WO2019213509A1 - Compositions et méthodes pour le traitement du cancer - Google Patents

Compositions et méthodes pour le traitement du cancer Download PDF

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WO2019213509A1
WO2019213509A1 PCT/US2019/030584 US2019030584W WO2019213509A1 WO 2019213509 A1 WO2019213509 A1 WO 2019213509A1 US 2019030584 W US2019030584 W US 2019030584W WO 2019213509 A1 WO2019213509 A1 WO 2019213509A1
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cells
cancer
hsv
oncovex
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Howard L. KAUFMAN
Praveen BOMMAREDDY
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Rutgers, The State University Of New Jersey
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    • 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
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
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    • 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
    • A61K31/495—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
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    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Melanoma is a metastatic tumor arising from melanocytes located in the stratum basale of the epidermis, mucosal membranes, and middle layer of the uvea. Cutaneous melanoma is the most common variant, and arises in skin, typically undergoing an initial radial growth phase during which complete surgical excision is often curative. If not treated, melanoma enters a vertical growth phase during which tumor cells may enter subdermal lymphatics, and can metastasize to regional lymph nodes, and eventually to almost any visceral organ, including the central nervous system.
  • Metastatic melanoma has historically been associated with dismal prognoses but systemic therapy has been transformed over the past decade, largely by advances in molecular therapy targeting the RAS-RAF-MEK-ERK mitogen activated protein kinase (MAPK) pathway in patients with tumors that harbor BRAF V600E/K mutations (about 40- 50% of cutaneous melanomas harbor mutations in BRAF, which serve as oncogenic drivers of the MAPK pathway promoting tumor progression), and by immunotherapy, most notably with immune checkpoint blockade targeting cytotoxic T lymphocyte antigen 4 (CTLA4; via ipilimumab) and programmed cell death 1 (PD-l; via nivolumab or pembrolizumab).
  • CTLA4 cytotoxic T lymphocyte antigen 4
  • PD-l programmed cell death 1
  • Combination approaches within drug classes have shown improved therapeutic benefit, but treatment is often associated with drug resistance and/or increased toxicity.
  • combination immune checkpoint blockade with ipilimumab and nivolumab results in improved progression-free and overall survival compared to ipilimumab alone, but was also associated with a 59% incidence of grade 3 or greater adverse events compared to 21% with nivolumab alone and 28% with ipilimumab alone.
  • Novel combination strategies with agents that enhance therapeutic responses, while limiting toxicity, have become a high priority for drug development in melanoma.
  • these methods include novel drug combination strategies encompassing certain drugs that target specific pathways. In other embodiments, these methods should be effective regardless of the BRAF status of the cancer.
  • the present invention meets these needs.
  • the present invention provides a method of treating or preventing a cancer in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of talimogene laherparepvec.
  • the method comprises administering to the subject a therapeutically effective amount of a mitogen-activated protein kinase kinase enzyme (MEK) inhibitor.
  • MEK mitogen-activated protein kinase kinase enzyme
  • the present invention further provides a method of treating or preventing a cancer in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of a HSV-l-based oncolytic virus lacking any copy of the ICP 34.5 neurovirulence gene.
  • the method comprises administering to the subject a therapeutically effective amount of a MEK inhibitor.
  • the method comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent.
  • the present invention further provides a method of determining if a subject afflicted with a cancer and being treated with a combination of a HSV-l -based oncolytic virus and a MEK inhibitor will benefit from administration of an immune checkpoint blockade agent.
  • the method comprises administering to the subject a therapeutically effective amount of a HSV-l -based oncolytic virus and a therapeutically effective amount of a MEK inhibitor.
  • the method comprises measuring and/or determining, in a sample of the subject’s cancerous tissue expression, levels of at least marker selected from the group consisting of CD-8, Gzmb, IFNy, PRF1, and TNFa.
  • the subject is counseled to be further administered an immune checkpoint blockade agent if the levels of the at least one marker in the sample of the subject’s cancerous tissue is higher than levels of the at least one marker in a control sample, the subject is counseled to be further administered an immune checkpoint blockade agent.
  • FIGs. 1A-1G illustrate the finding that MEK inhibition augments talimogene laherparepvec-mediated cell lysis in vitro and increases viral replication. Increased tumor cell lysis was demonstrated by MTS assay when cells were treated with combination of talimogene laherparepvec and trametinib (FIGs. 1 A-1D, left panels, black bars) compared to talimogene laherparepvec alone (FIGs. 1A-1D, left panels, gray bars).
  • SK-MEL-28 FIG.
  • SK-MEL-5 (FIG. 1B) are BRAF-mutant human melanoma cell lines
  • SK- MEL-2 (FIG. 1C) is BRAF wild-type
  • D4M3A (FIG. 1D) is a BRAF-mutant spontaneous murine melanoma cell line.
  • Cells (7.5 x 10 3 ) were seeded on 96-well plates, treated with vehicle or MEKi (Trametinib) (10 nM for SK-MEL-28, 5 nM for SK-MEL:5, 1.25 nM for SK-MEL-2, and 5 nM for D4M3A).
  • talimogene laherparepvec Six to eight hours later, cells were treated with talimogene laherparepvec at the indicated MOI. After 5 days (SK-MEL-28, SKMEL:5, SK- MEL-2) or 3 days (D4M3A) of incubation, MTS assay was performed to measure cell viability.
  • the right panels show HSV-l titers as measured by standard plaque assay from total cell lysates collected from each cell line 24 hrs. post infection with talimogene laherparepvec at 0.1 MOI (gray bars) or combination of talimogene laherparepvec and trametinib (black bars).
  • FIG. 1 A Cells (3.5 x 10 5 ) were seeded in 6-well plate, treated with vehicle or MEKi as in FIG. 1 A. Six to eight hours later, cells were inoculated with talimogene laherparepvec (MOI 0.1). 24 h post-viral infection, total cell lysates were harvested and talimogene laherparepvec titers were determined by plaque assay in Vero cells *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. Only significant differences are indicated. FIG.
  • 1F Infection metric analysis by Lumacyte (left panel) of SK-MEL-28 cells untreated (mock), treated with 10 nM trametinib (MEKi), 1 MOI talimogene laherparepvec or trametinib and talimogene laherparepvec (talimogene laherparepvec + MEKi).
  • the infection metric is derived from Lumacyte assessment of cell size and velocity (see FIGs. 10A-10C for velocity histograms) and detects viral-infected cells as larger and hence with higher infection metric.
  • the right panel shows a time course for untreated cells (black line), or those treated with 0.1 MOI of talimogene laherparepvec (dotted red line) or 1 MOI of talimogene laherparepvec (solid red line).
  • Data shows the infection metric is higher for cells treated at the higher viral dose, while the lower dose increases over time due to viral replication and matches the higher viral dose at 36 hours.
  • FIG. 1G Principle component analysis (PCA) of the infection metric for SK-MEL-28 cells shown in FIG. 1F, left panel.
  • Fl and F2 represented the largest possible variance in the data. PCA was able to distinguish cells based on treatment conditions. Each experiment was repeated two or more times with similar results.
  • T.E. talimogene laherparepvec.
  • FIGs. 2A-2F illustrate the finding that MEK inhibition enhances talimogene laherparepvec-induced tumor regression of human xenograft melanoma in vivo and promotes tumor cell apoptosis.
  • FIG. 2A Treatment schema; panel indicating treatment schedule, red arrows indicate days on where talimogene laherparepvec was injected and top blue bar indicated days of trametinib (MEKi) treatment.
  • MEKi trametinib
  • FIGs. 2C-2F Immunohistochemical staining of Ki67 (FIG. 2C), HSV-l gD (FIG. 2D), pERKl/2 (FIG. 2E), and cleaved caspase 3 (FIG. 2F) respectively in the tumor.
  • HSV-l staining was quantified as an average brown staining intensity over the selected area. Each experiment is repeated two or more times with similar results. Data are presented as mean ⁇ SEM and statistical differences between groups was measured by student’s t test. *p ⁇ 0.05, **p ⁇
  • T.E. talimogene laherparepvec.
  • FIGs. 3A-3H illustrate the finding that MEK inhibition enhances OncoVEX mGM CSF - induced tumor regression in an immune competent murine melanoma model, promotes recruitment of CD8 + T cells, and establishes long-term memory.
  • FIG. 3A Treatment schema, red arrows indicate days on where OncoVEX mGM CSF was injected and top blue bar indicated days of trametinib (MEKi) treatment (FIG. 3A-3B).
  • FIG. 3B Mean tumor area of mock-treated mice was compared with
  • FIG. 3E Bar graphs show the percentages of CD8 + ,
  • FIG. 3F Immunohistochemi cal staining of CD8 + T cells in the tumor.
  • B6 mice implanted with 3 x 10 3 D4M3A cells on day 0, treated with sterile water or OncoVEX mGM CSF (1 x 10 6 pfu) on days 15, 19 and 22 and MEKi (trametinib; 0.5 mg/kg) or vehicle gavaged orally once daily from days 15-19 (n 5/group), and mice euthanized on day 24.
  • tumor sections were collected, stained as indicated, and imaged at 10X using Olympus VS 120 whole slide scanner. Representative images with positive cells stained brown are shown.
  • FIG. 3G Bar graph indicating quantification of positive cells. Annotated whole tumor regions were subjected to unsupervised quantification using VisioPharm quantitative digital pathology software. Positive cell density was computed as positive cell count / mm 2 tissue area for CD8.
  • FIG. 3H Bar graph indicating CD4 + and CD4 + FoxP3 + (regulatory T cells; Tregs) and ratio of CD8 + T cells to Tregs. Each experiment was repeated three times with similar results. Data are presented as mean ⁇ SEM and statistical differences between groups was measured by student’s t test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. Only significant differences are indicated.
  • mT.E. OncoVEX mGM CSF .
  • FIGs. 4A-4E illustrate the finding that depletion of CD8 + T cells abrogates the effects of OncoVEX mGM CSF and MEKi combination therapy.
  • FIG. 4A Treatment schema; panel indicating treatment schedule, red arrows indicate days on where OncoVEX mGM CSF was injected, top blue bar indicated days of trametinib (MEKi) treatment and black arrows indicating days where depletion antibodies against CD4, CD8 and clodronate was injected.
  • MEKi trametinib
  • HPMC hydroxypropyl methyl cellulose
  • mice administered via intraperitoneal (i.p.) injection anti-mouse CD8a (clone 2.43; 10 mg/kg), anti-mouse CD4 (clone GK1.5; 10 mg/kg), or clodronate liposomes (first injection 50 mg/kg, followed by 25 mg/kg) on days 12, 15, 18, 21, 25, 28, and 32.
  • Mock group received sterile water (i.t.) + vehicle (0.2% Tween 80 and 0.5%
  • Isotype group received OncoVEX mGM CSF (i.t.) + MEKi (i.p.) + control rat IgG (i.p.) + empty liposomes (i.p.) as above.
  • aCD4 or aCD8 group received OncoVEX mGM CSF + MEKi + aCD4 or aCD8 + empty liposomes
  • Clodronate group received OncoVEX mGM CSF + MEKi + control rat IgG + clodronate liposomes.
  • FIGs. 4D-4E Flow cytometric analysis of tumor infiltrating T cells.
  • B6 mice implanted subcutaneously with 3 x 10 3 D4M3A murine melanoma cells in the right flank on day 0, treated with OncoVEX mGM CSF (1 x 10 6 pfu) or sterile water administered via intratumoral injection on days 15, 19, and 22 and MEKi (trametinib; 0.5 mg/kg) or vehicle was gavaged from days 15-19.
  • mice were injected i.p.
  • CD45 CD3 CD4 + (FIG. 4D) and CD45 + CD3 + CD8 + cells (FIG. 4E).
  • Each experiment is repeated two or more times with similar results.
  • Each experiment is repeated three times with similar results.
  • Data are presented as mean ⁇ SEM and the statistical differences between groups was measured by using two-tailed student t test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. Only significant differences are indicated.
  • FIGs. 5A-5C illustrate the finding that combination of OncoVEX mGM CSF and MEK inhibition induces viral-specific CD8 + T cells and increases melanoma antigen-specific CD8 + T cell responses.
  • C57BL/6J mice implanted subcutaneously in the right flank with 3 x 10 5 D4M3A cells and treated with OncoVEX mGM CSF (1 x 10 6 pfu) or sterile water for 3 doses on days 15, 19 and 22 and or trametinib (0.5 mg/kg) or vehicle (0.2% Tween 80 and 0.5% Hydroxypropyl methyl cellulose) orally once daily on days 15-19.
  • Tumors were harvested on day 24, cells dissociated and stained with fluorochrome-conjugated anti-mouse antibodies, and multicolor Flow cytometry was performed. Percentages of live CD45+ cells, CD3+ cells, and CD3+ sorted CD4+ and CD8 + subsets from the Mock, OncoVEX mGM CSF monotherapy, MEKi monotherapy, and OncoVEX mGM CSF + MEKi combination groups were analyzed and compared. Tumor-infiltrating CD8 + T cells were analyzed with HSV-l -specific H-2K b -HSV- lgB dextramer (FIG.
  • FIGs. 6A-6F illustrate the finding that Batf3 + dendritic cells play a role in the anti tumor activity and antigen spreading associated with combination of OncoVEX mGM CSF and MEK inhibition treatment.
  • FIG. 6A top panel: Treatment schema, red arrows indicate days on where OncoVEX mGM CSF was injected and top blue bar indicated days of trametinib (MEKi) treatment.
  • OncoVEX mGM CSF and MEKi combination therapy was compared to Batf3 /_ mice treated with OncoVEX mGM CSF and MEKi (p ⁇ 0.0l).
  • OncoVEX mGM CSF and MEKi combination therapy were compared to Batf3 /_ (p ⁇ 0.000l) mice treated with OncoVEX mGM CSF and MEKi at 45 days post tumor
  • Trametinib 0.5 mg/kg
  • vehicle 0.2% Tween 80 and 0.5% Hydroxypropyl methyl cellulose
  • combination group received both OncoVEX mGM CSF and trametinib.
  • the mock group received sterile water via intratumoral injection and vehicle via oral gavage. Mice euthanized on day 24, tumors were harvested, dissociated cells stained with fluorochrome-conjugated anti-mouse antibodies, and multicolor flow cytometry was performed. Percentages of live CD45 + cells, CD3 + cells, and CD3 + sorted CD4 + and CD8+ subsets from the B6 mice were analyzed and compared with Batf3 /_ mice. FIG.
  • FIG. 6C Bar graph indicating the percentage (first panel) and number (second panel) of tumor-infiltrating total CD8 + T cells and the frequency of CD8 IFN-y' (third panel) and CD8 + GranzymeB + T cells (fourth panel).
  • FIG. 6D Proliferating CD8 + Ki67 + T cells.
  • FIG. 6E Bar graph indicating the percentage of CD4 + FoxP3 + Tregs.
  • FIG. 6F Bar graph indicating the percentage of HSV-gB-, murine gplOO- and TRP2-specific CD8 + T cells as assessed by dextramers as described in FIGs. 5A- 5C.
  • FIGs. 7A-7D illustrate the finding that OncoVEX mGM CSF and MEK inhibition reprograms immune silent“COLD” tumors into immune inflamed“HOT” tumors and induces expression of PD-l and PD-L1.
  • B6 mice were implanted subcutaneously in the right flank with 3 x 10 5 D4M3A cells and treated with OncoVEX mGM CSF (1 x 10 6 pfu) intratumoral for 3 doses on days 15, 19 and 22 and or trametinib (0.5 mg/kg) orally once daily on days 15- 19. Tumors were harvested on day 24, total RNA was isolated and gene expression analysis performed using the NanoString PanCancer Immune panel as described in the Methods.
  • OncoVEX mGM CSF alone red
  • OncoVEX mGM CSF and MEKi purple
  • FIG. 7B A selected 5-gene expression signature represented by genes highly associated with CD8 + T cell activation, was also assessed and showed similar responses with increased gene expression with OncoVEX mGM CSF alone, but greatest expression in tumors treated with
  • FIGs. 8A-8H illustrate the finding that triple combination treatment with
  • OncoVEX mGM CSF , MEK inhibition, and PD-l blockade improves therapeutic treatment of melanoma, and outcomes are associated with decreased accumulation of exhausted CD8 + T cells.
  • FIG. 8A Treatment schema; panel indicating treatment schedule, red arrows indicate days on where OncoVEX mGM CSF was injected, top blue bar indicated days of trametinib (MEKi) treatment and brown arrows indicating days where aPD-l antibody was given.
  • FIG. 8A Treatment schema; panel indicating treatment schedule, red arrows indicate days on where OncoVEX mGM CSF was injected, top blue bar indicated days of trametinib (MEKi) treatment and brown arrows indicating days where aPD-l antibody was given.
  • FIG. 8C Median survival was improved with triple therapy. Mock-treatment (35 days) was compared with
  • FIG. 8D Rechallenge of mice cured from FIG. 8B.
  • FIG. 8E-8G Flow cytometry of tumors at day 24. Bar graph indicating percentage of positive (FIG. 8E) CD45 + PD-l + cells (left panel) and CD8 + PD-l + cells (right panel), (FIG. 8F) CD4 + FoxP3 + (left panel) and ratio of effector T cells (Teff) to Tregs (right panel).
  • FIG. 8G CD8 + T cells, granzyme B + CD8 + T cells, and Ki67 + CD8 + T cells, respectively.
  • FIG. 8H Evaluation of triple combination in the CT26 murine colon carcinoma model. Each experiment was conducted at least twice with similar results. Data are presented as means ⁇ SEM, and statistical differences between groups were measured by two-tailed Student’s t test. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • mT.E. OncoVEX mGM CSF .
  • FIGs. 9A-9G illustrate the finding that talimogene laherparepvec kills human melanoma cell lines and murine D4M3A cells, and BRAF inhibition enhances oncolysis in tumor cells harboring BRAF V600E mutations. Cytotoxic effects of talimogene
  • FIG. 9C mouse D4M3A
  • FIG. 9D mouse D4M3A melanoma cell lines.
  • Cells (7.5 x 10 3 ) were seeded on 96- well plates and treated with talimogene laherparepvec at the indicated MOI or control (sterile water). After 5 days (SK-MEL-28, SK-MEL-5, SK-MEL-2) or 3 days (D4M3A) of incubation, an MTS assay was performed to measure cell viability. Cytotoxic effects of talimogene laherparepvec and V emurafenib (BRAF inhibitor) in human SK-MEL-28 (FIG. 9E), SK-MEL-5 (FIG.
  • BRAF inhibitor V emurafenib
  • FIGs. 10A-10C illustrate how lumacyte laser flow cytometry analysis uses single cell size and velocity to determine viral infection.
  • SK-MEL-28 cells were infected with talimogene laherparepvec at 0.1 MOI and subjected to single cell laser flow cytometry.
  • FIG. 10A Photomicrograph showing that a virus -infected cell is larger and has a corresponding lower velocity through single cell capillary chamber (787 pm/s; left panel) compared to un infected cells that are smaller and have higher velocity (1028 pm/s; right panel).
  • FIG. 10B Standard velocity histograms generated at 12 h. (top), 24 h. (middle) and 36 hrs. (bottom) for uninfected cells (black [white filled] bars) or 1 MOI talimogene laherparepvec (red bars). Talimogene laherparepvec-infected cells exhibited slower velocity compared to uninfected cell.
  • FIG. 10C Standard velocity histograms show indicated time points same as FIG.
  • FIGs. 11 A-l 1D illustrate the finding that talimogene laherparepvec and MEK inhibition induces apoptosis in melanoma cells.
  • FIG. 11 A Flow cytometry analysis showing Annexin V staining in SK-MEL-28 cells treated with either talimogene laherparepvec or MEKi or both. SK-MEL-28 cells were treated with vehicle or MEKi (trametinib; 10 nM) for 6-8 hours. Afterwards, PBS or talimogene laherparepvec (MOI 1) was added to cells and cells were cultured for 24 h, stained for Annexin V (apoptosis), and analyzed by flow cytometry.
  • FIG. 11 A Flow cytometry analysis showing Annexin V staining in SK-MEL-28 cells treated with either talimogene laherparepvec or MEKi or both. SK-MEL-28 cells were treated with vehicle or MEK
  • FIG. 11B Quantitative analysis of FIG. 11A.
  • FIG. 11C Annexin-V staining could be blocked by treatment with Z-VAD FMK (20 mM).
  • FIG. 11D Western blot analysis of cleaved PARP.
  • SK-MEL-28 Cells (3.5 x 10 5 ) were seeded in 6-well plate, treated with vehicle or MEKi (trametinib; 10 nM) as in FIG. 11 A.
  • MEKi trametinib; 10 nM
  • FIG. 11 A Six to eight hours later cells were inoculated with talimogene laherparepvec (MOI 1). 24 h post-viral infection, total cell lysates were harvested and cleaved PARP level is detected by immunoblotting. This experiment was repeated twice, similar results were obtained. Data are presented as mean ⁇ SEM and statistical differences between groups were measured by two-tailed student t test.
  • T.E. talimogene laherparepvec.
  • FIGs. 12A-12B illustrate characterization of murine D4M3A cells.
  • FIG. 12A left panel: The murine B16-F10 melanoma cell line is not susceptible to HSV-l infection because it lacks HSV entry receptors. As shown by MTS assay measuring cell viability, of B16-F10 cells are resistant to talimogene laherparepvec infection (red) but Nectin-l- transduced B16-F10 cells (blue) are sensitive to talimogene laherparepvec infection at viral doses above 0.1 MOI.
  • FIG. 12A left panel: The murine B16-F10 melanoma cell line is not susceptible to HSV-l infection because it lacks HSV entry receptors. As shown by MTS assay measuring cell viability, of B16-F10 cells are resistant to talimogene laherparepvec infection (red) but Nectin-l- transduced B16-F10 cells (blue) are sensitive to
  • FIG. 12A right panel: MTS assay shows that D4M3A cells infected with increasing MOI of talimogene laherparepvec at 3 days post infection are sensitive to talimogene laherparepvec-mediated lysis.
  • FIG. 12B D4M3A cells also exhibit higher levels of phosphorylated ERK1/2 by immunoblot assay compared to native B16-F10 and B16-F10- Nectin-l cells. These data support the ability of talimogene laherparepvec to infect and kill D4M3A cells.
  • FIGs. 13A-13B illustrate validation of immune cell depletion.
  • mice were sacrificed 24 h after the last dose, splenocytes isolated, and stained with or without anti mouse CD4 and CD8a antibodies, or anti-mouse CDl lb and F4/80 antibodies, and analyzed by flow cytometry. Flow cytometry analysis confirmed depletion of targeted immune cell populations.
  • FIGs. 14A-14C illustrate characterization of D4M3A tumor cells in Batf3 knockout mice.
  • age-matched C57BL/6J and Batf3 / mice were implanted subcutaneously in the right flank with 3 x 10 5 D4M3A murine melanoma cells on day 0.
  • Tumor growth was monitored and the mean tumor area was similar in C57BL/6J and Batf3 /_ mice (FIG. 14A). Survival was also monitored and no differences were seen between the two types of mice (FIG. 14B).
  • FIGs. 15A-15C illustrate NanoString gene expression heat maps for all genes profiled and by gene function.
  • FIG. 15 A Heatmap representing normalized gene expression of all genes included in the NanoString PanCancer Immune panel.
  • FIG. 15 A Heatmap representing normalized gene expression of all genes included in the NanoString PanCancer Immune panel.
  • FIG. 15B Heatmap representing the normalized gene expression signature of genes associated with innate anti-viral immune responses.
  • FIG. 15C Heatmap representing the normalized gene expression signature of genes associated with specific immune cell function.
  • mT.E. OncoVEX mGM CSF .
  • FIGs. 16A-16B illustrates time course analysis of tumor-infiltrating CD8 + T cells during OncoVEX mGM CSF treatment.
  • Day 19 Mice bearing D4M3 A tumors were treated with 1 x 10 6 pfu of OncoVEX mGM CSF on days 15 and 18 and tumors collected on day 19. Bar graph indicating the % CD8 + antigen specific T cells as indicated.
  • Day 24 Mice bearing D4M3A tumors were treated with 1 x 10 6 pfu of OncoVEX mGM CSF on days 15, 18, 21, and 23 and tumors harvested on day 24. Bar graph indicating the % CD8 + antigen specific T cells as indicated.
  • FIGs. 17A-17C illustrate analysis of CD8 + T cells from spleen during
  • OncoVEX mGM CSF + MEKi treatment C57BL/6J mice implanted s.c. in the right flank with 3 x 10 5 D4M3A cells and treated with OncoVEX mGM CSF (1 x 10 6 pfu) or sterile water i.t. for 3 doses on days 15, 19 and 22 and or trametinib (0.5 mg/kg) or vehicle (0.2% Tween 80 and 0.5% Hydroxypropyl methyl cellulose) orally once daily on days 15-19. Spleens were harvested on day 24 and flow cytometry was performed. Cells were gates on live, CD45 + , CD3 + , CD8 + and further analyzed for antigen specificity. FIG.
  • FIG. 17A Representative plots and bar graph showing quantification of HSV-l -specific H-2Kb-HSV-lgB dextramer positive CD8 T cells from spleen.
  • FIG. 17B Representative plots and bar graph showing quantification of melanoma antigen specific H-2Db-gpl00 dextramer positive CD8 T cells from spleen.
  • FIG. 17C Representative plots and bar graph showing quantification of melanoma antigen specific H-2Kb-TRP2 dextramer positive CD8 T cells from spleen. Data presented as mean ⁇ SEM and the statistical differences between groups was measured by one-way ANOVA. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. Only significant values are indicated.
  • FIGs. 18A-18B illustrate MFI expression of PD-l expression and frequency of PD-l + cells.
  • OncoVEX mGM CSF lxlO 6 pfu
  • trametinib 0.5 mg/kg
  • vehicle control on days 15-19 via oral gavage
  • aPD-l antibody (clone: RMP1-14, 10 mg/kg) via i.p. injection on days 15, 19 and 22.
  • FIG. 18A Bar graph indicating the mean fluorescence intensity (MFI) of CD45 + PD-l + (right panel) and
  • FIG. 18B Bar graph indicating the percent positive CD45 + PD-l + (right panel) and percent positive CD45 + CD8 + PD-l + (left panel). Each experiment was repeated at least two times with similar results. Data are presented as mean ⁇ SEM and statistical differences between groups were measured by using one-way ANOVA *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. Only significant differences are indicated.
  • FIG. 19 illustrates individual tumor growth curves of BALB/c mice bearing CT26 tumors. Growth curves from two individual experiments.
  • groups received equivalent doses of double combination treatment with
  • OncoVEX mGM CSF , MEKi and aPD-l antibody Mean tumor area across groups was calculated on day 26 and statistical differences between groups were measured by one-way ANOVA, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. Only significant differences are shown.
  • the invention relates in part to the unexpected discovery that a MEK inhibitor and talimogene laherparepvec work synergistically to treat or prevent melanoma in a subject in need thereof.
  • the subject is administered a MEK inhibitor and talimogene laherparepvec.
  • the subject is administered a MEK inhibitor, a HSV-l-based oncolytic virus lacking any copy of the ICP 34.5 neurovirulence gene (such as, for example, talimogene laherparepvec), and a PD-1/PD-L1 checkpoint blockade agent (also known as checkpoint inhibitor).
  • the dual or triple treatment of the present invention can be used to treat or prevent cancers such as melanoma, breast cancer, head and neck cancer, lung cancer, urothelial (bladder) cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, hepatocellular cancer, lung cancer, non-melanoma skin cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, glioblastoma, and some forms of hematologic cancers, such as lymphoma and multiple myeloma.
  • cancers such as melanoma, breast cancer, head and neck cancer, lung cancer, urothelial (bladder) cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, hepatocellular cancer, lung cancer, non-melanoma skin cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, glioblastoma, and some forms of hematologic cancers, such as lymphoma and multiple myeloma.
  • Combination therapies have the potential to effectively target cancers, as the individual agents can act at different parts of the cancer-immunity cycle. Observation of unique and unexpected results provides an opportunity for a novel combination of cancer therapeutic agents to more effectively combat cancer. That said, it is important to test such combination therapies in vivo, as cancer progression often relies on multiple complex biological pathways.
  • MAPK inhibition can improve oncolytic virus responses, because viral infection can help activate a more robust immune response.
  • the present invention relates, in one aspect, to the combination of talimogene laherparepvec and MAPK-targeted therapy to enhance therapeutic activity.
  • the administration of talimogene laherparepvec and MEK inhibitors increases the sensitivity of the host to treatment with PD-1/PD-L1 blockade, and the talimogene laherparepvec and MEK inhibitor treatment is thus followed by administration of a PD-1/PD-L1 checkpoint blockade agent.
  • the subject is not administered a BRAF inhibitor.
  • Certain methods of the invention are exemplified herein with melanoma, which is characterized by frequency of BRAF mutations, availability of approved MAPK inhibitors, and an oncolytic virus for clinical testing.
  • melanoma which is characterized by frequency of BRAF mutations, availability of approved MAPK inhibitors, and an oncolytic virus for clinical testing.
  • talimogene laherparepvec was observed in both human xenograft and immune competent melanoma models.
  • Therapeutic activity was dependent on T cell activation, and presence of melanoma antigen specific CD8 + T cells and basic leucine zipper transcription factor ATF-like 3 (Batf3 + ) CDl03 + / CD8 + dendritic cells. Emergence of HSV-specific CD8 + T cells and antigen spreading with melanoma antigen-specific (gplOO and TRP-2) effector CD8 + T cells accumulating within the tumor microenvironment was demonstrated. Treatment was also associated with a decrease in regulatory CD4 + FoxP3 + T cells. The combination was associated with increased HSV-l replication and induced apoptotic cell death at higher rates than treatment with monotherapy in vitro and in vivo.
  • Talimogene laherparepvec increased an interferon-y-inflammatory gene expression, including increased expression of PD-l and PD-L1, and triple therapy with talimogene laherparepvec, trametinib, and PD-l blockade further enhanced tumor rejection and host survival.
  • These agents are all approved as monotherapies in melanoma, and these data support triple combination therapy with MEK inhibition, oncolytic viruses, and PD-1/PD-L1 checkpoint blockade for the treatment of melanoma.
  • the therapy can be used in patients with advanced melanoma (i.e.. melanoma that has metastasized to some extent through the body, and includes stage III and stage IV melanoma), independent of BRAF mutation status.
  • the present invention contemplates the use of an oncolytic virus (OV), which preferentially infects and kills cancer cells.
  • Oncolytic viruses OVs
  • OVs are a new class of cancer drugs that utilize native or genetically modified viruses to selectively replicate in tumor cells. OVs mediate therapeutic activity through multiple mechanisms, including direct immunogenic tumor cell killing, release of soluble tumor antigens, danger signals, and type 1 interferons, and induction of host anti-tumor immunity.
  • the OVs contemplated in the present invention are HSV-l based.
  • Talimogene laherparepvec is an oncolytic herpes simplex virus, type 1 (HSV-l), wherein the genes encoding ICP 34.5 and ICP 47 are deleted, and the gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) is inserted.
  • HSV-l herpes simplex virus
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • OncoVEX mGM CSF is an HSV-l modified similarly to talimogene laherparepvec, except that murine GM-CSF is inserted instead of human GM-CSF.
  • Talimogene laherparepvec is approved for the local treatment of advanced melanoma that has recurred after initial surgery. Talimogene laherparepvec treatment has had a tolerable safety profile, with most adverse events limited to low-grade constitutional symptoms and local injection site reactions. Based on the release of tumor antigens and favorable safety profile, talimogene laherparepvec has been combined with immune checkpoint inhibitors (such as pembrolizumab and ipilimumab), and preliminary data suggest that combination treatment is associated with improved response rates without an increase in immune-related adverse events.
  • immune checkpoint inhibitors such as pembrolizumab and ipilimumab
  • the OV useful within the invention includes talimogene laherparepvec, which is described in U.S. Patent Nos. 7,063,835; 7,223,593; 7,537,924; 8,277,818; and 8,680,068; all of which are included herein in their entireties.
  • the OV comprises a herpes simplex virus which: (i) comprises a gene encoding human GM-CSF; (ii) lacks a functional ICP34.5 encoding gene and a functional ICP47 encoding gene; and (iii) is replication competent in infected tumor cells.
  • the herpes simplex virus is HSV1 strain JSI as deposited in the European Collection of Cell Cultures (ECACC) under accession number 01010209, or an HSV1 strain derived therefrom.
  • the OV comprises a herpes simplex virus 1 (HSV1) strain, which is modified such that it lacks one or more of a functional ICP34.5-encoding gene, a functional ICP6- encoding gene, a functional glycoprotein H-encoding gene and a functional thymidine kinase encoding gene, and which is derived from HSVl strain JS1 as deposited at the ECACC under accession number 01010209.
  • the OV comprises a herpes simplex virus which: (i) comprises a gene encoding an immunostimulatory protein; (ii) lacks a functional ICP34.5 encoding gene and a functional ICP47 encoding gene; (iii) is replication competent in tumor cells; and (iv) is derived from HSV1 JS1 as deposited at the ECAAC under accession number 01010209.
  • the immunostimulatory protein is GM-CSF.
  • the OV is part of a pharmaceutical composition.
  • the present invention contemplates the use of a MEK (mitogen-activated protein kinase kinase enzyme) inhibitor, such as, but not limited to, a MEK allosteric inhibitor.
  • the MEK inhibitor is trametinib, or a salt or solvate thereof.
  • Drug resistance to single-agent BRAF inhibitors frequently emerged with most patients relapsing within 7 months of initial treatment response. In some cases, resistance was associated with transcriptional alterations or secondary downstream driver mutations, such as in the mitogen/extracellular signaling regulated kinase (MEK).
  • dabrafenib and trametinib, vemurafenib and cobimetinib) of combined treatment were approved for treatment-naive, metastatic melanoma patients with BRAF-mutated melanoma.
  • MAPK inhibitors such as MEK inhibitors
  • immunotherapy acts on immune cells to promote innate and adaptive immune responses and/or prevent suppression of host anti -tumor immunity.
  • the MEK inhibitor useful within the invention can be for example an allosteric inhibitor of MEK.
  • MEK inhibitors include, but are not limited to, the following examples (or a salt or solvate thereof): trametinib (GSK1120212, MEKINIST®, or N-(3- ⁇ 3-cyclopropyl-5-[(2-fluoro-4- iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin- 1 (277) -yl ⁇ pheny l)acetamide);
  • cobimetinib (XL518, or ( ⁇ S)-[3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl][3- hydroxy-3-(piperidin-2-yl)a/etidin-l -yl] methanone);
  • binimetinib (MEK162, ARRY-162, or 5-((4-bromo-2-fluorophenyl)amino)-4-fluoro-N- (2-hydroxyethoxy)-l-methyl-lH-benzo[d]imidazole-6-carboxamide);
  • selumetinib (AZD6244, or 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3- methylbenzimidazole-5-carboxamide);
  • PD-325901 N-[(27?)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl) amino] -benzamide
  • TAK-733 (3-[(27?)-2,3-dihydroxypropyl]-6-fluoro-5-[(2-fluoro-4-iodophenyl)amino]-8- methyl-pyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione).
  • checkpoint inhibitors contemplated in the present invention include, but are not limited to: ipilimumab (target: CTLA-4); nivolumab (target: PD-l); pembrolizumab (target: PD-l); atezolizumab (target: PD-L1); avelumab (target: PD-L1); cemiplimab (target: PD- 1); and durvalumab (target: PD-L1).
  • talimogene laherparepvec increases melanoma tumor cell killing through increased viral replication and apoptosis. While BRAF inhibition had some impact on talimogene laherparepvec oncolysis, this effect was seen only in BRAF -mutant tumor cells (FIG. 9G). In contrast, MEK inhibition was able to improve talimogene laherparepvec replication and oncolytic activity in both BRAF-mutant and BRAF wild-type cell lines (FIGs. 1C & 1E). In certain non-limiting embodiments, MEK inhibitors are better than BRAF inhibitors in combination with HSV- based oncolytic virus therapy. In other non-limiting embodiments, MEK inhibitors are active regardless of BRAF mutation status.
  • FIGs. 2A-2F demonstrate induction of apoptotic tumor cell death in vitro and in vivo with talimogene laherparepvec and MEK inhibition. While apoptosis was greatest with the combination treatment, increased apoptosis was also observed with talimogene laherparepvec monotherapy (FIGs. 2F and 11 A-l 1D), indicating that apoptosis can be associated with induction of immune responses and can promote therapeutic anti -tumor activity using oncolytic HSV-l viruses (such as, but not limited to, talimogene laherparepvec).
  • oncolytic HSV-l viruses such as, but not limited to, talimogene laherparepvec
  • the present data indicate that enhancing apoptosis is a useful adjunct for driving more potent oncolytic virus activity.
  • Apoptosis can help promote the release of soluble tumor-associated antigens and block cell proliferation, allowing increased viral replication.
  • FIG. 3H CD4 + FoxP3 + regulatory T cells
  • FIG. 4E Selective immune cell depletion studies confirmed the importance of CD8 + T cells.
  • FIGs. 4A-4E the combination of OncoVEX mGM CSF and MEK inhibition induced viral-specific CD8 + T cells and increased melanoma antigen-specific CD8 + T cell responses as well (FIGs. 5A-5C), suggesting this combination is effective at initiating an anti-tumor immune response.
  • OncoVEX mGM CSF and MEK inhibition treatment Over 80% complete tumor eradication and increased survival were observed without overt signs of toxicity (FIGs. 8A-8E).
  • the lack of exhausted CD8 + T cells in the tumors of mice treated with the triple therapy supports a mechanism wherein MEK inhibition promotes viral replication and soluble antigen release, while OncoVEX mGM CSF provides interferon and anti-viral T cells to transform an
  • the treatment can be sequential with limited OncoVEX mGM CSF and MEK inhibitor treatment followed by prolonged PD-l inhibition.
  • the articles“a” and“an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • “an element” means one element or more than one element.
  • the term“about” is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term“about” is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • cancer as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to melanoma, breast cancer, head and neck cancer, lung cancer, urothelial (bladder) cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, hepatocellular cancer, lung cancer, non-melanoma skin cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, glioblastoma and some forms of hematologic cancers, such as lymphoma and multiple myeloma.
  • melanoma breast cancer, head and neck cancer
  • lung cancer urothelial (bladder) cancer
  • colorectal cancer colorectal cancer
  • endometrial cancer ovarian cancer
  • cervical cancer hepatocellular cancer
  • lung cancer non-melanoma skin cancer
  • prostate cancer renal cell carcinoma
  • hyperproliferative disorders are referred to as a type of cancer including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
  • a“disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • the terms“effective amount” or“therapeutically effective amount” or “pharmaceutically effective amount” of a compound are used interchangeably to refer to the amount of the compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • the term“pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each salt or carrier must be“acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;
  • Ringer s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof.
  • “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • pharmaceutically acceptable salt refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.
  • the term“pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound include, but are not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • the term“prevent” or“prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease. Disease and disorder are used interchangeably herein.
  • prodrug refers to a pharmacological substance, drug, formulation or compound that is administered to a subject in an inactive form. Once administered, the prodrug is metabolized in vivo into an active metabolite. In certain embodiments, a prodrug should undergo chemical conversion by metabolic processes before becoming an active pharmacological agent.
  • A“therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
  • Treating means reducing the frequency with which symptoms are experienced by a patient or subject, or administering an agent or compound to reduce the severity with which symptoms are experienced by a patient or subject.
  • An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • Batf3 + basic leucine zipper transcription factor ATF-like 3
  • BRAF serine/threonine-protein kinase B-Raf
  • CTLA4 or CTLA-4 cytotoxic T-lymphocyte-associated protein 4, also known as CD152 (cluster of differentiation 152)
  • MAPK mitogen activated protein kinase
  • OV oncolytic virus
  • PD-l programmed cell death protein 1
  • PD-L1 programmed death-ligand 1
  • pfu plaque-forming unit.
  • the invention provides methods of treating or preventing a cancer in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of talimogene laherparepvec and a therapeutically effective amount of a mitogen-activated protein kinase kinase enzyme (MEK) inhibitor.
  • the talimogene laherparepvec and the MEK inhibitor are administered concomitantly or approximately concomitantly to the subject.
  • the talimogene laherparepvec and the MEK inhibitor are not administered concomitantly to the subject.
  • the talimogene laherparepvec and the MEK inhibitor are administered at different times to the subject.
  • the talimogene laherparepvec and the MEK inhibitor are administered separately to the subject.
  • the subject is not administered a BRAF inhibitor.
  • the BRAF inhibitor is at least one selected from the group consisting of vemurafenib (RG7204 or PLX4032; N-(3- ( 15 -f 4-Chl oropheny l )- 1 //-py rrol o [ 2.3 -b
  • the cancer is selected from the group consisting of melanoma, breast cancer, head and neck cancer, lung cancer, urothelial (bladder) cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, hepatocellular cancer, lung cancer, non-melanoma skin cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, glioblastoma, and some forms of hematologic cancers, such as lymphoma and multiple myeloma.
  • the cancer is melanoma.
  • the melanoma is advanced melanoma.
  • the cancer is driven by a BRAF mutation.
  • the BRAK mutation comprises a V600E or V600K mutation.
  • the cancer is not driven by a BRAF mutation.
  • the MEK inhibitor is an allosteric inhibitor. In other embodiments, the MEK inhibitor is selected from the group consisting of: trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, 0-1040, and TAK-733.
  • the talimogene laherparepvec is administered through a route selected from the group consisting of intratumoral, cutaneous, subcutaneous, into nodal lesions, or deep/visceral injection through image-guided injection.
  • the MEK inhibitor is administered through a rote selected from the group consisting of oral, rectal, parenteral, intravenous, and intragastrical.
  • the method comprises administering to the subject a therapeutically effective amount of a HSV-l -based oncolytic virus lacking any copy of the ICP 34.5 neurovirulence gene, a therapeutically effective amount of a MEK inhibitor, and a therapeutically effective amount of an immune checkpoint blockade agent.
  • the virus, the MEK inhibitor, and/or immune checkpoint blockade agent are administered concomitantly or approximately concomitantly to the subject.
  • the virus, the MEK inhibitor, and/or immune checkpoint blockade agent are not administered concomitantly to the subject.
  • the virus, the MEK inhibitor, and/or immune checkpoint blockade agent are administered at different times to the subject.
  • the virus, the MEK inhibitor, and/or immune checkpoint blockade agent are administered separately to the subject.
  • the virus is talimogene laherparepvec.
  • the MEK inhibitor and the virus are administered to the subject for about 4 to about 6 months.
  • administration of the MEK inhibitor and the virus is at least partially concomitant with administration of the immune checkpoint blockade agent.
  • administration of the immune checkpoint blockade agent to subject is initiated at about the same time as the initial administration of the MEK inhibitor and the virus to the subject.
  • administration of the immune checkpoint blockade agent to subject is initiated a given period of time after the initial administration of the MEK inhibitor and the virus to the subject.
  • administration of the immune checkpoint blockade agent to subject is initiated about 2 to 6 weeks after the initial administration of the MEK inhibitor and the virus to the subject.
  • the immune checkpoint blockade agent targets at least one selected from the group consisting of CTLA4, PD-L1, and PD-l. In other embodiments, the immune checkpoint blockade agent is at least one selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, cemiplimab, and durvalumab.
  • the dose of the virus administered to the subject ranges from about 10 6 pfu/mL to 10 8 pfu/mL. In other embodiments, the volume of each dose of the virus ranges from about 0.1 mL to about 4 mL.
  • the dose of the MEK inhibitor administered to the subject ranges from about 1 mg/ day to 2 mg/day, wherein the inhibitor is trametinib.
  • the subject is a mammal. In other embodiments, the subject is a human.
  • the invention further provides a method of determining if a subject afflicted with a cancer and being treated with a combination of a HSV-l -based oncolytic virus and MEK inhibitor will benefit from administration of an immune checkpoint blockade agent.
  • the HSV-l -based oncolytic virus lacks any copy of the ICP 34.5 neurovirulence gene.
  • the method comprises administering to the subject a therapeutically effective amount of the virus and a therapeutically effective amount of a MEK inhibitor.
  • the method comprises measuring and/or determining, in a sample of the subject’s cancerous tissue expression, levels of at least marker selected from the group consisting of CD-8, Gzmb, IFNy, PRF1, and TNFa.
  • the subject if the levels of the at least one marker in the sample of the subject’s cancerous tissue is higher than levels of the at least one marker in a control sample, the subject is counseled to be further administered an immune checkpoint blockade agent. In yet other embodiments, the subject is further administered the immune checkpoint blockade agent.
  • control sample is a sample of the subject’s cancerous tissue before administration of the virus and MEK inhibitor.
  • the levels of the at least one marker in the sample of the subject’s cancerous tissue is at least 20% higher than the levels of the at least one marker in the control sample.
  • the levels of the at least one marker in the sample of the subject’s cancerous tissue is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% higher than the levels of the at least one marker in the control sample.
  • the compounds of the present invention are useful in the methods of present invention in combination with one or more additional compounds useful for treating the diseases or disorders contemplated within the invention.
  • additional compounds may comprise compounds of the present invention or compounds, e.g., commercially available compounds, known to treat, prevent, or reduce the symptoms of the diseases or disorders contemplated within the invention.
  • agents contemplated in the present invention can be used in combination with other treatment regimens, including surgery, radiation therapy, cytotoxic chemotherapy, molecularly targeted therapy, adoptive T cell or chimeric antigen receptor (CAR) T cell therapy, vaccines, and other forms of immunotherapy.
  • other treatment regimens including surgery, radiation therapy, cytotoxic chemotherapy, molecularly targeted therapy, adoptive T cell or chimeric antigen receptor (CAR) T cell therapy, vaccines, and other forms of immunotherapy.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigrnoid-E max equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol. Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.
  • suitable methods such as, for example, the Sigrnoid-E max equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol. Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.
  • concentration-effect curve concentration-effect curve
  • isobologram curve concentration-effect curve
  • combination index curve concentration-effect curve
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions useful within the present invention may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder in the patient.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a non-limiting example of an effective dose range for a therapeutic compound of the present invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • herpes viruses are dosed as plaque-forming units (PFU) per milliliter (mL) with dose ranges of about 1 x 10 4 to about 1 x 10 8 pfu/mL.
  • PFU plaque-forming units
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.
  • compositions useful within the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound useful within the invention and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or poly alcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions useful within the invention are:
  • compositions useful within the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions useful within the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
  • Compounds for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments thereinbetween.
  • the dose of a compound is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the present invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
  • the present invention is directed to a packaged
  • composition comprising a container holding a therapeutically effective amount of a compound of the present invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other cognition improving agents.
  • the term“container” includes any receptacle for holding the pharmaceutical composition.
  • the container is the packaging that contains the pharmaceutical composition.
  • the container is not the packaging that contains the pharmaceutical composition, /. e.. the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.
  • compositions of the present invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical.
  • the compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual,
  • transbuccal (trans)urethral, vaginal (e.g, trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, cutaneous, subcutaneous, into nodal lesions, deep/visceral injection through image-guided injection, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients which are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or
  • the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRYTM OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorb
  • the compounds may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790.
  • Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.
  • the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method of the present invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds of the present invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the disease or disorder in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
  • a suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day.
  • the amount of each dosage may be the same or different.
  • a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a l2-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on
  • the compounds for use in the method of the present invention may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • Human melanoma cells SK-MEL-28, SK-MEL-2 and SK-MEL-5 were cultured in monolayers using RPMI supplemented with 10% heat inactivated bovine serum (Thermo Fisher Scientific), lOmM L-glutamine (Coming), and 0.5% penicillin G- streptomycin sulfate (Coming). Cells were detached using 0.25% trypsin EDTA (Coming) for passaging and were cultured at 37°C in 5% C0 2 .
  • the murine melanoma cell line D4M3A was generated from Tyr: :CreER;Braf A ;Pten !ox/!ox mice (Jenkins, el al, 2014, Pigment Cell Melanoma Res. 27:495-501).
  • D4M3A cells were cultured in advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% heat inactivated bovine serum (Thermo Fisher Scientific), lOmM L-glutamine (Coming), and 0.5% penicillin G-streptomycin sulfate (Coming). Cells were detached with 0.25% trypsin supplemented with 0.53 mM EDTA (Coming) for passaging. Cells were cultured at 37°C in 5% C0 2. All cells were low-passage and confirmed to be mycoplasma-free (LookOut mycoplasma kit; Sigma).
  • Talimogene laherparepvec is a modified JS1 strain of herpes simplex type 1 (HSV-l).
  • HSV-l herpes simplex type 1
  • both copies of the ICP 34.5 neuro virulence genes are deleted to limit neurotoxicity and enhance cancer cell-specific replication (Bommareddy, et al., 2017, Am. J. Clin. Dermatol. 18: 1-15).
  • HSV-l ICP47 gene which inhibits the transporter associated with antigen processing and presentation, is also deleted to promote antigen processing and presentation.
  • two copies of the human GM-CSF gene have been engineered in place of each ICP 34.5 gene to promote dendritic cell infiltration and maturation.
  • Talimogene laherparepvec is commercially available.
  • a modified virus (OncoVEX mGM CSF ) in which the human GM-CSF gene was replaced by murine GM-CSF, was used and generously provided by Amgen Inc., Thousand Oaks, CA. All human cell lines and xenograft experiments were performed using talimogene laherparepvec with human GM-CSF. All syngeneic experiments (using C57BL/6J mice) were performed using OncoVEX mGM CSF .
  • the MEK inhibitor trametinib (GSK1120212) and BRAF inhibitor vemurafenib were purchased from MedChem Express (Monmouth Junction, NJ). Both drugs were dissolved in DMSO to make a l0-mM stock solution for in vitro studies. The highest DMSO
  • trametinib powder (0.5 mg/kg) was dissolved in 0.5% hydroxypropyl methylcellulose and 0.2% Tween-80 (Sigma Aldrich) and was administered by oral gavage.
  • talimogene laherparepvec For plaque assays, cells were plated and treated with talimogene laherparepvec alone, or talimogene laherparepvec and trametinib, as reported elsewhere herein. Cells were treated with trametinib 6-8 h before talimogene laherparepvec infection.
  • talimogene laherparepvec infection the virus was diluted using RPMI and seeded over a cell monolayer at the indicated MOI for 2 hours (plates were gently rocked every 15 min to ensure even spread of virus).
  • Whole cell lysates were collected at indicated times and viral titers obtained by plaque assay on a monolayer of Vero cells. Each experimental condition was performed in triplicate and all experiments were repeated at least two independent times.
  • SK-MEL-28 cells were seeded into 24-well plates at a density of 500,000 cells/mL and infected with talimogene laherparepvec or trametinib or both as described elsewhere herein. At the specified time points, cells were detached from the wells using TrypLE (Thermo Fisher Scientific), resuspended in culture medium, and then analyzed using a Radiance instrument (LumaCyte). Radiance uses Laser Force Cytology, a combination of advanced optics and microfluidics to analyze cells based upon their intrinsic properties, while simultaneously taking high resolution video of each cell.
  • Radiance is similar in form and function to a modem flow cytometer but employs optical force (laser photon pressure) and image capture together with microfluidics to image, analyze, and characterize cell populations in a fundamentally distinct way.
  • the threshold velocity (which in this case defines the infection metric), was calculated based on the velocity at which approximately 5% of the control cells have a velocity above the infection metric. This is a similar to gating for fluorescence in flow cytometry.
  • a principal component analysis (PCA) was performed using XLStat. input data included the infection metric, average velocity, and average size of each sample. This combination resulted in components (FI and F2) that represented the largest possible variance the data.
  • mice Male C57BL/6J mice (stock: 000664), NOD scid gamma, NOD-vcv / IL2R «' m ".
  • NOD- scid IL2Rgamma nu11 (NSG) mice stock:005557) and BatG mice,
  • Batf3 tmlKmm /J (stock:0l3755) were used at 8-9 weeks of age. All mice were obtained from Jackson Labs (Farmington, CT). All syngeneic C57BL/6J mice studies with D4M3A tumors were treated using OncoVEX mGM CSF .
  • mice were monitored for tumor-growth and euthanized before tumors reached 400 mm 2 . Kaplan-Meier curves were used to calculate survival. Mice were weighed twice a week and no weight loss was observed during the treatment.
  • SK-MEL-28 cells (8 x 10 6 ) were injected into the right flank of NSG mice in 100 m ⁇ PBS. Mice were treated with either talimogene laherparepvec (1 x 10 5 pfu) or sterile water via intratumoral injection on days 35, 40, and 45. Trametinib (0.5 mg/kg) or vehicle control was given on days 35-43 via oral gavage.
  • mice in the combination treatment group received both talimogene laherparepvec and trametinib at the above doses and schedule.
  • the vehicle control comprised a mixture of 0.2% Tween 80 and 0.5% hydroxypropyl methylcellulose (HPMC).
  • mice 7 from 2 independent experiments
  • Tumors were harvested and FACS was performed as described elsewhere herein.
  • D4M3A cells (6 x 10 5 ) in the contralateral flank (left) at day 96.
  • Batf3 /_ mouse studies Batf3 /_ or C57BL/6J mice were implanted with (3 x 10 5 ) D4M3A cells on day 0 and treated with OncoVEX mGM CSF or sterile water via i.t. injection and/or trametinib or vehicle control via oral gavage.
  • Murine anti-PD-l (rat clone RMP1-14; 10 mg/kg), and isotype control antibodies, rat IgG2a (clone LTF-2), were obtained from BioXcell and administered 4 times via intraperitoneal (i.p.) injection.
  • mice 10 from 2 independent experiments
  • OncoVEX mGM CSF + MEKi + aPD-l therapy were rechallenged on day 130 with a two-fold increased number of D4M3A cells (6 x 10 5 ) in the contralateral (left) flank.
  • groups received double combination treatment with OncoVEX mGM CSF EC and MEKi (and rat IgG isotype control) or triple. Tumors were collected on day 24 and flow cytometry analysis of tumor infiltrating lymphocytes was performed as described elsewhere herein.
  • groups received double treatment with OncoVEX mGM CSF + aPD-l, MEKi + aPD-l,
  • OncoVEX mGM CSF , MEKi and aPD-l antibody OncoVEX mGM CSF , MEKi and aPD-l antibody.
  • tumor growth was measured in two dimensions recording the greatest length and width using digital calipers. Tumor area was calculated by multiplying length and width. Tumor sizes were plotted as average size for each group. For survival experiments mice were monitored for tumor-related death and euthanized when they reached experimental endpoints. Kaplan-Meier curves were used to document survival.
  • mice were weighed twice a week and no weight loss was observed during the treatment.
  • Mock group received sterile water (i.t.) + vehicle (0.2% Tween 80 and 0.5% HPMC) + control rat IgG (i.p.) + empty liposomes (i.p.).
  • Isotype group received talimogene laherparepvec (i.t.) + MEKi (i.p.) + control rat IgG (i.p.) + empty liposomes (i.p.) as elsewhere herein.
  • Anti-CD4 or anti-CD8 group received talimogene laherparepvec + MEKi + anti-CD4 or anti-CD 8 + empty liposomes
  • Clodronate group received talimogene laherparepvec + MEKi + control rat IgG + clodronate liposomes.
  • Tumors were harvested at indicated time points and sections were deparaffimzed using Xylene twice for 10 min each, followed by gradual rehydration using 100%, 90% and 70% ethanol treatment (5 mm each). Sections were left in distilled water for 10 mm, followed by dipping sections in a hematoxylin container for 1 min, washing in tap water for 5 min, dipping in Eosm Y (1 % alcoholic) for 30 s. This was followed by gradual dehydration using 95% ethanol (twice 5 mm each) and 100% ethanol (twice 5 min each), treating with Xylene twice tor 10 min each, and mounting in Xylene-based media (Cytoseai XYL; Thermo Scientific).
  • mice (n ::: 5) were implanted with human melanoma SK-MEL-28 cells (8 x 10 6 ) on day 0 and treated with either talimogene laherparepvec (5 x 10 5 pfu) or sterile water for injection via intratumoral injection on days 30 and 34 or trametinib (0.5 mg/kg) or vehicle control on days 30-34 via oral gavage.
  • mice in the combination group received both talimogene
  • B6 mice were implanted with 3 x 10 5 D4M3A cells and treated with OncoVEX mGM CSF (10 6 pfu i.t.) for 3 doses on days 15, 19 and 22 and/or trametinib (0.5 mg/kg) orally once daily on days 15-19.
  • mice were euthanized on day 24 and tumors were removed and fixed in 10% formalin for 24-36 h, embedded in paraffin, and 5 pm-sections subjected to immunohistochemistry with indicated antibodies (Key Resources Table, IHC), followed by incubation with appropriate secondary antibodies (Vector Laboratories) as described above.
  • IHC Intracellular Immunosorbent
  • secondary antibodies Vector Laboratories
  • Positive cell density was computed as positive cell count / mm 2 tissue area for cleaved caspase 3, Ki67, pERKl/2 and CD8.
  • HSV-l staining was quantified as an average brown staining intensity over the selected area.
  • Annexin-V expression was detected on SK-MEL-28 ceils after culture for 24 h with or without talimogene laherparepvec at the indicated MOI and/or trametinib at 5 nM. Ceils were centrifuged, counted, re-suspended in FACS buffer (2% inactivated fetal calf serum in PBS), incubated with 7-AAD (BD Biosciences) and FITC-conjugated antibody for 30 min, washed, fixed in 4% paraformaldehyde, washed, re-suspended in FACS buffer, and analyzed using an LSRII flow cytometer (BD Biosciences) and FlowJo software (v.10.4; Tree Star).
  • mice with D4M3A flank tumors from treated groups were harvested, mechanically dissociated using a gentleMacs Octo Dissociator (Miitenyi), incubated with co!lagenase (1 mg/ml, Sigma Aldrich) and DNase I (10 U/tnl; Promega) for 30 minutes with rocking at 37°C, mechanically dissociated again, passed through a 40 pm screen re-suspended in FACS buffer and stained with fluorochrome- conjugated anti-mouse antibodies, as well as appropriate isotype control antibodies. Fixable live/dead viability Kit (Invitrogen) was used to stain dead ceils.
  • spleens from naive B6 nnce were treated with ACK.
  • Lysis Buffer Sigma Aldrich
  • Single cells were stained with each of ten fl uorescen t-conj ugated antibodies according to manufacturer’s instructions. All samples were analyzed using a BD LSRH flow cytometer. Data were analyzed with FlowJo software (v.10.4; Tree Star). Technicians acquiring and analyzing the data were blinded to the treatments.
  • B6 mice implanted with D4M3A cells (3 x 10 5 ) were treated with OncoVEX mGM CSF (10 6 pfu) via i.t. injection for 3 doses on days 15, 19 and 22 and/or trametinib (0.5 mg/kg) orally once daily on days 15-19. Tumors were harvested on day 24, and total RNA was isolated using a Qiagen RNAeasy kit. Gene expression analysis was performed using the NanoString PanCancer Immune panel. Per sample, 50 ng of total RNA in a final volume of 5 m ⁇ was mixed with a 3' biotinylated capture probe and a 5' reporter probe tagged with a fluorescent barcode from the custom gene expression code set.
  • Probes and target transcripts were hybridized at 65°C for 12-16 h. Hybridized samples were run on the NanoString nCounter preparation station using the recommended manufacturer protocol, in which excess capture and reporter probes were removed and transcript-specific ternary complexes were immobilized on a streptavidin-coated cartridge. The samples were scanned at maximum scan resolution on the nCounter Digital Analyzer. Data w ere processed using nSolver Analysis Software and the nCounter Advanced Analysis module. For gene expression analysis data were normalized using the geometric mean of housekeeping genes selected by the GeNorm algorithm (Vandesompele, et al., 2002, Genome Biol. 3:RESEARCH0034).
  • Example 1 Combination of MEK Inhibition and Oncolytic Virus Treatment Augments Oncolytic Activity and Viral Replication in Human and Mouse Melanoma Cell Lines.
  • Talimogene laherparepvec is an oncolytic HSV-l wherein the genes encoding ICP 34.5 and ICP 47 are deleted, and the gene encoding GM-CSF is inserted.
  • Talimogene laherparepvec exhibits selective replication in human tumor cells and is the first oncolytic virus approved by the FDA as a treatment for melanoma.
  • talimogene laherparepvec alone exhibit long-term benefit.
  • melanoma patients experience only a short-term benefit from therapy focused on targeting cells with BRAF or N-Ras mutations common to melanoma.
  • Talimogene laherparepvec was found to be able to replicate in and kill both melanomas that harbor BRAF V600E mutations but have wild-type N-Ras (SK-MEL-28 and SK-MEL-5; FIGs. 9A-9B) and those with wild-type BRAF but an N-Ras Q61R mutation (SK-MEL-2; FIG. 9C).
  • Lumacyte single-cell laser radiance-based quantitative technology was utilized. This is a system that can detect virally infected single cells based on their size and velocity in a single-cell capillary flow chamber (in single-cell level). Cells infected with virus are larger, and hence, move at a slower velocity through the system (FIG. 10A). A set of velocity histograms are generated at various time points (12, 24 and 36 hours) after viral infection and cells subjected to radiance cytometric analysis (FIG. 10B-10C). Based on the velocity and cell size, an infection metric is calculated for each cell population and this value is higher in virally infected cells. As shown in FIG.
  • the infection metric was increased at 18 hours for virally infected cells, with the highest metric seen in cells treated with talimogene laherparepvec and MEK inhibitor (FIG. 1F, left).
  • a time-course analysis was performed in cells infected at low MOI (0.01) or high MOI (1.0) of talimogene laherparepvec or uninfected control cells. Infection metric was highest for cells infected with 1 MOI but cells infected with 0.01 MOI demonstrated an increase in infection metric at 36 hours when more virus had replicated (FIG. 1F, right).
  • PCA principal component analysis
  • Example 2 Combination of Talimogene laherparepvec and MEK Inhibition Induces Tumor Cell Apoptosis and Inhibits Tumor Growth in Melanoma Xenograft Models.
  • talimogene laherparepvec and MEK inhibition had therapeutic activity against melanoma tumor growth in vivo.
  • a murine xenograft model challenged with SK-MEL-28 was utilized (FIG. 2A).
  • NSG mice were transplanted with SK-MEL-28 (8 x 10 6 ) cells in the right flank and allowed to establish palpable tumors, typically around day 30.
  • These mice were treated with 0.5 mg/kg of trametinib by oral gavage daily for 7 days from day 35-43 alone or with the addition of talimogene laherparepvec (1 x 10 5 pfu) given via intratumoral injection on days 35, 40 and 45.
  • the talimogene laherparepvec group received talimogene laherparepvec and vehicle (0.5% hydroxypropyl methylcellulose and 0.2% Tween-80) via oral gavage, and the mock group received sterile water via intratumoral injection and vehicle via oral gavage.
  • MEKi group received trametinib via oral gavage and sterile water via intratumoral injection.
  • FIG. 2A a delay in tumor growth was observed for either MEK inhibition alone or talimogene laherparepvec alone, but combination treatment was associated with a significant decrease in tumor growth (p ⁇ 0.0001) compared to mock or monotherapy treatment (FIG. 2B).
  • HSV-l was present in tumor cells as shown by immunostaining for the HSV-l glycoprotein D, which was seen in the talimogene laherparepvec alone-treated tumors and significantly increased in tumors exposed to combination of talimogene laherparepvec and trametinib (FIG. 2D).
  • Decreased levels of phosphorylated (p)-ERK were observed in tumor cells treated with trametinib, but levels of p- ERK were significantly reduced in tumors treated with talimogene laherparepvec alone and even further decreased in tumors treated with combination of talimogene laherparepvec and trametinib (FIG. 2E).
  • SK-MEL-28 cells were treated in vitro with talimogene laherparepvec, trametinib, both or mock controls, and an increase in Annexin-V staining was found in cells treated with combination therapy (FIGs. 11A-11B), and this effect could be partially blocked by Z-VAD (FIG. 11C). Further, an increase in cleaved PARP was shown in tumor cells treated with both talimogene laherparepvec and trametinib by Western blot analysis (FIG. 11D).
  • OncoVEX mGM CSF (1 x 10 6 pfu) or trametinib (0.5 mg/kg) or both.
  • OncoVEX mGM CSF was given via intratumoral injection for 6 doses on days 15, 19, 22, 26, 29 and 33 post tumor implantation. Trametinib was given, from days 15 to 27 once daily by oral gavage. Tumors were measured twice weekly using calipers and tumor area calculated.
  • Combination of OncoVEX mGM CSF and trametinib was associated with significant tumor inhibition and improved host survival compared to mock- and
  • mice that exhibited complete tumor eradication were re-challenged with twice the number of D4M3A cells (6 x 10 5 ) implanted in the opposite flank (left) of cured mice. In this setting, most mice (5/7) 70% completely rejected the re-challenged tumor (FIG. 3D).
  • OncoVEX mGM CSF and MEK inhibition is associated with significant therapeutic activity against melanoma in immune competent mice and treatment results in durable memory responses.
  • tumors were harvested from treated mice at day 24 and tumors evaluated for infiltrating CD8 + T cells by flow cytometry.
  • FACS analysis revealed a significant increase in tumor infiltrating CD8 + T cell during combination therapy (FIG. 3E).
  • the increased CD8 + T cell population was further characterized as having increased levels of interferon-g, Granzyme B, and Ki-67 positive CD8 + T cells, all consistent with an activated cytotoxic phenotype (FIG. 3E).
  • Increase in CD8 + T cells is also confirmed by
  • Example 4 Depletion of CD8 + T cells Abrogates the Effects of OncoVEX mGM CSF and MEKi Combination Therapy; OncoVEX mGM CSF and MEKi Combination is CD8 + T cell-Dependent
  • OncoVEX mGM CSF and MEKi combination therapy the in vivo tumor experiments were repeated in the setting of CD4 + or CD8 + cell depletion. It was also investigated whether macrophage populations play a role by depleting these cells with liposomal clodronate. All cell depletions were confirmed by FACS analysis of splenocytes derived from mice treated with specific depletion antibodies for CD4 or CD8 (FIG. 13 A) or clodronate for macrophages (FIG. 13B). Mice bearing D4M3A tumors were treated as described in the survival experiments in FIGs. 3A-3H.
  • Example 5 Combination of OncoVEX mGM CSF and MEK Inhibition Induces Viral- specific CD8 + T Cell Responses and Promotes Anti-Tumor Immunity.
  • OncoVEX mGM CSF treatment resulted in detectable HSV-l gB-specific CD8 + T cells (8%) while combination of OncoVEX mGM CSF and MEK inhibition resulted in a significant increase in tumor-infiltrating HSV-l gB-specific CD8 + T cells to 17% of the CD8 + T cell pool (FIG. 5 A).
  • combination treatment also enhanced melanoma-specific T cell responses, the presence of CD8 + T cells specific against known melanoma antigens from the same tumor-infiltrating lymphocyte bulk population was evaluated.
  • Example 6 Combination of OncoVEX mGM CSF and MEK Inhibition is Dependent on Batf3 + Dendritic Cells to Augment Anti-Tumor Activity and Antigen Spreading.
  • mice Batf3 /_ mice, and once tumors were established (usually around day 15) mice were treated with OncoVEX mGM CSF (1 x 10 6 pfu) by intratumoral injection on days 15, 19, 22, 26, 29 and 33 (or mock sterile water injection), trametinib (0.5 mg/kg) by oral gavage on days 15-27 (or vehicle), or both OncoVEX mGM CSF and trametinib as described in FIGs. 3A-3H. While combination treatment resulted in delayed tumor growth in C57BL/6J mice, as previously seen, this effect was significantly diminished in Batf3 /_ mice (FIG. 6A) and the host survival benefit observed in B6 wild-type mice treated with OncoVEX mGM CSF and MEK inhibition was also reduced in Batf3 /_ mice (FIG. 6B).
  • OncoVEX mGM CSF and MEKi tumors harvested at day 24 and were subjected to flow cytometry analysis.
  • Treated Batf3 mice demonstrated a significant decrease in the percentage and number of CD8 + T cells compared to C57BL/6J (B6) (FIG. 6C).
  • OncoVEX mGM CSF and MEK inhibition and that these cells are involved in priming viral- specific CD8 + T cells and promoting cross presentation of melanoma antigens to generate melanoma-specific CD8 + T cells within the tumor microenvironment of treated animals.
  • Example 7 Combination of OncoVEX mGM CSF and MEK Inhibition Induces an
  • PD-l checkpoint blockade There is an inflammatory gene signature in patients responding to PD-l checkpoint blockade, comprising interferon-g responsive genes associated with antigen presentation, chemokine expression, cytotoxic lymphocyte function and adaptive resistance.
  • interferon-g responsive genes associated with antigen presentation, chemokine expression, cytotoxic lymphocyte function and adaptive resistance.
  • tumors are more susceptible to immunotherapy.
  • IFN-g in this process indicates that counter- regulatory expression of checkpoint inhibitors, such as PD-L1, can further increase sensitivity to immunotherapy.
  • OncoVEX mGM CSF is associated with interferon release and CD8 + T cell recruitment to the tumor microenvironment
  • the tumors from treated mice were evaluated for evidence of an increased inflammatory gene expression profile.
  • NanoString gene expression analysis a 16-gene inflammatory expression profile was increased in tumors treated with OncoVEX mGM CSF compared to both mock and trametinib treatment, and that this profile was highest in tumors treated with combination of OncoVEX mGM CSF and trametinib (FIG. 7A).
  • OncoVEX mGM CSF the combination of OncoVEX mGM CSF and MEKi resulted in increased gene expression compared to OncoVEX mGM CSF alone, except for interleukin-34 ( IL34 ) and NKG2D ligand [UL16 binding protein 1 (Ulbpl) ⁇ .
  • IL34 interleukin-34
  • Ulbpl UL16 binding protein 1
  • OncoVEX mGM CSF and MEK inhibitor-treated animals (FIG. 7C), this was further confirmed through flow cytometric analysis of CD45 + cells harvested from the tumor microenvironment at day 24 (FIG. 3D).
  • Flow cytometry analysis demonstrated an increase in both PD-l and PD-L1 levels in tumors treated with OncoVEX mGM CSF alone but levels of both PD-l and PD- Ll were highest in tumors treated with combination of OncoVEX mGM CSF and MEK inhibition (FIG. 7D).
  • These data indicate that combination of OncoVEX mGM CSF and trametinib increase expression of interferon-y-regulated genes related to immune cell function and most notably T cell activation. Further, treatment enhances expression of T cell checkpoints, such as PD-l and PD-L1, which can make tumors more sensitive to treatment with immune checkpoint blockade.
  • Example 8 Triple Treatment with OncoVEX mGM CSF , MEK Inhibition and PD-l Blockade Further Enhances Therapeutic Activity and Limits Accumulation of
  • OncoVEX mGM CSF and MEK inhibition reduced tumor burden and enhanced survival of treated mice (FIGs. 3B-3C), but tumors were completely eradicated in only 30-40% of mice.
  • FIG. 7D Based on the flow cytometry analysis and the gene expression profiling showing an increase in PD-l and PD-L1 expression in the tumor microenvironment (FIG. 7D), that therapeutic activity can be further expanded by addition of PD-l blockade to the combination regimen.
  • D4M3A tumor-bearing mice were treated with OncoVEX mGM CSF , trametinib or both as described in FIG. 3C with or without anti-PD-l antibody (10 mg/kg twice weekly on days 15, 19, 22, and 26).
  • OncoVEX mGM CSF and trametinib (FIG. 8C). Triple therapy also resulted in a significant improvement in survival (FIG. 8C). All mice who cleared primary tumors with mT- VEC/MEKi/aPD-l therapy rejected subsequent tumor rechallenge (FIG. 8D). Flow cytometry analysis performed on tumors showed a significant decrease in CD45 + PD-l + and CD8 + PD-l + cells in mice treated with triple therapy compared to OncoVEX mGM CSF and MEKi (FIG. 8E and FIGs. 18A-18B). No significant changes were observed in Tregs or the CD8 + /Treg ratio (FIG. 8F).
  • OncoVEX mGM CSF , MEK inhibition, and PD-l blockade in melanoma indicate that treatment is associated with considerable survival benefit in an immune competent murine model and correlates with accumulation of fewer exhausted CD8 + PD-l + T cells in the tumor microenvironment.
  • the present studies demonstrate that the combination of T-VEC and MEK inhibition increases melanoma tumor cell killing through increased viral replication and apoptosis in vitro and enhances melanoma-specific adaptive immune responses in vivo.
  • a factor known to influence the replicative ability of viruses is the status of the antiviral machinery, which is composed of intracellular factors that detect viral nucleic acids and molecular elements that promote viral clearance, such as type 1 IFN, and viral DNA sensors such as cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes).
  • the expression of the antiviral machinery is typically defective in tumor cells, which allows preferential replication for many oncolytic viruses.
  • STING inhibition in tumor cells may promote apoptosis and drive immunogenic cell death, MEK inhibition may also block T cell activation. It was thus unexpected that strong antiviral and antitumor CD8+ T cell responses were observed herein (FIGs. 3A-3H and 5A-5C). Without wishing to be limited by any theory, increased expression of STING and TLRs can also promote recruitment of antitumor CD8 + T cells because induction of these innate immune sensors restores effective antitumor immunity. In certain embodiments, STING expression is a predictive biomarker of talimogene
  • melanoma cell sensitivity to talimogene laherparepvec is inversely related to STING expression.
  • talimogene laherparepvec promotes innate and adaptive anti-tumor immunity and induces therapeutic responses in low STING expressing tumors, such as but not limited to certain melanomas.
  • BRAF inhibition enhanced talimogene laherparepvec oncolysis only in BRAF-mntmt tumor cells.
  • MEK inhibition improved talimogene laherparepvec replication and oncolytic activity in both BRAF-rmtmt and BRAF wild-type cell lines.
  • MEK inhibition is more efficient than BRAF inhibition in combination with HSV-based oncolytic viral therapy and is active regardless of BRAF mutation status.
  • Therapeutic effectiveness was seen herein in both human xenograft and immune- competent melanoma models. Combination of talimogene laherparepvec and MEK inhibition is also associated with increased accumulation of activated CD8 + T cells, characterized byproduction of IFN-g and granzyme B, within the tumor microenvironment as well as an increase in CD8 + /Treg ratio.
  • CD8 + T cells was confirmed through selective immune cell depletion studies.
  • HSV-l can promote IFN production, and an increase in PD-l and PD-L1 expression was observed within the tumor microenvironment, and this may be in certain embodiments related to the counter-regulatory induction of immune checkpoints in the setting of excessive IFN-g.
  • the Batf3 + (CD8 + CDl03 + ) DC population is thought as being critical for priming antiviral CD8+ T cell responses. These DCs are also critical for antitumor immunity and recruitment of lymphocytes through chemokines, such as CXCL9.
  • the present experiments showed that Batf3 + DCs are also critical for the recruitment of CD8 + T cells in melanoma models after treatment with talimogene laherparepvec and trametinib and an increase in CXCL9 expression was observed.
  • the present studies evaluated the combination of MAPK inhibition and talimogene laherparepvec in murine and human melanoma cell lines and showed a synergistic effect between talimogene laherparepvec and MEK inhibition regardless of BRAF mutation status. Therapeutic responses were further improved by addition of anti-PD-l therapy. The present studies did not indicate overt signs of toxicity in mice, supporting an improved therapeutic window. Collectively, these data provide rationale for triple-combination treatment of talimogene laherparepvec, MEK inhibition, and PD-l blockade in patients with melanoma.
  • Embodiment 1 provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of talimogene laherparepvec and a therapeutically effective amount of a mitogen- activated protein kinase kinase enzyme (MEK) inhibitor.
  • MEK mitogen- activated protein kinase kinase enzyme
  • Embodiment 2 provides the method of Embodiment 1, wherein the cancer is selected from melanoma, breast cancer, head and neck cancer, lung cancer, urothelial (bladder) cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, hepatocellular cancer, lung cancer, non-melanoma skin cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, glioblastoma, lymphoma, or multiple myeloma.
  • the cancer is selected from melanoma, breast cancer, head and neck cancer, lung cancer, urothelial (bladder) cancer, colorectal cancer, endometrial cancer, ovarian cancer, cervical cancer, hepatocellular cancer, lung cancer, non-melanoma skin cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, glioblastoma, lymphoma, or multiple myeloma.
  • Embodiment 3 provides the method of any of Embodiments 1-2, wherein the cancer is melanoma.
  • Embodiment 4 provides the method of any of Embodiments 1-3, wherein the melanoma is advanced melanoma.
  • Embodiment 5 provides the method of any of Embodiments 1-4, wherein the cancer is driven by a BRAF mutation.
  • Embodiment 6 provides the method of Embodiment 5, wherein the BRAF mutation comprises a V600E or V600K mutation.
  • Embodiment 7 provides the method of any of Embodiments 1-4, wherein the cancer is not driven by a BRAF mutation.
  • Embodiment 8 provides the method of any of Embodiments 1-7, wherein the MEK inhibitor is an allosteric inhibitor.
  • Embodiment 9 provides the method of any of Embodiments 1-8, wherein the MEK inhibitor is selected from trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI- 1040, or TAK-733.
  • the MEK inhibitor is selected from trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI- 1040, or TAK-733.
  • Embodiment 10 provides the method of any of Embodiments 1 -9, wherein the talimogene laherparepvec is administered through a route selected from intratumoral, cutaneous, subcutaneous, into nodal lesions, or deep/visceral injection through image-guided injection.
  • Embodiment 11 provides the method of any of Embodiments 1-10, wherein the MEK inhibitor is administered through a rote selected from oral, rectal, parenteral, intravenous, intraperitoneal, intrapleural, or intragastrical.
  • Embodiment 12 provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a HSV-l -based oncolytic virus lacking any copy of the ICP 34.5 neurovirulence gene, a therapeutically effective amount of a MEK inhibitor, and a therapeutically effective amount of an immune checkpoint blockade agent.
  • Embodiment 13 provides the method of Embodiment 12, wherein the HSV-l -based oncolytic virus is talimogene laherparepvec.
  • Embodiment 14 provides the method of any of Embodiments 12-13, wherein the MEK inhibitor and the HSV-l -based oncolytic virus are administered to the subject for about 4 to about 6 months.
  • Embodiment 15 provides the method of any of Embodiments 12-14, wherein administration of the MEK inhibitor and the HSV-l -based oncolytic virus is at least partially concomitant with administration of the immune checkpoint blockade agent.
  • Embodiment 16 provides the method of any of Embodiments 12-15, wherein administration of the immune checkpoint blockade agent to subject is initiated at about the same time as the initial administration of the MEK inhibitor and the HSV-l -based oncolytic virus to the subject.
  • Embodiment 17 provides the method of any of Embodiments 12-15, wherein administration of the immune checkpoint blockade agent to subject is initiated a given period of time after the initial administration of the MEK inhibitor and the HSV-l -based oncolytic virus to the subject.
  • Embodiment 18 provides the method of any of Embodiments 12-15 and 17, wherein administration of the immune checkpoint blockade agent to subject is initiated about 2 to 6 weeks after the initial administration of the MEK inhibitor and/or the HSV-l-based oncolytic virus to the subject
  • Embodiment 19 provides the method of any of Embodiments 12-18, wherein the immune checkpoint blockade agent targets at least one selected from CTLA-4, PD-L1, or PD-l.
  • Embodiment 20 provides the method of any of Embodiments 12-19, wherein the immune checkpoint blockade agent is at least one selected from ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, cemiplimab, or durvalumab.
  • the immune checkpoint blockade agent is at least one selected from ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, cemiplimab, or durvalumab.
  • Embodiment 21 provides the method of any of Embodiments 12-20, wherein the dose of the HSV-l-based oncolytic virus administered to the subject ranges from about 10 6 pfu/mL to 10 8 pfu/mL.
  • Embodiment 22 provides the method of any of Embodiments 12-21, wherein the volume of each dose of the HSV-l-based oncolytic virus ranges from about 0.1 mL to about 4 mL.
  • Embodiment 23 provides the method of any of Embodiments 12-22, wherein the dose of the MEK inhibitor administered to the subject ranges from about 1-2 mg/day, wherein the MEK inhibitor is trametinib, or a salt or solvate thereof.
  • Embodiment 24 provides the method of any of Embodiments 12-23, wherein the subject is a mammal.
  • Embodiment 25 provides the method of any of Embodiments 12-24, wherein the subject is a human.
  • Embodiment 26 provides a method of determining if a subject afflicted with a cancer and being treated with a combination of a HSV-l -based oncolytic virus and a MEK inhibitor will benefit from administration of an immune checkpoint blockade agent, the method comprising: administering to the subject a therapeutically effective amount of a HSV-l - based oncolytic virus and a therapeutically effective amount of a MEK inhibitor; measuring and/or determining, in a sample of the subject’s cancerous tissue expression, levels of at least marker selected from CD-8, Gzmb, IFNy, PRF1, or TNFa; wherein, if the levels of the at least one marker in the sample of the subject’s cancerous tissue is higher than levels of the at least one marker in a control sample, the subject is counseled to be further administered an immune checkpoint blockade agent.
  • Embodiment 27 provides the method of Embodiment 26, wherein the HSV-l -based oncolytic virus is talimogene laherparepvec.
  • Embodiment 28 provides the method of any of Embodiments 26-27, wherein the subject is further administered the immune checkpoint blockade agent if the levels of the at least one marker in the sample of the subject’s cancerous tissue is higher than levels of the at least one marker in a control sample.
  • Embodiment 29 provides the method of any of Embodiments 26-28, wherein the control sample is a sample of the subject’s cancerous tissue before administration of the HSV-l -based oncolytic virus and MEK inhibitor.
  • Embodiment 30 provides the method of any of Embodiments 26-29, wherein the levels of the at least one marker in the sample of the subject’s cancerous tissue is at least 20% higher than the levels of the at least one marker in the control sample.

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Abstract

L'invention concerne en partie la découverte inattendue selon laquelle un inhibiteur de MEK et du talimogène laherparepvec agissent de manière synergique pour traiter ou prévenir un mélanome chez un sujet en ayant besoin. Dans certains modes de réalisation, un agent de blocage du point de contrôle PD-1/PD-L1 est également administré au sujet.
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