a “tumor” is an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, and is also referred to as a neoplasm. Tumors can be either benign (not cancerous) or malignant.
- the abnormal proliferation of cells may result in a localized lump or tumor, be present in the lymphatic system, or may be systemic.
- the neoplastic disease is benign.
- the neoplastic disease is pre-malignant, i.e. , potentially malignant neoplastic disease.
- the neoplastic disease is malignant, i.e., cancer.
- the neoplastic disease is a refractory cancer, i.e., a cancer or tumor thai does not respond to treatment, such as surgery, radiation or chemotherapy.
- the cancer or tumor may be resistant at the beginning of treatment or it may become resistant during treatment.
- the cancer or tumor is a metastasis of an original cancer, i.e., formed by cells that have spread from the original site of the cancer or tumor.
- the cancer can include, without limitation, breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, leukemia, myeloma, lymphoma, glioma, and multidrug resistant cancer.
- the neoplastic disease is Kaposi's sarcoma, Merkel cell carcinoma, hepatocellular carcinoma (liver cancer), cervical cancer, anal cancer, penile cancer, vulvar cancer, vaginal cancer, neck cancer, head cancer, multicentric Castleman's disease, primary effusion lymphoma, tropical spastic paraparesis, adult T-cell leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, post-transplantation lymphoproliferative disease, nasopharyngeal carcinoma, pleural mesothelioma (cancer of the lining of the lung), osteosarcoma (a bone cancer), ependymoma and choroid plexus tumors of the brain, and non-Hodgkin's lymphoma.
- Kaposi's sarcoma Merkel cell carcinoma, hepatocellular carcinoma (liver cancer), cervical cancer, anal cancer, penile cancer, vulvar cancer, vaginal cancer,
- the cancer may be a systemic cancer, such as leukemia.
- the cancer is a human glioblastoma.
- the cancer is a prostate adenocarcinoma.
- the cancer is a lung adenocarcinoma.
- the cancer is non-small cell lung adenocarcinoma (NSCLC).
- the cancer is squamous cell carcinoma.
- the cancer is liver cancer.
- the cancer is a multidrug resistant cancer.
- the cancer is a drug resistant cancer.
- the cancer is melanoma. In another embodiment, the cancer is a metastatic or refractory melanoma. In another embodiment, the cancer is a breast cancer. In another embodiment, the cancer is a metastatic or refractory breast cancer, e.g., adenocarcinoma.
- the methods of inhibiting growth or spread of a cancer are practiced when the subject has an established malignancy, or a refractory cancer, or a metastatic cancer. Similarly such methods are useful when the subject is newly diagnosed and prior to treatment.
- reduce refers to the ability of the components (a) and/or (b) or composition described herein to inhibit, retard, suppress, or reduce the growth, proliferation, spread, mestatasis, or refractory behavior of the cancer cells or tumor in the subject.
- treating or “treatment” is meant to encompass administering to a subject one or more compounds or compositions described herein for the purposes of amelioration of one or more symptoms of a cancer.
- a treatment includes inhibiting the growth, proliferation, size and/or spread of the cancer or tumor.
- administering or “route of administration” is delivery of the small molecule kinase inhibitor of a target enzyme of the MEK/MAPK pathway and/or the molecule that induces T cell proliferation, with or without a pharmaceutical carrier or excipient, or with or without another chemotherapeutic agent into the subject systemically or into the environment of the cancer cell or tumor microenvironment of the subject.
- Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, systemic routes, such as intraperitoneal, intravenous, intranasal, intravenous, intramuscular, intratracheal, subcutaneous, and other parenteral routes of administration or intratumoral or intranodal administration.
- the route of administration is oral.
- the route of administration is
- the route of administration is intraperitoneal.
- the route of administration is intravascular. Routes of administration may be combined, if desired. In some embodiments, the administration is repeated periodically.
- chemotherapeutic agent or therapy is meant a drug or therapy designed for using in treating cancers.
- chemotherapeutic agent or therapy is meant a drug or therapy designed for using in treating cancers.
- One of skill in the art would readily be able to select a chemotherapeutic for formulations with or for administration with the small molecule inhibitor of a target enzyme of the MEK/MAPK pathway and/or the molecule that induces T cell proliferation based on consideration of such factors as the cancer being treated and stage of the cancer, the subject's age and physical condition, among others factors.
- chemotherapeutics which may be utilized as described herein include, without limitation,cisplatin, carboplatin, 5-fluorouracil, cyclophosphamide, Oncovin, vincristine, prednisone, rituximab, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine, lomustine, semustine,
- thriethylenemelamine triethylene thiophosphoramide, hexamethylmelamine altretamine, busulfan, triazines dacarbazine, methotrexate, trimetrexate, fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5-azacytidine, 2,2'-difluorodeoxycytidine, 6- mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin,
- erythrohydroxynonyladenine fludarabine phosphate, 2-chlorodeoxyadenosine, camptothecin, topotecan, irinotecan, paclitaxel, vinblastine, vincristine, vinorelbine, docetaxel, estramustine, estramustine phosphate, etoposide, teniposide, mitoxantrone, mitotane, or aminoglutethimide.
- Other therapies for use with the methods and compositions using the small molecule inhibitor of a target signaling molecule/enzyme of the MEK/MAPK pathway and/or the molecule that induces T cell proliferation as described herein include non-chemical therapies.
- the additional or adjunctive therapy includes, without limitation, radiation, acupuncture, surgery, chiropractic care, passive or active immunotherapy, X-ray therapy, ultrasound, diagnostic measurements, e.g. , blood testing.
- these therapies are be utilized to treat the patient.
- these therapies are utilized to determine or monitor the progress of the disease, the course or status of the disease, relapse or any need for booster administrations of the compounds discussed herein.
- MEK/MAPK pathway or “MAPK signaling pathway” refers to a chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell and play a key role in the regulation of gene expression, cellular growth, and survival.
- MAPK signaling is initiated by receptor tyrosine kinases upon their activation by growth factors in the
- Adaptor molecules that interact directly with the intracellular portion of the receptor mediate the recruitment and activation of signaling molecules of this pathway.
- target signaling molecule/enzyme of the MEK/MAPK pathway includes Ras, Raf, MEK, and ERK, the latter also known as MAPK.
- MEK 1 and 2 are dual specificity threonine/tyrosine kinases often upregulated in various cancer cell types and play a key role in the activation of the signaling pathway that regulates cell growth.
- inhibitor or antagonist of a target signaling molecule or enzyme of the
- MEK/MAPK pathway includes small chemical/pharmaceutical molecules, peptides, nucleotide sequences, e.g., siRNA or shRNAs, and intracellular antibodies that have the ability to penetrate the cell and bind or interact with the targeted MEK/MAPK pathway gene or its expression product so as to prevent or inhibit or oppose the normal expression or activity of the targeted member of the pathway or to interrupt, prevent or inhibit or oppose the normal activity of the pathway itself.
- This inhibition suppresses or retards for a certain time period the biological activity that is normally facilitated by the targeted kinase or the entire pathway.
- the inhibitor targets multiple signaling molecules of the MEK/MAPK pathway.
- the inhibitor targets a single signaling molecule/enzyme of that pathway.
- trametinib also known as GSK1120212, is a small molecule with the chemical formula C2 6 H2 3 FIN5O4 . It is an orally bioavailable inhibitor of mitogen-activated protein kinase (MEK MAPK/ERK kinase). Trametinib specifically binds to and inhibits MEK 1 and 2, resulting in an inhibition of growth factor-mediated cell signaling and cellular proliferation in various cancers. Trametinib is a MEK inhibitor in clinical trials.
- MEK inhibitors include AZD6244 (selumetinib; C 17 H 15 BrCIFN 4 C>3 as described in MCE MedChem Express; and in Huynh H et al, Mole Cancer Ther. 2007, 6 (1): 138-146 among other publications),
- GDC0973 C21H21F 3 IN 3 O2 as described in MCE MedChem Express; and Hoeflich KP, et al. Cancer Res. 2012 Jan l ;72(l):210-9
- GDC0623 C1 6 H14FIN4O 3 as described in MCE MedChem Express; and Hatzivassiliou G, et al. 2013 Nature. 501(7466):232-6
- refametinib as described in Canadian Patent Appln CA2923990
- Binimetinib Array BioPharma
- MK1833 balamapimod; PubChem ID No. 11478684, C 30 H 3 2CIN7OS
- an ERK inhibitor is SCH772984 (C 33 H 33 N 9 S2; PubChem ID Nol. 24866313).
- trametinib is the prototype of an inhibitor or antagonist of the prototype target, MEK, of the MEK/MAPK pathway for use in the methods and compositions described herein. It should be understood that one of skill in the art given the teachings of this specification may readily select another signaling molecule of MEK/MAPK and an appropriate inhibitor of that target.
- molecules/enzymes in the MEK/MAPK pathway include those known in the art to antagonize the indicated pathway targets and their salts derived from pharmaceutically or physiologically acceptable acids, bases, alkali metals and alkaline earth metals. Still other inhibitors useful in the methods described herein may be found in the catalogs of various biochemical and pharmaceutical suppliers.
- Physiologically acceptable acids include those derived from inorganic and organic acids. A number of inorganic acids are known in the art and include, without limitation, hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric, and phosphoric acid.
- organic acids include, without limitation, lactic, formic, acetic, fumaric, citric, propionic, oxalic, succinic, gly colic, glucuronic, maleic, furoic, glutamic, benzoic, anthranilic, salicylic, tartaric, malonic, mallic, phenylacetic, mandelic, embonic, methanesulfonic,
- Inhibitor compound salts can be also in the form of esters, carbamates, sulfates, ethers, oximes, carbonates, and other conventional "pro-drug" forms, which, when administered in such form, convert to the active moiety in vivo.
- the prodrugs are esters.
- the inhibitor compounds discussed herein also encompass "metabolites" which are unique products formed by processing the selected inhibitor compound by the cell or subject. In one embodiment, metabolites are formed in vivo.
- antisense nucleotide sequence or a small nucleic acid molecule having a complementarity to a target nucleic acid sequence e.g., Ras, MEK, etc. It can also comprise a nucleic acid sequence having
- the composition comprises a nucleic acid construct comprising a sequence that reduces or suppresses the expression of one of the targets or a combination thereof in the target cancer cells.
- the inhibiting composition can include a nucleic acid construct comprising a short nucleic acid molecule selected from the group consisting of a short hairpin RNA (shRNA), a short interfering RNA (siRNA), a double stranded RNA (dsRNA), a micro RNA, and an interfering DNA (DNAi) molecule, optionally under the control of a suitable regulatory sequence.
- a molecule that induces T cell proliferation or "proliferative cytokine” as used herein is meant a molecule or proliferative cytokine or an agonist of a proliferative cytokine that can stimulate proliferation, differentiation, and cell migration of T cells.
- the T cells are CD4 T cells.
- the T cells are CD8 T cells.
- this stimulation occurs in the presence of the pathway inhibitors described above. In one embodiment, this stimulation occurs both in the absence and presence of the pathway inhibitors described above.
- the function of the cytokines or cytokine agonist can depend on the target cell, environment of the target cell, culture conditions, cofactors or synergistic effects.
- Representative proliferative cytokines for use in the methods and compositions herein include IL-15, IL-2, or IL-7 used independently.
- the useful cytokines are a combination of IL-15 and IL-2 or a combination of IL-15 and IL-7.
- the useful cytokines are a combination of IL-2 and IL-7.
- the useful cytokines are a combination of IL-15, IL-2 and IL-7.
- IL-15 is one of the proliferative cytokines, and is an essential survival factor for natural killer (NK), natural killer-like T (NKT), and CD44hi memory CD8 T cells.
- NK natural killer
- NKT natural killer-like T
- CD44hi memory CD8 T cells CD44hi memory CD8 T cells.
- the bioactivity of IL-15 in vivo is conferred mainly through a trans -presentation mechanism in which IL-15 is presented in complex with the a-subunit of soluble IL-15 receptor (IL- 15R) to NK, NKT or T cells rather than directly interacts with membrane-bound IL- 15R.
- an IL-15 agonist refers to a molecule capable of stimulating or mimicking the biological activity in vivo and/or binding with the cytokine receptor on a cell.
- the agonist is an IL-15 agonist.
- IL-15 agonists are those which consist of IL-15 and partial or whole sequence of soluble IL- 15R.
- an IL-15 agonist is a molecule generated from a pre- association of IL-15 and its soluble receptor a-subunit-Fc fusion to form IL-15:IL-15Ra- Fc complex.
- an IL-15 agonist is a molecule generated by the expression of the hyperagonist IL-15-sIL-15Ra-sushi fusion protein consisting of IL-15 and the recombinant soluble sushi domain of IL-15Ra which was identified to have the most binding affinity for IL-15.
- the triple fusion protein combining Apolipoprotein A-I, IL-15 and 15Ra-sushi is similarly useful (see, on-line publication by DIGNA Biotech).
- an IL-15 agonist is a fusion protein of human IL-15 mutant IL-15N72D (residue substitution at position 72) with the soluble domain of IL-15Ra.
- ALT-803 is a fusion protein of IL-15N72D and IL- 15RaSu/Fc as a stable soluble complex (Altor Bioscience). As used throughout this specification, ALT-803 is the prototype of a proliferative cytokine agonist for use in the methods and compositions described herein. Still other IL-15 agonists include heterologous forms of IL-15 and its fragments (see, e.g., US Patent Application
- Suitable IL-15 agonists such as the above are described in below-listed References 43 (Wu) and 45(Zho) and the documents cited therein, all incorporated herein by reference.
- the agonist is an IL-2 agonist. In another embodiment, the agonist is an IL-7 agonist. Agonists of the proliferative cytokines for use in the methods and compositions herein include agonists of IL-15, IL-2, or IL-7 used independently. In another embodiment, the useful agonists are a combination of IL-15 agonist and IL-2 agonist or a combination of IL-15 agonist and IL-7 agonist. In another embodiment, the useful cytokine agonists are a combination of IL-2 agonist and IL-7 agonist. In still a further embodiment, the useful cytokine agonists are a combination of IL-15 agonist, IL-2 agonist and IL-7 agonist.
- pharmaceutically acceptable carrier or excipient is meant a solid and/or liquid carrier, in in dry or liquid form and pharmaceutically acceptable.
- compositions are typically sterile solutions or suspensions.
- excipients which may be combined with the small molecule inhibitor of the MEK/MAPK pathway and/or the molecule that induces T cell proliferation include, without limitation, solid carriers, liquid carriers, adjuvants, amino acids (glycine, glutamine, asparagine, arginine, lysine), antioxidants (ascorbic acid, sodium sulfite or sodium hydrogen-sulfite), binders (gum tragacanth, acacia, starch, gelatin, polyglycolic acid, polylactic acid, poly-d,l- lactide/glycolide, polyoxaethylene, polyoxapropylene, polyacrylamides, polymaleic acid, polymaleic esters, polymaleic amides, polyacrylic acid, polyacrylic esters, polyvinylalcohols, polyvinylesters, polyvinylethers, polyvinylimidazole,
- polyvinylpyrrolidon, or chitosan buffers (borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (mannitol or glycine), carbohydrates (such as glucose, mannose, or dextrins), clarifiers, coatings (gelatin, wax, shellac, sugar or other biological degradable polymers), coloring agents, complexing agents (caffeine, polyvinylpyrrolidone, ⁇ -cyclodextrin or hydroxypropyl- -cyclodextrin), compression aids, diluents, disintegrants, dyes, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents (peppermint or oil of wintergreen or fruit flavor), glidants, granulating agents, lubricants, metal chelators (ethylenediamine tetraacetic acid (EDTA)), osmo- regulators, pH adjustors
- Solid carriers include, without limitation, starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, calcium carbonate, sodium carbonate, bicarbonate, lactose, calcium phosphate, gelatin, magnesium stearate, stearic acid, or talc.
- Fluid carriers without limitation, water, e.g., sterile water, Ringer's solution, isotonic sodium chloride solution, neutral buffered saline, saline mixed with serum albumin, organic solvents (such as ethanol, glycerol, propylene glycol, liquid polyethylene glycol, dimethylsulfoxide (DMSO)), oils (vegetable oils such as fractionated coconut oil, arachis oil, corn oil, peanut oil, and sesame oil; oily esters such as ethyl oleate and isopropyl myristate; and any bland fixed oil including synthetic mono- or diglycerides), fats, fatty acids (include, without limitation, oleic acid find use in the preparation of injectables), cellulose derivatives such as sodium carboxymethyl cellulose, and/or surfactants.
- organic solvents such as ethanol, glycerol, propylene glycol, liquid polyethylene glycol, dimethylsulfoxide (DMSO)
- oils vegetable oils such as
- the effective amount of the small molecule inhibitor of the MEK/MAPK pathway is meant the amount or concentration (by single dose or in a dosage regimen delivered per day) sufficient to retard, suppress or kill cancer or tumor, while providing the least negative side effects to the treated subject.
- the effective amount of the inhibitor is within the range of 0. 01 mg/kg body weight to 10 mg/kg body weight in humans including all integers or fractional amounts within the range.
- the effective amount is at least 0.01 , 0.03, 0.05, 0.07, 0.09, 0.1, 0.25, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mg/kg body weight, including all integers or fractional amounts within the range.
- the effective amount is about 0.3 mg/kg (or 2 mg/day/adult patient).
- the effective total daily dose per adult is at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or at leastl O or more mg per adult.
- these amounts represent a single dose.
- the above amounts define an amount delivered to the subject per day.
- the above amounts define an amount delivered to the subject per day in multiple doses.
- these amounts represent the amount delivered to the subject over more than a single day.
- the effective amount of the small molecule inhibitor when the inhibitor and cytokine agonist are administered together or sequentially, is an amount larger than that required when the inhibitor is administered to retard cancer cell growth and spread in a subj ect in the absence of the cytokine agonist. In another embodiment of the method and compositions described herein, the effective amount of the small molecule inhibitor is an amount lesser than that required when the inhibitor is administered to retard cancer cell growth and spread in a subject in the absence of the cytokine agonist. In another embodiment of the method and compositions described herein, the effective amount of the small molecule inhibitor is less than that required to to retard cancer cell growth in a subject in the absence of the cytokine agonist.
- the effective amount of the molecule that induces T cell proliferation is meant the amount or concentration (by single dose or in a dosage regimen delivered per day) sufficient to stimulate and enhance T cell proliferation in the presence of the small molecule inhibitor of the target member of the MEK/MAPK pathway.
- the effective amount of the proliferative cytokine or cytokine agonist is within the range of at least 0.1 ⁇ g/kg body weight to 70 ⁇ g/kg body weight in humans including all integers or fractional amounts within the range.
- the effective amount of IL-15 or IL-15 agonist is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more ⁇ g/kg body weight including all integers or fractional amounts within the range.
- the effective total daily dose per adult is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more ⁇ g daily including all integers or fractional amounts within the range.
- the above amounts represent a single dose.
- the above amounts define an amount delivered to the subject per day.
- the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than a single day.
- Effective amounts of IL-2 in certain embodiments can be administered at the same dosages as discussed above for IL-15.
- Such doses include at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more ⁇ g/kg body weight including all integers or fractional amounts within the range.
- the effective total daily dose per adult is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more ⁇ g daily including all integers or fractional amounts within the range.
- the above amounts represent a single dose.
- the above amounts define an amount delivered to the subject per day.
- the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than 2 days. In another embodiment, much higher dosages than those identified above for IL-15 cytokine or its agonist are suitable for administration of IL-2.
- known dosages of IL-2 include two 5 day cycles (600,000
- dosages for administration of IL-7 or IL-7 agonists may be the same as those for IL-15 described.
- Such doses include at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more ⁇ g/kg body weight including all integers or fractional amounts within the range.
- the effective total daily dose per adult is at least 0.1 , 0.2, 0.3, 0.4, 0.5,
- the above amounts represent a single dose. In another embodiment, the above amounts define an amount delivered to the subject per day. In another
- the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than a week. In yet another embodiment, effective amounts of IL-7 may be similar to the high doses as described above for IL-2. One of skill in the art may readily select a suitable effective amount of IL-7 or an agonist thereof by resort to known pharmaceutical literature.
- cytokine/agonist are administered together or sequentially, the effective amount of the cytokine/agonist is an amount larger than that required when the cytokine/agonist is administered to stimulate T cell production in a subject in the absence of the inhibitor. In another embodiment of the method and compositions described herein, the effective amount of the cytokine/agonist is the same as that required to stimulate T cell production in a subject in the absence of the inhibitor. In another embodiment of the method and compositions described herein, the effective amount of the cytokine/agonist is less than that required to stimulate T cell production in a subject in the absence of the inhibitor.
- the combination of the inhibitor and cytokine/agonist with yet another pharmacological agent or treatment protocol permits lower than usual amounts of the inhibitor and/or cytokine/agonist and additional chemotherapeutic agent to achieve the desired therapeutic effects.
- the combination of the pathway inhibitor and cytokine agonist with another chemotherapy treatment protocol permits adjustment of the additional protocol regimen to achieve the desired therapeutic effect.
- a method of treating a mammalian subject with cancer comprises, in one aspect, administering to the subject having a cancer, a combination therapy.
- the combination therapy involves administering (a) a small molecule inhibitor of the MEK/MAPK pathway and (b) a molecule that induces T cell proliferation in the presence of the inhibitor.
- the small molecule inhibitor i.e., component (a) is trametinib and the inducing molecule is the proliferative IL-15 agonist, i.e., component (b) is ALT-803.
- Other selections of components (a) and (b) may be selected from those mentioned or incorporated by reference into this application or those indicated in the examples and Table 1.
- the combination of components (a) and (b) reduce the proliferation of the cancer and tumor cells in vivo.
- a method of treating a mammalian subject with cancer comprises, in one aspect, administering to the subject having a metastatic or refractory cancer or tumor, a combination therapy.
- the combination therapy involves administering a small molecule inhibitor of the MEK/MAPK pathway and a molecule that induces T cell proliferation in the presence of the inhibitor.
- the administration of (a) occurs before the administration of (b). In another embodiment of the method, the administration of (a) occurs after the administration of (b). In still another embodiment of the method, the administration of (a) occurs substantially simultaneously with the administration of (b).
- a component (a), trametinib was administered once daily from days 3-13 and a component (b), ALT-803, administered on days 3, 8, and 13.
- the method of delivering the pathway inhibitor in concert or sequentially with the proliferative cytokine/agonist further includes administering repeated doses of one or more of component (a) or component (b) to the subject. As illustrated in the examples below, this method of treatment prevents recurrence of aggressive tumors through the generation of protective immunity.
- the methods of treatment of cancer described herein further includes
- compositions or kits containing components (a) and (b) are further described herein for use in the treatment of cancer, including a metastatic or refractory cancer or tumor.
- a pharmaceutical composition contains a combination of (a) an effective amount of a small molecule inhibitor of a target member of the MEK/MAPK pathway to reduce, inhibit or suppress growth of the cancer or tumor; and (b) an effective amount of a molecule that induces T cell proliferation in the presence of said inhibitor. This induction is in the microenvironment of the tumor or in occurs systemically in vivo.
- the components (a) and (b) in one embodiment are admixed. In another embodiment, they are separately available, e.g., as in separate ampoules.
- the components (a) and (b) are individually or together formulated with a pharmaceutically acceptable carrier or diluent.
- the pharmaceutical composition contains, e.g. , about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
- composition or kit may also contain components (a) and
- the composition comprises the inhibitor, trametinib, admixed or separately associated with a proliferative cytokine or an agonist of a proliferative cytokine, such as a cytokine/agonist specified above.
- the cytokine is
- the cytokine agonist is an IL-15 agonist, such as ALT-803.
- the cytokine is IL-2.
- compositions and/or a kit may provide the components (a) and (b), with or without an additional chemotherapeutic.
- the compositions and/or kits may provide suitable pharmaceutically acceptable diluents, carriers or other pharmaceutical ingredients for admixture with component (a) or component (b) before administration.
- the following examples elucidate the effects of multiple targeted therapies on the tumor immunoenvironment and, subsequently, anti-tumor immunity.
- the inventors analyzed a panel of molecules for activity on human T cells and then focused on dissecting the role of an exemplary kinase inhibitor, trametinib, in restricting the growth of a KRas-driven tumor cell line in immune-competent mice.
- the results indicate that, in vivo, trametinib exerts divergent effects on at least 3 different cells types, resulting in significant differences in the accumulation of suppressive myeloid cells at tumor beds and therefore, despite a direct suppressive effect on T cells, a boost in anti-tumor immunity.
- trametinib acts on multiple tumor and non-tumor cells in the tumor microenvironment that, overall, make its effects very different in vitro and in vivo.
- One of these effects involves alterations in the secretion of cytokines by intrinsically inflammatory, tumor cells.
- osteopontin has been implicated in the recruitment of macrophages into tumors 10 and its expression is positively correlated with CD204 + M2-like macrophages 11 .
- Osteopontin secreted by tumor cells also drives the expansion of MDSCs in the spleens of tumor bearing mice through activation of the ERK1/2-MAPK pathway in myeloid progenitors 12 .
- ALT-803 has a beneficial effect on the anti-tumor immune response when used in combination with the IL-15 agonist ALT-803.
- ELISPOT analysis measured IFN-gamma and GranzymeB responses of tumor specific T cells. Disease progression in tumor bearing mice treated with trametinib, BKM-
- ALT-803 synergizes with the MEK inhibitor to provide enhanced therapeutic effect.
- mice - WT C57BL/6 and congenic Ly5.1 female 6-8 week old mice were procured from the National Cancer Institute or Charles River Laboratory.
- OTl C57BL/6- Tg (TcraTcrb)l 100 Mjb/J transgenic mice were obtained from Jackson Labs.
- Transgenic KRastm4Tyj and Trp53tmlBrn mice 28 29 were obtained from NCI Mouse Models of
- the Brpkpl 10 primary mammary tumor cell line was generated by culturing a mechanically dissociated B6 L-Stop-L-KRas G12D p53 flx/flx L-Stop- L-Myristoylated pi 10a-GFP/+ primary breast tumor mass 27 . Tumor cells were passaged a total of 10 times before deriving the Brpkpl 10 cell line.
- A2780 cells were obtained from AddexBio Technologies. ID8 cells 39 were provided by K. Roby (Department of Anatomy and Cell Biology, University of Kansas, Kansas City, KS) and retrovirally transduced to express Defi29 and Vegf-a 40 or OVA 25 . Lewis Lung Carcinoma cells (LLC) were obtained from ATCC.
- A2780 cells were cultured in D10 (DMEM (CellGro, with HEPES, glucose, and L-glutamine, without sodium pyruvate), 10% FBS, penicillin (100 I.U./ml), streptomycin (100 ⁇ g/ml), L- glutamine (2 mM), sodium pyruvate (0.5 mM), ⁇ -mercaptoethanol (50 ⁇ )).
- D10 DMEM (CellGro, with HEPES, glucose, and L-glutamine, without sodium pyruvate
- 10% FBS penicillin
- streptomycin 100 ⁇ g/ml
- L- glutamine 2 mM
- sodium pyruvate 0.5 mM
- ⁇ -mercaptoethanol 50 ⁇
- All other cell lines and lymphocytes were cultured in R10 (RPMI-1640 (CellGro, with L- glutamine), 10% FBS, penicillin (100 I.U./ml), streptomycin (100 ⁇ g/ml), l-glutamine (2 mM), sodium pyruvate (0.5 mM), ⁇ -mercaptoethanol (50 ⁇ )).
- R10 RPMI-1640 (CellGro, with L- glutamine), 10% FBS, penicillin (100 I.U./ml), streptomycin (100 ⁇ g/ml), l-glutamine (2 mM), sodium pyruvate (0.5 mM), ⁇ -mercaptoethanol (50 ⁇ )).
- mice were injected with anti-CD8a (BioXcell, clone YTS 169.4) on day 3 (500 ⁇ g/mouse) and day 10 (250 ⁇ g/mouse) post tumor inoculation. All antibodies, including isotype control (BioXcell, clone LTF-2), were injected i.p. in sterile PBS.
- Tumor inoculation - Brpkpl 10 tumors were initiated by injecting 5 x 10 5 cells subcutaneously into the axillary region. LLC tumors were initiated by injection of 2 x 105 cells either intraperitoneally or into the axillary region subcutaneously. Tumor volume was calculated as: 0.5 x (L x W 2 ), where L is length, and W is width.
- T cell stimulation For human T cell proliferation assays, peripheral blood lymphocytes were obtained by leukapheresis/elutriation and Miltenyi bead-purified and K562 cells expressing human CD32 were generated as previously described 41 . K32 were ⁇ -irradiated (100 Gy) and loaded with anti-CD3 (500 ng/ml, clone OKT3; eBioscience) plus anti-CD28 (500 ng/ml, clone 15E8; EMD Millipore) antibodies at room temperature for 10 minutes.
- anti-CD3 500 ng/ml, clone OKT3; eBioscience
- anti-CD28 500 ng/ml, clone 15E8; EMD Millipore
- PBMCs were labeled with CELLTRACE Violet stain (Invitrogen) according to the manufacturer's instructions and cocultured with loaded K32 cells at a 10: 1 T cells:K32 ratio or activated with ConA (2 ⁇ g/ml, Sigma). Proliferation of stimulated T cells was determined 7 days later by FACS and Division Index was calculated using FlowJo software.
- pan-T cells were negatively purified with antibodies to B220 (RA3), Mac-1 (Ml 70.13), and MHC-II (M5/114) using magnetic beads.
- T cells were labeled with CELLTRACE Violet (Invitrogen) and stimulated with either agonistic CD3/CD28 beads (Dynabeads, Life Technologies) or tumor-pulsed bone marrow dendritic cells (BMDCs) and analyzed for proliferation by FACS either 3 days (CD3/CD28 beads) or 7 days (BMDCs) later.
- BMDCs Day 7 BMDCs were generated as previously described 44 and cultured overnight with double-irradiated (y-irradiated,100 Gy; and UV, 30 min) ID8-Defb29/Vegf-A cells. BMDCs were added to cultures of T cells at a 10: 1 (T cell:BMDC) ratio.
- T cell:BMDC BMDC
- ELISpot assays mouse T cells were primed with tumor-pulsed BMDCs plus IL-2 (30 U/ml) and IL-7 (5 ng/ml), and restimulated 7 days later with fresh tumor-pulsed BMDCs at a 10: 1 ratio in an IFN- ⁇ ELISpot (eBioscience).
- Cell proliferation assays - Cells were plated in 96-well plates. Compounds were added the next morning, and cell proliferation was measured 48 hrs later with the
- CD8+ cells were sorted from PBMCs and rested overnight in RIO. T cells (0.5 x 10 6 per condition) were stained with OKT3-biotin (BioLegend, 10 ⁇ g/ml) for 15 mins on ice, and washed in cold PBS. TCR ligation was performed by adding streptavidin (Promega, 25 ⁇ g/ml) and anti-CD28 (Millipore, clone 15E8, 1 ⁇ g/ml) in the presence of indicated inhibitors for 10 min at 37°C.
- mice In vivo OT-I proliferation - Congenic Ly5.1 mice were injected with 1.5 x 10 6 ID8-OVA cells i.p 25 . On day 10, mice were injected i.p. with 1.5 x 10 6 CELLTRACE Violet-labeled, naive OT-I T cells, and administered ALT-803 (0.2 mg/kg) or PBS i.p. Mice were gavaged with trametinib or vehicle on days 9-13. On day 14, mice were euthanized and peritoneal washes were analyzed for proliferating OT-I T cells.
- MDSCs were added to 2 x 10 5 CellTrace-labelled, WT splenocytes simultaneously activated with anti-CD3 (500 ng/ml, clone 2C11, Tonbo) and anti-CD28 (1 ⁇ g/ml, clone 37.51, Tonbo) in 96 well plates. Proliferation of T cells was measured 3 days later.
- MDSCs were separated into Ly6G+ and Ly6G- fractions with anti-Ly6G MicroBeads (Miltenyi) according to the manufacturer's protocol. Chemotaxis was measured toward recombinant carrier-free osteopontin (R&D
- Proteins were quantified by BCA assay (Thermo Scientific), diluted in reducing lamelli buffer, denatured by incubation at 95°C, run on mini Protean TGX Ready Gels (Bio-Rad Laboratories), transferred to a nitrocellulose membrane, blocked, and incubated with primary antibodies for p-ERKl/2 (Cell Signaling Technologies, clone D13.14.4E), p-AKT (Cell Signaling Technologies, clone D9E), beta- tubulin (Cell Signaling Technologies, clone 9F3), beta-actin (Sigma, clone AC- 15), or IGF-1R (Cell Signaling Technologies, #3027). Immunoreactive bands were developed using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad Laboratories) and ECL substrate (GE Healthcare).
- Immunohistochemistry - Tissues were embedded in Tissue-Tek OCT and frozen. Endogenous peroxidases were quenched from acetone-fixed sections (8 ⁇ ) by incubating in 0.3% H2O2 for 10 minutes at room temperature. Following quenching, sections were blocked using 3% goat serum followed by staining with antibodies against Ki-67 (clone D3B5, Cell Signaling Technology). Immunohistochemistry using the ABC Kit (Vector labs) was performed according to the manufacturer's instructions, and sections were counter stained with hematoxylin. Slides were then imaged at 10X objective magnifications on a Nikon E600 Upright microscope with a Nikon DS-Ril
- LC-MS/MS - Brpkpl 10 cells were cultured in serum-free RPMI with DMSO or trametinib for 40hrs. Supernatants were collected, centrifuged, passed through a 0.22 ⁇ filter, and concentrated by centrifugation in Amicon 3000 MWCO tubes (EMD).
- MS data were analyzed with MaxQuant 1.5.2.8 software 42 .
- MS/MS data were searched against the mouse UniProt protein database (July 2014) using full trypsin specificity with up to two missed cleavages, static carboxamidomethylation of Cys, and variable oxidation of Met and protein N-terminal acetylation. Consensus identification lists were generated with false discovery rates of 1% at protein and peptide levels.
- ELISA - Mouse and human osteopontin concentrations were measured using ELISA kits (Ray Biotech) according to the manufacturer's instructions. Plasma was isolated from peripheral blood of mice by centrifugation in lithium heparin tubes (Becton Dickinson).
- EXAMPLE 2 SMALL MOLECULE INHIBITORS SUPPRESS HUMAN T CELL ACTIVATION IN VITRO.
- trametinib the MEK inhibitor approved by the FDA for BRAF -mutant melanoma
- trametinib the MEK inhibitor approved by the FDA for BRAF -mutant melanoma
- trametinib the MEK inhibitor approved by the FDA for BRAF -mutant melanoma
- the observed EC50 of every molecule with some activity on A2780 cells was lower for human T cells than A2780 cells, as shown in Table 1.
- Table 1 lists the small molecule names, targets, and EC50 values on T cells and A2780 cells for each compound. EC50 values were calculated from normalized percent inhibition data using non-linear curve fitting in PRISM software. N/A indicates ambiguous curve fits or EC50 values greater than the highest concentration tested (25 ⁇ ).
- GDC0973 GDC0973 inhibitors also restricted the proliferation of human T cells in response to artificial antigen presenting cells (aAPC) coated with agonistic CD3 and CD28 antibodies 15 (FIG IB). Importantly, these effects were consistent among 3 different donors (FIG 1C). Comparable results were obtained with the MEK inhibitor trametinib (FIG ID). As expected, T cells were more sensitive to the kinase inhibitors when they were stimulated with weaker signals (aAPCs lacking anti-CD28 and lower concentrations of anti-CD3), as could occur in tumor-bearing hosts (FIG. 8).
- pan-PI3K and MEK inhibitors completely abrogated the initial priming response of murine T cells activated with tumor lysate-pulsed dendritic cells (DCs) 16 ' 17 (FIG IE and IF). More importantly, the direct suppressive effects of pan-PI3K and MEK inhibitors were not restricted to proliferative responses because the frequency of murine tumor-primed T cells secreting IFN- ⁇ in response to re-stimulation with fresh tumor lysate-pulsed DCs was also significantly reduced when either pan-PI3K or MEK were inhibited (FIGs. 1G and 1H).
- trametinib is approved for clinical use, we were particularly interested in whether methods to selectively rescue T cells from potent MEK inhibition could improve the therapeutic efficacy of trametinib.
- IL-15 because it provides strong memory signaling to CD8 T cells without inducing Treg expansion, as compared to IL-2 19 ' 20 .
- IL-15 can rescue early (within 10 minutes) TCR-induced MAPK signaling from MEK inhibition, as shown by ERK1/2 phosphorylation (FIG 2C).
- OT-I T cells CELLTRACE Violet-labelled (Ovalbumin (OVA)-specific) OT-I T cells into mice growing OVA-transduced syngeneic ID8 ovarian tumors, a system that allows the recovery of tumor microenvironmental lymphocytes through peritoneal wash 25 16 . After 4 days, we found that in mice treated with trametinib, the OT-I T cells proliferated significantly less than in mice gavaged with vehicle (FIGs 2G and 2H).
- ALT-803 when ALT-803 was co-administered with the OT-I T cells (only one dose), proliferation was dramatically enhanced and was not restricted by trametinib, indicating that therapeutic activation of IL-15 signaling can restore CD8 T cells suppressed by trametinib in vivo.
- Brpkpl 10 cells have identifiable signaling through MEK that can be inhibited with trametinib (FIG 3 A) and generate aggressive tumors when grown subcutaneously.
- mice that rejected their tumors in the trametinib/ ALT-803 combination group developed immunological memory, because they rejected subsequent re-challenge with Brpkpl lO cells in the opposite side over 30 days after initial tumor rejection, whereas naive control mice developed tumors (FIG 3E).
- FOG 3E naive control mice developed tumors
- trametinib treatment of tumor bearing mice did not decrease the frequencies of CD8 or CD4 tumor-infiltrating T cells able to produce IFN- ⁇ when restimulated ex vivo (FIG 41).
- these results support that the therapeutic activity of trametinib is not determined solely by tumor cell intrinsic mechanisms, and that the tumor microenvironment has a large impact on the response to trametinib treatment.
- trametinib requires an intact CD8 T cell compartment for optimal effectiveness against KRas-driven Brpkpl lO tumors in vivo.
- EXAMPLE 6 TRAMETINIB REDUCES THE ACCUMULATION OF LY6C+ MDSCS IN TUMOR-BEARING MICE.
- CD1 lb+MHC-II-Ly6CloLy6G+ (granulocytic) MDSCs were surprisingly unaffected, whether analyzed as a proportion of total leukocytes (FIG 5B), or as a proportion of CD1 lb+MHC-II- myeloid cells (FIG 5C, and 5D). Supporting a broad effect on monocytic MDSCs, corresponding reductions in Ly6C+ MDSCs were found in trametinib-treated, subcutaneous LLC tumors (FIG 5E). As expected, this MDSC population expresses high levels of the immunosuppressive molecules Arginasel and
- trametinib reduced MEK signaling in myeloid precursors differentiated with Brpkpl 10 conditioned media (FIG 6F).
- these results indicate that trametinib, by inhibiting the Ras-MAPK pathway in myeloid precursors, selectively decreases the accumulation of immunosuppressive Ly6C+ MDSCs, both at tumor beds and systemically.
- trametinib overall, enhances protective immunity by decreasing monocytic MDSCs, so that its full anti-tumor efficacy paradoxically requires CD8 T cells.
- trametinib impairs tumor progression by inhibiting myeloid cell mobilization, despite direct (IL-15-reversible) suppressive effects on lymphocytes.
- trametinib may also alter the composition of the tumor microenvironment by modulating the KRas-dependent secretion of inflammatory factors by tumor cells.
- LC-MS/MS analysis of culture supernatants from Brpkpl 10 cells revealed multiple cytokines dramatically altered in response to trametinib treatment.
- a number of factors known to be involved in recruitment of myeloid cells such as CSF1, CCL2, and CX3CL1 (FIG 7A) were decreased after trametinib treatment.
- osteopontin SPP1 was decreased >18-fold upon trametinib treatment, which was confirmed by ELISA in independent experiments (FIG 7B).
- We focused on osteopontin because it has been reported to induce the expansion of MDSCs 12 and the recruitment of macrophages into the tumor 11 .
- Vella LJ et al. MEK inhibition, alone or in combination with BRAF inhibition, affects multiple functions of isolated normal human lymphocytes and dendritic cells. Cancer Immunol Res. 2014;2:351-60.
- Lin CN et.al. The significance of the co-existence of osteopontin and tumor-associated macrophages in gastric cancer progression. BMC Cancer. 2015;15: 128.
- Marigo I et al. Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity. 2010;32:790-802.
- MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS- mutant advanced non-small-cell lung cancer (NSCLC)dagger.
- NSCLC advanced non-small-cell lung cancer
- Rutkowski MR et al. Microbially driven TLR5-dependent signaling governs distal malignant progression through tumor-promoting inflammation. Cancer Cell. 2015;27:27-40.
- Huarte E, et al. PILAR is a novel modulator of human T-cell expansion. Blood. 2008;112: 1259-68.
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US10973917B2 (en) | 2016-05-18 | 2021-04-13 | Modernatx, Inc. | MRNA combination therapy for the treatment of cancer |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
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EP3468581A1 (en) | 2016-06-13 | 2019-04-17 | Torque Therapeutics, Inc. | Methods and compositions for promoting immune cell function |
US20190048055A1 (en) * | 2017-03-31 | 2019-02-14 | Altor Bioscience Corporation | Alt-803 in combination with anti-cd38 antibody for cancer therapies |
CN109306340B (en) * | 2017-07-27 | 2023-06-06 | 上海细胞治疗研究院 | Artificial antigen presenting cell for efficiently amplifying whole T cells and application thereof |
CN109337872B (en) * | 2017-07-27 | 2023-06-23 | 上海细胞治疗研究院 | Artificial antigen presenting cell for efficiently amplifying CAR-T cells and application thereof |
CN111655716B (en) * | 2017-08-28 | 2024-03-08 | 艾尔特生物科技公司 | IL-15 based fusion with IL-7 and IL-21 |
AU2018328209A1 (en) | 2017-09-05 | 2020-04-23 | Torque Therapeutics, Inc. | Therapeutic protein compositions and methods of making and using the same |
TW201938165A (en) * | 2017-12-18 | 2019-10-01 | 美商輝瑞股份有限公司 | Methods and combination therapy to treat cancer |
WO2020054791A1 (en) * | 2018-09-14 | 2020-03-19 | 住友化学株式会社 | Composition useful for producing acidic gas separation membrane |
CA3126153A1 (en) * | 2019-01-11 | 2020-07-16 | Memorial Sloan Kettering Cancer Center | Multimerization of il-15/il-15r-alpha-fc complexes to enhance immunotherapy |
EP4188431A4 (en) * | 2020-07-31 | 2024-07-24 | Univ Leland Stanford Junior | Combination therapy for cancer |
CN113234674B (en) * | 2021-03-15 | 2023-02-07 | 青岛华赛伯曼医学细胞生物有限公司 | T cell activation and amplification method and application thereof |
CN113667637B (en) * | 2021-08-05 | 2023-06-16 | 大连医科大学 | Method for in vitro induction of differentiation and amplification of human peripheral MDSCs |
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