MICROENVIRONMENT

AND RELATED METHODS OF TREATING CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application No. 62/725,834, filed on August 31, 2018, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates generally to methods of treating cancer. More specifically, this disclosure relates to treatment of cancer in a patient by administering a tumor-targeted gene vector encoding a cytocidal inhibitor of human cyclin G1 and at least one cancer immunotherapy agent to evoke a more robust antitumor immunity and a greater antitumor response.

BACKGROUND

[0003] The tragedy of metastatic cancer is immense principally because of its grim prognosis. ¹ Despite numerous approved treatment regimens presently in place, more novel therapies need to be added to the oncologists' arsenal. "Pathotropic (disease-seeking) targeting" has recently shown great promise in the clinic. Since 2003, DeltaRex-G (former names: Mx-dnGl, Rexin G) - a tumor targeted retrovector - has been improving overall survival in intractable cancers such as pancreatic cancer, malignant melanoma, bone and soft tissue sarcoma, breast cancer, renal cell carcinoma, and B-cell lymphoma. ² ' ^3,4,5

[0004] By inhibiting the "human cyclin G1 (CCNG1)" cell cycle control pathway, DeltaRex-G arrests the proliferative cell cycle in G1 phase and, consequently, causes apoptosis of tumor cells. ⁶ The elegance of DeltaRex-G as a promising cancer gene therapy lies in its tumor-targeted "nanoparticles" that selectively seek out and accumulate in the tumor microenvironment (TME) by binding to abnormally exposed collagenous proteins caused by the invading tumor. ^3,4,6

[0005] In addition to killing the cancer cells and associated tumor vasculature, DeltaRex- G enables 1) the entry of immune cells and immune checkpoint inhibitors into the tumor microenvironment by destroying the extracellular matrix-producing stromal fibroblasts and 2) the preservation of a patient's innate tumor surveillance function by sparing the bone marrow and the immune system from collateral damage. Hence, DeltaRex-G can be combined synergistically with immunotherapy, such that identification of strategic combinatorial regimens would advance the clinical utility of DeltaRex-G in the management of metastatic disease.

[0006] Previous studies, scientific reports, and intellectual property development focused primarily on the unprecedented tumor targeting capabilities of the DeltaRex-G gene vector encoding a cytocidal construct of the CCNG1 (Cyclin Gl) gene product. However, among the more unusual clinical responses to this precision tumor targeted cancer gene therapy - observed upon critical analysis of both "good clinical responders" and long-term survivors - is the innovative concept of DeltaRex-G treatment-evoked Immune Response Activation. This is of particular importance in non-immunogenic cancers such as pancreatic cancer and sarcoma, where patient anti-cancer immune responses are often found to be deficient. Moreover, DeltaRex-G treatment-evoked Immune Response Activation may be of considerable scientific and clinical utility as a treatment adjuvant or a primer, for optimizing existing cancer immunotherapy approaches. These would include FDA approved immunotherapies, CAR-T cell therapy, and experimental autologous and heterologous natural killer cell therapies. This invention relates to the molecular characterization and clinical utility of DeltaRex-G when used in combination with known cancer immunotherapy agents.

[0007] The current disclosure is supported by the following lines of reasoning: (i) a critical analysis of the available histological immunostaining and documentation in patients treated with DeltaRex-G provides cellular/mechanistic validation of overt immune response activation; (ii) analysis of the objective "immunological' tumor responses in Stage 4 pancreatic cancer, B-cell lymphoma and metastatic melanoma in light of the latest clinical long-term survival data (> 10 years cancer free); (iii) the notorious lack of appreciable anticancer immunity (and/or associated adoptive immune responses) seen in Stage 4 pancreatic cancer patients, and (iv) the natural conversion of B cell lymphoma, otherwise non-immunogenic, into an immunogenic phenotype and long term survival by treatment with DeltaRex-G followed by DeltaVax (a tumor-targeted gene vector encoding GM-CSF) therapy.

[0008] Importantly, DeltaRex-G treatment-evoked Immune-Response-Activation that, taken together with the direct killing of proliferative tumor cells, as demonstrated previously (i.e., cancer cells, stromal cells, and tumor associated neo-vasculature), is remarkable in both magnitude and breadth of the exceedingly robust cellular immune responses observed and defined within the lesions, suggesting that the previously noted yet incompletely characterized molecular and cellular mechanisms of Immune Response Activation following DeltaRex-G infusions is a valuable discovery of complementary immunological mechanisms that may be of considerable clinical utility in managing metastatic cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The embodiments disclosed herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompany drawings.

[0010] FIG. 1 Immunohistochemical characterization of tumor-infiltrating lymphocytes in tumor a: Tumor nodule with a cytokeratin-17 immunostain; H&E. b: Collagenous material (bluish material); trichome stain c: Cells expressing the leukocyte common antigen (reddish material). Immunohistochemistry for d: CD35+ dendritic cells, e: CD20+ B cells, f: CD4+ helper T cells, and g: CD8+ killer T cells. ECM, extracellular matrix; fib, fibrosis; im, immune cells; tu, tumor.

[0011] FIG. 2 Histological section of excised liver tumor a: This shows preponderance of fibrosis (fib) with moderately differentiated epithelioid tumor cells (tu) arrayed in columnar/ductal structures, seen in various stages of degeneration; H&E stain, pancreatic cancer cells marked by a cytokeratin-17 immunostain (inset) b. Abundant fibrosis is observed throughout the tumor nodule, as shown by Masson's trichrome stain for ECM, c, d, e. Extensive apoptosis of the tumor vasculature and tumor cells, stromal fibroblasts, as well as visible karyorrhexis are evident along the borders of the glandular structures.

[0012] FIG. 3 Further characterization of the immune infiltrate in the nodule in FIG. 2. a: The immune cells are interspersed within the reactive/reparative fibrosis (fib) that surrounds the tumor cells (tu) of the excised nodule with the reddish material being keratin and the bluish material, ECM; Masson's trichrome stain. This characterization reveals that the cadre of recruited immune cells, collectively marked by the b: CD45 common leucocyte antigen, contains both c: CD4+ helper T-cells and d: CD8+ killer T-cells; the latter of which are selectively cytotoxic, adaptive components of cell-mediated tumor immunity.

[0013] FIG. 4 Immunohistochemical characterization of tumor infiltrating lymphocytes (TILs) in metastatic tumor nodules excised from a DeltaRex-G-treated patient with pancreatic cancer. Representative tissue sections of residual tumor nodules within the biopsied liver show significant TIL infiltration with a functional complement of immunoreactive T and B cells. Clockwise from upper left: helper T cells (CD4+), killer T cells (CD8+), B cells (CD20+), monocyte/macrophages (CD45+), dendritic cells (CD35+), and natural killer cells (CD56+). Note, the presence (i.e., migration) of a cadre of TILs that function in the context of cell-mediated and humoral immunity suggest the potential for cancer immunization in an immune-competent host.

[0014] FIG. 5 Histological characterizations and analysis of anti-cancer immune responses observed in pancreatic cancer biopsies following repeated infusions of DeltaRex- G demonstrates a previously unrecognized "Immune sensitization/activation".

[0015] FIG. 6 Intravenous DeltaRex-G induces necrosis, apoptosis and fibrosis in a cancerous lymph node of a patient with metastatic melanoma. (A) H&E-stained tissue sections of inguinal lymph node revealing extensive necrosis (n), apoptosis (indicated by arrows) and fibrosis (f) of cancer cells with a rim of viable tumor cells in the periphery (t). (B) Higher magnification (xlOO) of sections of A showing numerous cells undergoing apoptosis indicated by small cells with pyknotic or fragmented nuclei. (C) Higher magnification (xlOO) of A revealing golden-yellow hemosiderin-laden macrophages. (D) Representative tissue sections of inguinal lymph node showing significant infiltration with immunoreactive CD35+ dendritic cells, (E) CD68+ macrophages and (F) CD8+ killer T cells.

[0016] FIG. 7 Histologic evidence of long lasting anti-tumor immunity in a patient with B-Cell Lymphoma. An indicator cervical lymph node was resected at 8 weeks and one year, respectively, after treatment using DeltaRex-G + DeltaVax, GM-CSF cytokine bearing retroviral vector. (A-B) H&E shows complete effacement of lymph node architecture and replacement with tumor infiltrating lymphocytes (TILs); (C, Boxed from A) CD8+ killer T cells; (A-left inset) Chronic inflammatory cells in lymph node aspirate with no malignant cells one year later. This patient is alive today, 10 years later.

DETAILED DESCRIPTION

[0017] Enhanced immune cell trafficking in excised tumors of DeltaRex-G-treated patients suggest an underlying immunologic mechanism for the noted improvements in survival reported in these patients. In fact, three patients, each with a different type of intractable cancer (e.g., metastatic pancreatic cancer, B-cell lymphoma, and metastatic osteosarcoma), are still alive 9-10 years following courses of CCNG1 inhibitor therapy; to date, their chemo-resistant cancers have not recurred, nor did they require additional cancer treatments. ³

[0018] In the cytological characterizations (Fig. 1-3), anti-tumor immune cells must be differentiated from pro-tumor immune cells. ^4,7 Agents proven to be on the anti-tumor arm of the immune system include dendritic cells, natural killer cells, helper T cells, and killer T cells. The killer T cells are invaluable in controlling tumor growth and metastasis. These lymphocytes - with the aid of antigen presenting cells (e.g., dendritic cells) - are recruited when tumor cells first appear. The majority of the helper T cells work in coordination with the killer T cells. Natural killer cells act as support for killer T cells by attacking the tumor cells that are rendered undetectable to the latter by the lack of major histocompatibility complexes.

[0019] Regulatory T cells, "tumor-associated macrophages (TAMs)", and B cells can abet the tumor cells and protect them against the immune system. Regulatory T cells can impede antigen presentation and killer T cell action. Ml-type TAMs can elicit anti-tumor inflammation, but they can be overpowered by M2-type TAMs that promote tumor pathogenicity; hence, the potential pro-tumor attribute of macrophages. B cells can also help recognize tumor cells for other immune cells to target, but a subtype (i.e., regulatory B cells), can foster tumor growth by producing "neo-angiogenesis" and immune suppressive cytokines. ^1,9

[0020] The ideal of prompting, and even enhancing, the body's own immune surveillance system in the clinical management of cancer is in complete accord with the stated goals of precision medicine. The cytocidal mechanisms of action of tumor-targeted DeltaRex-G, which appears to recruit the immune system into the TME, runs parallel to the principle of immunotherapy. Immunotherapy has achieved some success in the past years with checkpoint inhibitors/blockers: including the anti-Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4; ipilimumab) for metastatic melanoma; the anti-Programmed Cell Death protein 1 (PD-1; nivolumab, pembrolizumab, atezolizumab) for non-small cell lung cancer; and so-called adoptive T cell therapies, using customized genetically engineered antigen- specific T cell clones. Other types of investigational immunotherapies include cancer vaccines, oncolytic viruses, and monoclonal antibodies. ¹⁰ Together, the emerging diversity of immunomodulatory treatment options, each with very distinct mechanisms of action, represents a promising approach in terms of new combinatorial therapies.

[0021] To optimize cancer immunotherapy, the innate and adaptive components of the immune system must work together. ^10,11 Delta-Rex G can conceivably synergize with different types of immunotherapy to enhance the beneficial effects and reduce immune- related toxicities. For example, anti-PD-1 inhibitors work well with cancers that already have a pre-existing CD8+ T cell infiltrates; the anti-PD-1 inhibitors can be combined with treatment that can increase anti-tumor T cells. Targeted drug therapy with immunomodulators like lenalidomide can be combined along with natural killer T cell therapy to enhance the latter's ability to kill the tumor cells.

[0022] Targeted gene therapies can also be used to modulate the pro-tumor effects of specific immune cells, such as the regulatory B cells and macrophages (TAMs). DeltaVax, a tumor-targeted retrovector encoding the human granulocyte / macrophage colony- stimulating factor (GM-CSF) gene, was reported to extend the overall survival of cancer patients treated with DeltaRex-G followed by DeltaVax in an attempt to effectuate a longer lasting anti-tumor immunity. ¹²

[0023] The present disclosure provides methods of treating cancer. Such methods may include repeated infusions of a tumor targeted gene vector such as DeltaRex-G, resulting in enhanced immune cell trafficking in the tumor microenvironment, followed by one or more infusions of a cancer immunotherapy agent bearing a cytokine GM-CSF for recruitment of killer T cells, helper T cells, dendritic cells and natural killer cells for in situ vaccination. [0024] Without being bound by any particular theory, such methods may enhance or restore a cancer patient's innate tumor surveillance function by priming the tumor microenvironment (TME) for entry of the patient's own killer T cells that have been activated by (1) blockade of tumor "escape" checkpoints such as by inhibitors of programmed cell death protein 1 (PD-1), (2) depletion of growth promoting M2 macrophages in the TME, (3) polarization of macrophages to Ml killer macrophages, and (4) ex vivo manipulation, expansion and treatment with autologous natural killer cells. In certain embodiments, such methods of treating a cancer in a patient comprise administering to the patient a plurality of infusions of a pharmaceutical composition, wherein the pharmaceutical composition comprises a tumor-targeted gene vector encoding a cytocidal inhibitor of human cyclin G1 (e.g., DeltaRex-G), and administering to the patient at least one cancer immunotherapy, to evoke a more robust anti-tumor immunity and a greater antitumor response. The phrase "a plurality of" means "at least two."

[0025] The FDA approvals of a number of immune checkpoint inhibitors (PD-1, PD-L1) and CAR-T cell therapies represent a laudable advancement in the field of cancer therapy/gene therapy. Immunotherapy represents a conceptual shift in cancer treatment wherein activation of the innate immune response is the primary goal. Cytokines, cancer vaccines, natural killer (NK) cell therapy, and adoptive cell transfers, are other promising strategies for eradicating cancer. In certain embodiments, the cancer immunotherapy comprises a monoclonal antibody, an immune checkpoint inhibitor, a cytokine, a cancer vaccine, an oncolytic viruses, or a genetically engineered antigen-specific T cell.

[0026] We consistently observed that DeltaRex-G induces apoptosis in tumor cells, proliferative vasculature, and ECM producing stromal fibroblasts. Moreover, the direct cytocidal activity observed within the DeltaRex-G treated tumors was generally associated with a robust immune response that involves both cell-mediated and humoral immunity juxtaposed with overt apoptosis and necrosis of tumor cells. ^2,4,8 This underscores the advantageous pleotropic effects of DeltaRex-G: killing the tumor cells, and conceivably, promoting cancer immunization in situ.

[0027] In some embodiments, the cancer immunotherapy is an immune checkpoint inhibitor. The immune checkpoint inhibitor may be a Cytotoxic T-Lymphocyte-Associated

/ protein 4 inhibitor (e.g., ipilimumab), a Programmed Cell Death protein 1 inhibitor (e.g., nivolumab or pembrolizumab), a Programmed Cell Death-Ligandl inhibitor (e.g., atezolizumab), or a genetically engineered antigen-specific T cell clone.

[0028] In some embodiments, the cancer immunotherapy comprises an adoptive T cell therapy using customized genetically engineered antigen-specific T cell clones. In some embodiments, the cancer immunotherapy is a cancer vaccine. For example, a cancer vaccine that targets the brachyury protein may be used for a chordoma. As another example, a cancer vaccine that targets NYESO-1 may be used for a tumor that expresses NYESO-1.

[0029] In some embodiments, the immunotherapy strategy comprises one or more infusions of autologous or heterologous "off the shelf" natural killer (NK) cells with or without cytokines. In some embodiments, the cancer immunotherapy comprises a monoclonal antibody. In some embodiments, the comprises chimeric antigen receptor (CAR) T cells (CAR-T cell therapy).

[0030] In some embodiments, the immunotherapy comprises administration of one or more cytokines. In certain embodiments, the patient is administered one or more cytokines. In certain other embodiments, the patient is administered a vector comprising a cytokine gene. In some of these embodiments, the cancer immunotherapy comprises an oncolytic virus expressing a cytokine. In some embodiments, the cancer immunotherapy comprises a second tumor targeted gene vector encoding a cytokine gene. In certain embodiments, the cytokine polarizes macrophages into Ml killer macrophages (e.g. GM- CSF).

[0031] In some embodiments, the cancer immunotherapy comprises a chemotherapeutic agent that depletes the growth-promoting macrophages in the tumor microenvironment (e.g. trabectedin).

[0032] In some embodiments, a non-immunogenic cancer is converted into an immunogenic cancer for enhanced efficacy and long-lasting antitumor immunity. These cancers may, e.g., include pancreatic cancer, sarcoma, B-cell lymphoma, and breast cancer. [0033] In some embodiments, an immunogenic cancer becomes a more immunogenic cancer for enhanced efficacy and long-lasting antitumor immunity. These cancers may, e.g., include melanoma, renal cell carcinoma, urothelial carcinoma, and small cell lung cancer.

[0034] It will be readily understood that the embodiments, as generally described herein, are exemplary. This detailed description of various embodiments is not intended to limit the scope of the present disclosure but is merely representative of various embodiments. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified. EXAMPLES

[0035] The following examples are illustrative of disclosed methods and compositions.

In light of this disclosure, those of skill in the art will recognize that variations of these examples would be possible without undue experimentation.

Example 1 - DeltaRex-G induces immune cell trafficking in the tumor microenvironment

[0036] The inventors reviewed the published literature on immune cell trafficking in the tumor microenvironment (TME) of excised tumors following DeltaRex-G therapy in order to better understand the mechanisms of DeltaRex-G therapy, particularly its ability to increase immune cell entry, and to determine possible synergism with known cancer immunotherapies.

[0037] In addition to killing the cancer cells and associated tumor vasculature, DeltaRex- G would enable 1) the entry of immune cells and immune checkpoint inhibitors into the tumor microenvironment by destroying the extracellular matrix-producing stromal fibroblasts and 2) the preservation of a patient's innate tumor surveillance function by sparing the bone marrow and the immune system from collateral damage. Hence, DeltaRex- G can be combined synergistically with immunotherapy, such that identification of strategic combinatorial regimens would advance the clinical utility of DeltaRex-G in the management of metastatic disease.

[0038] The inventors reviewed the published literature that has discussed DeltaRex-G as a novel precision targeted gene therapy for cancer. The types of human cancers included metastatic pancreatic cancer, pancreatic B-cell lymphoma, malignant melanoma, breast adenocarcinoma, non-small cell lung carcinoma, and osteosarcoma.

[00B9] The immunohistochemical staining characteristics of excised tumors from DeltaRex-G-treated patients were extracted from the published sources and

summarized. ² ' ³ ' ⁴ ' ⁵ ' ⁶ ' ⁷ ' ⁸

[0040] Table 1 enumerates the tumor-infiltrating lymphocytes seen in excised tumors of DeltaRex-G-treated cancer patients. Visual examples from 2 patients with metastatic pancreatic cancer show a preponderance of immune cells interspersed with tumor destruction (Figs. 1-3). ^2,7

Ta ble 1 I m m u ne ce l l trafficking i n th e TM E of De lta Rex-G-treated tu mors.

Example 2 - Validation of DeltaRex-G (Tumoricidal Therapy) + DeltaVax (Immunotherapy)

[0041] A patient with B-lymphoma involving lymph nodes and pancreas received therapeutic doses of DeltaRex-G on Days 1,3, and 5, plus DeltaVax on Day 3, and valacyclovir from Days 6-19, comprising one cycle. This cycle was repeated up to 6 cycles for six months. An indicator cervical lymph node was resected at 8 weeks and one year, respectively, after treatment with the clinical vectors. Histologic evidence of long lasting anti-tumor immunity was shown in the resected lymph node as indicated in Figure 7. (A-B) H&E shows complete effacement of lymph node architecture and replacement with tumor infiltrating

lymphocytes (TILs); (C, Boxed from A) CD8+ killer T cells; (A-left inset) Chronic inflammatory cells in lymph node aspirate with no malignant cells one year later. This patient is alive today, 10 years later, with no evidence of cancer.

References

[0042] All references cited in this disclosure are incorporated by reference in their entirety.

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