Methods and compositions comprising tumor suppressor gene therapy and CD122 / CD132 agonists for cancer treatment.

Abstract

Provided herein are methods and compositions for treating cancer in an individual, comprising administering to the individual effective amounts of at least one CD122 / CD132 agonist, at least one immune checkpoint inhibitor, and a viral composition comprising one or more viruses modified to overexpress a tumor suppressor gene and / or an adenoviral death protein. Also provided herein are methods and compositions for treating cancer in an individual, comprising administering to the individual effective amounts of at least one oncolytic viral composition, at least one CD122 / CD132 agonist, and at least one immune checkpoint inhibitor. Also provided herein are methods for improving anti-tumor efficacy by administering the above-mentioned agents in combination with other cancer therapies. These treatments unexpectedly resulted in complete tumor remission and curative outcomes in highly aggressive forms of cancer that are generally known to be resistant to immunotherapy.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 645,022, filed Mar. 19, 2018, and U.S. Provisional Patent Application No. 62 / 803,887, filed Feb. 11, 2019, both of which are hereby incorporated by reference in their entirety.

[0002] Background Art 1. Field The present invention generally relates to the fields of biology and medicine. More specifically, the present invention relates to methods and compositions for restoring or amplifying tumor suppressor function in combination with a selective CD122 / CD132 agonist.

Background Art

[0003] 2. Description of Related Art Malignant cells are often resistant to DNA-damaging agents such as chemotherapy and irradiation-induced programmed cell death, i.e., apoptosis. Such resistance is generally the result of the aberrant expression of specific oncogenes or the loss of expression of tumor suppressor genes in the control of apoptosis. Methods for replacing defective tumor suppressor genes and expressing apoptosis-inducing genes hold promise for the restoration of this form of cell death in tumor cells.

[0004] Perhaps the most studied tumor suppressor gene is p53, which plays an important role in several processes including cell cycle regulation and the control of apoptosis (Hartwell et al., 1994). p53 mutations are frequent in tumor cells and are associated with cancer progression and the development of resistance to both chemotherapy and radiotherapy (Spitz et al., 1996). Restoration of wild-type (wt) p53 function has been shown in both in vitro and in vivo preclinical studies to be able to induce apoptosis in cancer cells. Intratumoral injection of retroviral or adenoviral wt-p53 constructs into animal models has resulted in tumor regression for various different tumor histologies including non-small cell lung cancer (NSCLC), leukemia, glioblastoma, and breast, liver, ovarian, colon, and kidney cancers (Fujiwara et al., 1994). Based on strong preclinical and clinical data, an international randomized phase II / III trial of a p53 gene therapy for first-line treatment of patients with ovarian cancer was initiated (Buller et al., 2002). However, this study was closed after the first interim analysis due to a lack of sufficient treatment effect (Zeimet and Marth, 2003).

[0005] Therefore, despite significant progress using tumor suppressor gene therapy, several barriers including non-specific expression, low efficiency of delivery, and biological safety still limit success in the clinic. Furthermore, there are multiple genetic changes in cancer and epigenetic dysregulation that result in abnormal silencing of genes. Therefore, single gene therapy cannot be a suitable method for cancer treatment. Thus, there is a need for methods to target multiple tumor suppressors in combination with other anti-cancer agents for improved anti-tumor activity and efficient delivery of gene therapy.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] In one embodiment, the present disclosure provides a method and composition for the treatment of cancer in a subject, comprising administering to the subject an effective amount of (1) a nucleic acid encoding p53 and / or a nucleic acid encoding MDA-7, and (2) at least one CD122 agonist and a CD132 agonist (e.g., a selective CD122 / CD132 agonist).

[0008] In some embodiments, a subject is administered a nucleic acid encoding p53. In certain embodiments, a subject is administered a nucleic acid encoding MDA7. In some embodiments, a subject is administered both a nucleic acid encoding p53 and a nucleic acid encoding MDA7.

[0009] In certain embodiments, the nucleic acid encoding p53 and / or MDA-7, and / or a CD122 / CD132 agonist are delivered in an amount effective to restore or amplify tumor suppressor function. In certain embodiments, the nucleic acid encoding p53 and / or MDA-7 are delivered to one or more tumor sites. In certain embodiments, two or more CD122 / CD132 agonists are administered. In certain embodiments, the subject is human.

[0010] In certain embodiments, CD122 / CD132 agonists selectively bind to the CD122 / CD132 receptor complex and have lower affinity binding to CD25 or IL15α receptors compared to affinity binding to the CD122 / CD132 receptor complex. In certain embodiments, one or more CD122 / CD132 agonists are IL-2 / anti-IL-2 immune complexes, IL-15 / anti-IL-15 immune complexes, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complexes, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein. CD122 / CD132 agonists can be IL-15 variants (e.g., IL-15N72D) bound to an IL-15 receptor α / IgG1 Fc fusion protein, such as ALT-803. In certain embodiments, IL-15 is pre-complexed with IL-15Rα and preferentially binds to CD122 / CD132. In certain embodiments, the IL-2 receptor agonist is not F42K. In some embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are delivered by a viral method and / or a nonviral method. In certain embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are delivered within an expression cassette such as a viral vector. In some embodiments, p53 and MDA-7 are under the control of a single promoter such as cytomegalovirus (CMV), SV40, or PGK. In certain embodiments, the viral vector is an adenovirus vector (e.g., an adenovirus vector overexpressing ADP), a retroviral vector, a vaccinia virus vector (e.g., a NIL-deficient vaccinia virus vector), an adeno-associated virus vector, a herpesvirus vector, a vesicular stomatitis virus vector, or a polyomavirus vector.

[0011] In some embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are delivered by gene editing methods such as zinc finger nucleases (ZFNs), transcription-activating effector nucleases (TALENs), or clustered, regularly spaced short palindromic repeats (CRISPR), for purposes such as restoring or amplifying the expression of tumor suppressor genes. Viral and non-gene-transfer delivery and expression methods, and / or combinations of gene editing methods are considered in this disclosure. In certain embodiments, the adenovirus p53 (Ad-p53) injection dose (mL) is 1 cm³. 3 At least 1 × 10⁻¹⁴ per tumor volume 11 Each tumor injury is caused by receiving an Ad-p53 dose of the viral particles (vp). In some embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are administered to the subject in a lipoplex. In some embodiments, the lipoplex comprises DOTAP and at least one of cholesterol, cholesterol derivatives and cholesterol mixtures.

[0012] In some embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 is administered to the target intravenously, intra-arterially, intravascularly, intrapleurally, intraperitoneally, intratracheally, intratumorally, intrathecally, intramuscularly, endoscopically, intrafocally, percutaneously, subcutaneously, locally, stereotactically, or by direct injection or perfusion. In certain embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 is administered to the target intratumor. In some embodiments, administration includes local or local injection. In some embodiments, administration is via serial infusion, intratumor injection, or intravenous injection.

[0013] In some embodiments, the subject is administered two or more doses of nucleic acid encoding p53 and / or nucleic acid encoding MDA-7. In certain embodiments, the subject is administered two or more doses of at least one CD122 / CD132 agonist. In some embodiments, the subject is administered p53-coding nucleic acid and / or MDA-7-coding nucleic acid before, simultaneously with, or after at least one CD122 agonist and CD132 agonist.

[0014] In some aspects, cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, hepatocellular carcinoma, retinoblastoma, astrocytoma, gliablastoma, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, genitourinary cancer, airway cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, carcinoma, sarcoma, central nervous system cancer, peripheral nervous system cancer, lymphoma, brain cancer, colon cancer, or bladder cancer. In certain aspects, cancer is metastatic.

[0015] In some embodiments, the method further comprises administering at least one additional anticancer therapy. In certain embodiments, the at least one additional anticancer therapy is surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, radioresection, or biological therapy. In some embodiments, the biological therapy is monoclonal antibody, siRNA, miRNA, antisense oligonucleotide, ribozyme, gene editing, cell therapy, or gene therapy.

[0016] In some embodiments, at least one additional anticancer treatment is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some embodiments, at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is tremelimumab or ipilimumab. In certain embodiments, at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. In some embodiments, the anti-KIR antibody is lirirumab. In some embodiments, the PD-L1 inhibitor is durvalumab, atezolizumab, or avelumab. In some embodiments, the PD-L2 inhibitor is rHIgM12B7. In some embodiments, the LAG3 inhibitor is IMP321 or BMS-986016. In some embodiments, the A2aR inhibitor is PBF-509. In some embodiments, at least one immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) axis-binding antagonist. In certain embodiments, the PD-1 axis-binding antagonist is selected from the group consisting of PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. In some embodiments, the PD-1 axis-binding antagonist is a PD-1 binding antagonist. In certain embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PDL1 and / or PDL2. In particular, the PD-1 binding antagonist is a monoclonal antibody or its antigen-binding fragment. In some embodiments, the PD-1 binding antagonist is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224.

[0017] In some embodiments, at least one additional therapeutic agent is a histone deacetylase (HDAC) inhibitor. In certain embodiments, the HDAC inhibitor is tractinostat (CHR-3996 or VRx-3996). In certain embodiments, the method further comprises providing an extracellular matrix-degrading protein such as relaxin, hyaluronidase, or decorin.

[0018] In some embodiments, at least one additional anticancer treatment is a tumor lysis virus. In some embodiments, the tumor lysis virus is modified to express p53, MDA-7, IL-12, a TGF-β inhibitor, and / or an IL-10 inhibitor. In certain embodiments, the tumor lysis virus is a single-stranded or double-stranded DNA virus, RNA virus, adenovirus, adeno-associated virus, retrovirus, lentivirus, herpesvirus, poxvirus, vaccinia virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, Epstein-Barr virus, influenza virus, reovirus, myxoma virus, marabavirus, rhabdovirus, enadenochusileb, or coxsackievirus. In some embodiments, the tumor lysis virus is modified to express granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-12. In some embodiments, the tumor lysis virus is further defined as talimogene-laharpalebeck (T-VEC). In some embodiments, tumor-disintegrating adenovirus vectors are derived from adenoviruses that are driven by E1b-deficient adenoviruses, as well as adenoviruses that have a modified E1a regulatory sequence, in which the Ad E1a gene is fully modified to express a therapeutic gene, such as α-fetoprotein (AFP) promoter tumor-disintegrating adenoviruses, modified TERT promoter tumor-disintegrating adenoviruses, HRE-E2F-TERT hybrid promoter tumor-disintegrating adenoviruses, and / or adenoviruses that have a modified E1a regulatory sequence in which at least one Pea3 binding site or its functional protein is deficient by an Elb-19K clone insertion site.

[0019] In certain embodiments, at least one additional anticancer treatment is a protein kinase or growth factor signaling pathway inhibitor. In certain embodiments, the protein kinase or growth factor signaling pathway inhibitor is afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, salakatinib, These include sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, vemurafenib, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridafololimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, or GW572016. In some embodiments, the protein kinase inhibitor is a PI3K inhibitor such as a PI3Kδ inhibitor.

[0020] In some embodiments, immunotherapy comprises cytokines such as GM-CSF, interleukins (e.g., IL-2), and / or interferons (e.g., IFNα) or heat shock proteins. In certain embodiments, immunotherapy comprises a costimulatory receptor agonist, a stimulator of innate immune cells, or an activator of innate immunity. In certain embodiments, the costimulatory receptor agonist is an anti-OX40 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-CD40 antibody, or an anti-CD27 antibody. In some embodiments, the stimulator of immune cells is an inhibitor of a cytotoxic inhibitory receptor or an agonist of an immunostimulatory Toll-like receptor (TLR). In some embodiments, the cytotoxic inhibitory receptor is an inhibitor of NKG2A / CD94 or CD96 TACTILE. In some embodiments, the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. In some embodiments, the immunotherapy comprises a combination of a PD-L1 inhibitor, a 4-1BB agonist, and an OX40 agonist. In certain embodiments, the immunotherapy comprises an interferon gene-stimulating factor (STING) agonist. In some embodiments, the activator of innate immunity is an IDO inhibitor, a TGFβ inhibitor, or an IL-10 inhibitor. In some embodiments, when these immunotherapies are proteins, the proteins may be administered as polypeptides or their corresponding nucleic acids, which are administered by replicable and / or non-replicable viral and / or non-viral gene therapies. In some embodiments, the chemotherapy comprises DNA damaging agents such as gamma irradiation, X-rays, ultraviolet irradiation, microwaves, electron emission, adriamycin, 5-fluorouracil (5FU), capecitabine, etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), or hydrogen peroxide.

[0021] In another embodiment, a method for treating cancer in a subject is provided, comprising administering an effective amount of at least one tumor lysis virus and at least one CD122 / CD132 agonist to the subject, and at least one immune checkpoint inhibitor. In some embodiments, the at least one tumor lysis virus is an adenovirus modified to overexpress adenovirus death protein (ADP), such as VirRx007. In some embodiments, the at least one tumor lysis virus is genetically modified to express p53, MDA-7, cytokines, and / or immunostimulatory genes. In certain embodiments, the cytokines are GM-CSF or IL-12. In some embodiments, the immunostimulatory genes are inhibitors of TGFβ or IL-10.

[0022] In some embodiments, at least one tumor lysis virus is selected from the group consisting of single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia viruses, vesicular stomatitis viruses, polioviruses, Newcastle disease viruses, Epstein-Barr viruses, influenza viruses, reoviruses, myxoma viruses, maraba viruses, rhabdoviruses, enadenochusileb, and coxsackieviruses.

[0023] In some embodiments, the viruses used in the above embodiments include replicable and / or non-replicable viruses. In certain embodiments, replicable or non-replicable viruses include single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia viruses, varicella stomatitis viruses, polioviruses, Newcastle disease viruses, myxoma viruses, Epstein-Barr viruses, influenza viruses, reoviruses, maraba viruses, rhabdoviruses, enadenochusileb, or coxsackieviruses. In certain embodiments, one or more viruses are used. In certain embodiments, the virus composition includes a combination of replicable and non-replicable viruses.

[0024] In a further embodiment, the replicable viruses in the above embodiments may be one or more tumor lysis viruses. These tumor lysis viruses can be recombinant to express p53 and / or IL24, and / or other genes other than p53 and / or IL24, such as cytokines (e.g., IL12) and / or other immunostimulatory genes (e.g., TGF-β inhibitors, or IL10 inhibitors, or heat shock proteins). In certain embodiments, tumor lysis viruses can be used in place of or in addition to p53 and / or IL24 tumor suppressor therapies. Examples of tumor lysis viruses include single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia viruses, vesicular stomatitis viruses, polioviruses, Newcastle disease viruses, Epstein-Barr viruses, influenza viruses and reoviruses, myxoma viruses, maraba viruses, rhabdoviruses, enadenochusileb, or coxsackieviruses. Examples of tumor lysis viruses include, but are not limited to, Ad5-yCD / mutTKSR39rep-hIL12, Cavatak®, CG0070, DNX-2401, G207, HF10, IMLYGIC®, JX-594, MG1-MA3, MV-NIS, OBP-301, Reolysin®, Toca 511, Oncorine(H101), Onyx-015, H102, H103, RIGVIR, adenoviruses overexpressing adenovirus death protein (ADP), such as VirRx007, N1L-deficient vaccinia virus, or N1L-deficient vaccinia virus expressing IL12.

[0025] In some embodiments, viral and nonviral nucleic acids and gene editing compositions induce local and / or systemic effects. In some embodiments, these compositions induce local and systemic effects.

[0026] In certain embodiments, the treated subject is a mammal or a human. In certain embodiments, the treatment is provided to prevent or treat a pre-malignant or malignant hyperproliferative state. In certain embodiments of prevention, the subject is a healthy subject. In other embodiments of prevention, the subject includes pre-malignant injuries such as leukoplakia or dysplasia. In other embodiments of prevention, the subject is at risk of cancer progression, for example, by being a smoker or having a family history of cancer. In certain embodiments, the treatment is for an early or recurrent hyperproliferative state. In some embodiments, the treatment is administered to enhance or reverse resistance to another treatment. In certain embodiments, resistance to treatment is known by medical history for a specific population of patients with hyperproliferative states. In certain embodiments, resistance to treatment is observed in individual patients with hyperproliferative states.

[0027] In certain embodiments of the above embodiments, the method further comprises providing an extracellular matrix-degrading protein. In some embodiments, the method comprises administering an expression cassette encoding an extracellular matrix-degrading protein. In some embodiments, the extracellular matrix-degrading protein is relaxin, hyaluronidase, or decorin. In certain embodiments, the extracellular matrix-degrading protein is relaxin. In some embodiments, the expression cassette is contained within a viral vector. In certain embodiments, the viral vector is an adenovirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus vector, a herpesvirus vector, a vesicular stomatitis virus vector, or a polyomavirus vector, or another type of viral or non-gene therapy vector.

[0028] In some embodiments, the expression cassette encoding extracellular matrix-degrading proteins is administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, stereotactically, or by direct injection or perfusion. In certain embodiments, the subject is administered at least one CD122 / CD132 agonist followed by a nucleic acid encoding p53 and / or MDA-7. In certain embodiments, the subject is administered at least one CD122 / CD132 agonist followed by a nucleic acid encoding p53 and / or MDA-7. In certain embodiments, the subject is administered at least one CD122 / CD132 agonist simultaneously with a nucleic acid encoding p53 and / or MDA-7. In certain embodiments, the adenovirus vector is administered to the subject intratumorally. In some embodiments, nucleic acids encoding p53 and / or MDA-7, along with at least one CD122 / CD132 agonist, induce an abscopal (systemic) effect in distant tumors where the nucleic acids encoding p53 and / or MDA-7 have not been injected.

[0029] In certain aspects, cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, hepatocellular carcinoma, retinoblastoma, astroglioma, gliablastoma, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, genitourinary cancer, airway cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, carcinoma, sarcoma, central nervous system cancer, peripheral nervous system cancer, lymphoma, brain cancer, colon cancer, or bladder cancer. In some aspects, cancer is metastatic.

[0030] In some embodiments, the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are located within an expression cassette. In certain embodiments, the expression cassette is located within a viral vector. In some embodiments, the viral vector is an adenovirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus vector, a herpesvirus vector, a vesicular stomatitis virus vector, or a polyomavirus vector. In certain embodiments, the viral vector is an adenovirus vector.

[0031] In a specific manner, the viral vector is approximately 10 3 ~about 10 13 The adenovirus vector is administered as individual virus particles. In some embodiments, the adenovirus vector is administered to the subject intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intratumorally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, stereotactically, or by direct injection or perfusion. In certain embodiments, the subject is administered the adenovirus vector two or more times.

[0032] In some embodiments, the subject is administered a nucleic acid encoding p53. In another embodiment, the subject is administered a nucleic acid encoding MDA-7. In a specific embodiment, the subject is administered both a nucleic acid encoding p53 and a nucleic acid encoding MDA-7. In some embodiments, p53 and MDA-7 are under the control of a single promoter. In some embodiments, the promoter is cytomegalovirus (CMV), SV40, or PGK.

[0033] In some embodiments, nucleic acids are administered to a subject encapsulated in a lipoplex. In certain embodiments, the lipoplex comprises DOTAP and at least one of cholesterol, cholesterol derivatives, and cholesterol mixtures. In some embodiments, nucleic acids are administered encapsulated in nanoparticles.

[0034] In certain embodiments, administration includes local or local injection. In other embodiments, administration is via serial infusion, intratumoral injection, or intravenous injection.

[0035] In some embodiments, the method further comprises administering at least one additional anticancer therapy. In certain embodiments, the at least one additional anticancer therapy is surgical therapy, chemotherapy (e.g., administration of protein kinase inhibitors or EGFR-targeted therapy), embolization therapy, chemoembolization therapy, radiotherapy, cryotherapy, hyperthermia therapy, phototherapy, radioresection therapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, or biological therapy (e.g., monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes, or gene therapy). In some embodiments, immunotherapy includes cytokines. In certain embodiments, cytokines are interleukins such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, and / or interferons such as IFN-α. Additional approaches to accelerate the immune response that targets tumors include additional immune checkpoint inhibition. In some embodiments, immune checkpoint inhibitors include anti-CTLA4, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-TIM-3, anti-LAG-3, anti-A2aR, or anti-KIR antibodies. In some embodiments, immunotherapy includes costimulatory receptor agonists such as anti-OX40 antibody, anti-GITR antibody, anti-CD137 antibody, anti-CD40 antibody, and anti-CD27 antibody. In certain embodiments, immunotherapy includes suppression of T regulatory cells (Treg), myeloid-derived immunosuppressive cells (MDSCs), and cancer-associated fibroblasts (CAFs).

[0036] In a further embodiment, immunotherapy involves stimulating innate immune cells such as natural killer (NK) cells, macrophages, and dendritic cells. Further immunostimulatory therapies include IDO inhibitors, TGF-β inhibitors, IL-10 inhibitors, interferon gene-stimulating factor (STING) agonists, Toll-like receptor (TLR) agonists (e.g., TLR7, TLR8, or TLR9), tumor vaccines (e.g., whole tumor cell vaccines, peptides, and recombinant tumor-associated antigen vaccines), and adoptive cell therapies (ACTs) (e.g., T cells, natural killer cells, TILs, and LAK cells), as well as ACTs with recombinant receptors (e.g., chimeric antigen receptors (CARs) and T cell receptors (TCRs)). In a particular embodiment, combinations of these activators can be used, such as immune checkpoint inhibitors, checkpoint inhibition + T cell costimulatory receptor agonism, and checkpoint inhibition + TIL ACT combinations. In certain embodiments, additional anticancer therapies include combinations of immune checkpoint inhibitors (e.g., avelumab), 4-1BB (CD-137) agonists (e.g., utomirumab), and OX40 (TNFRS4) agonists. In some embodiments, chemotherapy includes a DNA damaging agent. In some embodiments, the DNA damaging agent is gamma irradiation, X-rays, ultraviolet irradiation, microwaves, electron emission, adriamycin, 5-fluorouracil (5FU), capecitabine, etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), or hydrogen peroxide. In certain embodiments, the DNA damaging agent is 5FU or capecitabine.

[0037] In some embodiments, chemotherapy includes cisplatin (CDDP), carboplatin, procarbazine, mechloretamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxonbicin, bleomycin, precomycin, mitomycin, etoposide (VP16), tamoxifen, taxotere, taxol, trans-platinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, HDAC inhibitors, or any analogues or derivatives thereof.

[0038] In some embodiments, at least one additional cancer treatment is a protein kinase inhibitor or monoclonal antibody that inhibits a protein kinase or a receptor involved in the growth factor signaling pathway. For example, the protein kinase or receptor inhibitor may be an EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor, or BRAF inhibitor. In certain embodiments, the protein kinase inhibitor is a PI3K inhibitor. In some embodiments, the PI3K inhibitor is a PI3Kδ inhibitor. For example, protein kinase or receptor inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, and tra. The protein kinase inhibitor may be sutuzumab, vandetanib, AP23451, vemurafenib, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridafololimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or a mixture thereof. In certain embodiments, the protein kinase inhibitor is an AKT inhibitor (e.g., MK-2206, GSK690693, A-443654, VQD-002, miltefosine, or perifosine). In certain embodiments, EGFR-targeted therapies for use according to the embodiments include, but are not limited to, inhibitors of EGFR / ErbB1 / HER, ErbB2 / Neu / HER2, ErbB3 / HER3, and / or ErbB4 / HER4. A wide range of such inhibitors are known, including, but are not limited to, tyrosine kinase inhibitors that are active against receptors and EGFR-binding antibodies or aptamers.For example, EGFR inhibitors can be gefitinib, erlotinib, cetuximab, matuzumab, panitumumab, AEE788;CI-1033, HKI-272, HKI-357, or EKB-569. Protein kinase inhibitors can be BRAF inhibitors such as dabrafenib, or MEK inhibitors such as trametinib.

[0039] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while illustrating preferred embodiments of the present invention, are given only as examples, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. In certain embodiments, for example, the following are provided: (Item 1) A method for treating cancer in a subject, comprising administering to the subject an effective amount of (1) a nucleic acid encoding p53 and / or a nucleic acid encoding MDA-7, and (2) at least one selective CD122 / CD132 agonist. (Item 2) The method according to item 1, wherein the subject is administered nucleic acid encoding p53. (Item 3) The method according to item 1, wherein the subject is administered nucleic acid encoding MDA7. (Item 4) The method according to item 1, wherein the subject is administered nucleic acid encoding p53 and nucleic acid encoding MDA7. (Item 5) The method according to item 7, wherein the at least one CD122 / CD132 agonist is selected from the group consisting of IL-2 / anti-IL-2 immune complexes, IL-15 / anti-IL-15 immune complexes, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complexes, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, IL-15 mutein, and / or IL-15 variants that bind to the α / IgG1 Fc fusion protein of the IL-15 receptor. (Item 6) The method according to item 5, wherein the IL-15 is pre-complexed with IL-15Rα and selectively binds to CD122 / CD132. (Item 7) The method according to item 7, wherein one, two, three, or four types of CD122 / CD132 agonists are administered to the subject. (Item 8) The method according to item 1, wherein at least one of the CD122 agonists and / or CD132 agonists is not F42K. (Item 9) The method according to item 1, wherein the cancer is metastatic. (Item 10) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are contained within an expression cassette. (Item 11) The method described in item 10, wherein p53 and MDA-7 are under the control of a single promoter. (Item 12) The method according to item 11, wherein the promoter is cytomegalovirus (CMV), SV40, or PGK. (Item 13) The method described in item 10, wherein the expression cassette is located within the viral vector. (Item 14) The method according to item 13, wherein the viral vector is an adenovirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus vector, a herpesvirus vector, a vesicular stomatitis virus vector, or a polyomavirus vector. (Item 15) The method according to item 13, wherein the viral vector is an adenovirus vector or a vaccinia virus vector. (Item 16) The method according to item 15, wherein the vaccinia virus vector is further defined as an NIL-deficient vaccinia virus vector. (Item 17) The method according to item 15, wherein the adenovirus vector is further defined as an adenovirus vector in which ADP expression is increased. (Item 18) The aforementioned viral vector is approximately 10 3 ~about 10 13 The method described in item 13, administered with viral particles. (Item 19) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 is administered by a nonviral approach. (Item 20) The method according to item 1, wherein the method includes restoring and / or amplifying p53 and / or MDA-7 function by gene editing. (Item 21) The method according to item 20, wherein gene editing involves expressing p53 and / or MDA-7 using a zinc finger nuclease (ZFN), a transcription-activating effector nuclease (TALEN), or clustered, regularly spaced short palindromic repeats (CRISPR). (Item 22) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are administered via a viral vector and gene editing. (Item 23) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 is administered to the subject intravenously, intraarterially, intravascularly, intrapleurally, intraperitoneally, intratracheally, intratumorally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, locally, stereotactically, or by direct injection or perfusion. (Item 24) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 is administered intratumorally to the subject. (Item 25) The method according to item 1, wherein the subject is administered the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 two or more times. (Item 26) The method according to item 1, wherein the subject is administered at least one type of CD122 / CD132 agonist at least twice. (Item 27) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 is administered to the subject before, simultaneously with, or after the at least one CD122 / CD132 agonist. (Item 28) The method according to item 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are administered to the subject in a lipoplex. (Item 29) The method according to item 28, wherein the lipoplex comprises DOTAP and at least one of cholesterol, a cholesterol derivative, or a cholesterol mixture. (Item 30) The method according to item 1, wherein the administration includes local or local injection. (Item 31) The method according to item 1, wherein administration is via serial infusion, intratumor injection, or intravenous injection. (Item 32) The method described in item 1, wherein the subject is a human. (Item 33) The method according to item 1, wherein the cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, hepatocellular carcinoma, retinoblastoma, astroglioma, gliablastoma, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, genitourinary cancer, airway cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, carcinoma, sarcoma, central nervous system cancer, peripheral nervous system cancer, lymphoma, brain cancer, colon cancer, or bladder cancer. (Item 34) The method according to item 1, further comprising administering at least one additional anti-cancer treatment. (Item 35) The method according to item 34, wherein the at least one additional anticancer treatment is surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, radioresection, or biological therapy. (Item 36) The method according to item 34, wherein the at least one additional anti-cancer treatment is an immune checkpoint inhibitor. (Item 37) The method according to item 36, wherein the at least one checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. (Item 38) The method according to item 37, wherein the at least one checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA4 antibody, and / or an anti-KIR antibody. (Item 39) The method according to item 38, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224. (Item 40) The method according to item 38, wherein the anti-PD-L1 antibody is durvalumab, atezolizumab, or avelumab. (Item 41) The method according to item 38, wherein the anti-PD-L2 antibody is rHIgM12B7. (Item 42) The method according to item 37, wherein the inhibitor of LAG3 is IMP321 or BMS-986016. (Item 43) The method according to item 37, wherein the inhibitor of A2aR is PBF-509. (Item 44) The method according to item 38, wherein the anti-CTLA-4 antibody is tremelimumab or ipilimumab. (Item 45) The method according to item 38, wherein the anti-KIR antibody is lirilumab. (Item 46) The method described in item 36, wherein two or more checkpoint inhibitors are administered. (Item 47) The method according to item 36, wherein the immune checkpoint inhibitor is administered systemically. (Item 48) The method according to item 34, wherein the at least one additional anticancer treatment is a histone deacetylase (HDAC) inhibitor. (Item 49) The method according to item 48, wherein the HDAC inhibitor is tractinostat. (Item 50) The method according to item 1, further comprising providing an extracellular matrix degradation protein. (Item 51) The method according to item 50, wherein the extracellular matrix-degrading protein is relaxin, hyaluronidase, or decorin. (Item 52) The method according to item 34, wherein the biological therapy is a monoclonal antibody, siRNA, miRNA, antisense oligonucleotide, ribozyme, or gene therapy. (Item 53) The method according to item 34, wherein the at least one additional anti-cancer treatment is a tumor lysis virus. (Item 54) The method according to item 53, wherein the tumor lysis virus is modified to express p53, MDA-7, IL-12, at least one heat shock protein, a TGF-β inhibitor, and / or an IL-10 inhibitor. (Item 55) The method according to item 53, wherein the tumor lysis virus is a single-stranded or double-stranded DNA virus, RNA virus, adenovirus, adeno-associated virus, retrovirus, lentivirus, herpesvirus, poxvirus, vaccinia virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, Epstein-Barr virus, influenza virus, reovirus, myxoma virus, marabavirus, rhabdovirus, enadenochusileb, coxsackievirus, or E1b-deficient adenovirus. (Item 56) The method according to item 53, wherein the tumor lysis virus is a herpes simplex virus. (Item 57) The method according to item 53, wherein the tumor lysis virus is modified to express cytokines. (Item 58) The method according to item 57, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL12. (Item 59) The method according to item 53, wherein the tumor lysis virus is further defined as talimodyne laharpalebeck (T-VEC). (Item 60) The method according to item 34, wherein the at least one additional anticancer treatment is a protein kinase or a growth factor signaling pathway inhibitor. (Item 61) The protein kinase or growth factor signaling pathway inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, salakatinib, sorafenib, and sun The method described in item 60, which is tinib, trastuzumab, vandetanib, AP23451, vemurafenib, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridafololimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, or GW572016. (Item 62) The method according to item 60, wherein the protein kinase inhibitor is a PI3K inhibitor. (Item 63) The method according to item 62, wherein the PI3K inhibitor is a PI3Kδ inhibitor. (Item 64) The method described in item 35, wherein the immunotherapy includes cytokines. (Item 65) The method according to item 64, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL12. (Item 66) The method according to item 65, wherein the cytokine is interleukin and / or interferon. (Item 67) The method according to item 65, wherein the interleukin is IL-2. (Item 68) The method according to item 65, wherein the interferon is IFNα. (Item 69) The method according to item 35, wherein the immunotherapy comprises a costimulatory receptor agonist, a stimulator of innate immune cells, or an activator of innate immunity. (Item 70) The method according to item 69, wherein the costimulatory receptor agonist is an anti-OX40 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-CD40 antibody, or an anti-CD27 antibody. (Item 71) The method according to item 69, wherein the stimulating factor for immune cells is an inhibitor of a cytotoxic inhibitory receptor or an agonist of an immunostimulatory Toll-like receptor (TLR). (Item 72) The method according to item 69, wherein the cytotoxic inhibitory receptor is an inhibitor of NKG2A / CD94 or CD96 TACTILE. (Item 73) The method according to item 71, wherein the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. (Item 74) The method according to item 35, wherein the immunotherapy comprises a combination of a PD-L1 inhibitor, a 4-1BB agonist, and an OX40 agonist. (Item 75) The method described in item 35, wherein the immunotherapy comprises an interferon gene-stimulating factor (STING) agonist. (Item 76) The method according to item 75, wherein the activator of innate immunity is an IDO inhibitor, a TGFβ inhibitor, or an IL-10 inhibitor. (Item 77) The method according to item 35, wherein the chemotherapy includes a DNA damaging agent. (Item 78) The method according to item 77, wherein the DNA damaging agent is gamma irradiation, X-rays, ultraviolet irradiation, microwaves, electron emission, adriamycin, 5-fluorouracil (5FU), capecitabine, etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), or hydrogen peroxide. (Item 79) A method for treating cancer in a subject, comprising administering an effective amount of at least one tumor lysis virus and at least one CD122 / CD132 agonist to the subject. (Item 80) The method according to item 79, wherein at least one tumor lysis virus is modified to express p53, MDA-7, cytokines, and / or immunostimulatory genes. (Item 81) The method according to item 80, wherein the cytokine is GM-CSF or IL-12. (Item 82) The method according to item 80, wherein the immunostimulatory gene is a TGFβ inhibitor, an IL-10 inhibitor, or a heat shock protein. (Item 83) The method according to item 79, wherein the at least one tumor lysis virus is selected from the group consisting of single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia viruses, vesicular stomatitis viruses, polioviruses, Newcastle disease viruses, Epstein-Barr viruses, influenza viruses, reoviruses, myxoma viruses, maraba viruses, rhabdoviruses, enadenochusileb, and coxsackieviruses. (Item 84) The aforementioned at least one tumor lysis virus is Ad5-yCD / mutTKSR39rep-hIL12, CAVATAK(trademark), CG0070, DNX-2401, G207, HF10, IMLYGIC(trademark), JX-594, MG1-MA3, MV-NIS, OBP-301, REOLYSIN(registered trademark), Toca 511, Oncorine(H101), H102, H103, RIGVIR, adenovirus overexpressing adenovirus death protein (ADP), T-VEC, N1L-deficient vaccinia virus, E1b-deficient adenovirus, α-fetoprotein (AFP) promoter Ad The method according to item 79, wherein the adenovirus is an E1a gene-regulated adenovirus, a modified TERT promoter tumor-disintegrating adenovirus, an HRE-E2F-TERT hybrid promoter tumor-disintegrating adenovirus, and / or an adenovirus having a Pea3 binding site E1a regulatory sequence deletion and an Elb-19K clone insertion site. (Item 85) The method according to item 84, wherein the adenovirus that overexpresses ADP is ViRx007. (Item 86) The method according to item 84, wherein the N1L-deficient vaccinia virus is modified to express IL-12. (Item 87) The method according to item 79, wherein the at least one CD122 / CD132 agonist is selected from the group consisting of IL-2 / anti-IL-2 immune complexes, IL-15 / anti-IL-15 immune complexes, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complexes, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, IL-15 mutein, and / or IL-15 variants that bind to the α / IgG1 Fc fusion protein of the IL-15 receptor. (Item 88) The method according to item 79, wherein one, two, three, or four types of CD122 / CD132 agonists are administered to the subject. (Item 89) The method described in item 79, wherein at least one of the aforementioned CD122 / CD132 agonists is not F42K. (Item 90) The method according to item 79, wherein the subject is administered at least one of the CD122 / CD132 agonists at least twice. (Item 91) The method according to item 79, wherein the tumor lysis virus is administered to the subject before, simultaneously with, or after the at least one CD122 / CD132 agonist. (Item 92) The method according to item 79, wherein the administration includes local or local injection. (Item 93) The method according to item 79, wherein administration is via serial infusion, intratumor injection, or intravenous injection. (Item 94) The method described in item 79, wherein administration is via intratumor injection. (Item 95) The method described in item 79, wherein the subject is a human. (Item 96) The method according to item 79, wherein the cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, hepatocellular carcinoma, retinoblastoma, astrocytoma, gliablastoma, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, genitourinary cancer, airway cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, carcinoma, sarcoma, central nervous system cancer, peripheral nervous system cancer, lymphoma, brain cancer, colon cancer, or bladder cancer. (Item 97) The method according to item 79, further comprising administering at least one additional anti-cancer treatment. (Item 98) The method according to item 97, wherein the at least one additional anticancer treatment is surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, or biological therapy. (Item 99) The method according to item 97, wherein the at least one additional anti-cancer treatment is a dendritic cell vaccine. (Item 100) The method according to item 99, wherein the dendritic cell vaccine is modified to express p53 as a tumor-associated antigen. (Item 101) The method according to item 97, wherein the at least one additional anticancer treatment is an immune checkpoint inhibitor. (Item 102) The method according to item 101, wherein the at least one checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. (Item 103) The method according to item 102, wherein the at least one checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA4 antibody, and / or an anti-KIR antibody. (Item 104) The method according to item 103, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224. (Item 105) The method according to item 103, wherein the anti-PD-L1 antibody is durvalumab, atezolizumab, or avelumab. (Item 106) The method according to item 103, wherein the anti-PD-L2 antibody is rHIgM12B7. (Item 107) The method according to item 102, wherein the inhibitor of LAG3 is IMP321 or BMS-986016. (Item 108) The method according to item 102, wherein the inhibitor of A2aR is PBF-509. (Item 109) The method according to item 103, wherein the anti-CTLA-4 antibody is tremelimumab or ipilimumab. (Item 110) The method according to item 103, wherein the anti-KIR antibody is lirilumab. (Item 111) The method according to item 101, wherein two or more checkpoint inhibitors are administered. (Item 112) The method according to item 101, wherein the immune checkpoint inhibitor is administered systemically. (Item 113) The method according to item 97, wherein the at least one additional anticancer treatment is a histone deacetylase (HDAC) inhibitor. (Item 114) The method according to item 113, wherein the HDAC inhibitor is tractinostat. (Item 115) The method according to item 79, further comprising providing an extracellular matrix-degrading protein. (Item 116) The method according to item 115, wherein the extracellular matrix-degrading protein is relaxin, hyaluronidase, or decorin. (Item 117) (a) a nucleic acid encoding p43 and / or a nucleic acid encoding MDA-7; and (b) a pharmaceutical composition comprising at least one CD122 / CD132 agonist. (Item 118) (a) a tumor lysis virus; and (b) a pharmaceutical composition comprising at least one CD122 / CD132 agonist. [Brief explanation of the drawing]

[0040] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention will be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0041] [Figure 1]Effective Ad-p53 administration and tumor response. Waterfall plot of tumor response for a preferred tumor p53 biomarker patient subgroup treated with Ad-p53 at doses greater than 7 × 10¹⁰ viral particles / cm³ (left panel) compared with Ad-p53 at doses less than 7 × 10¹⁰ viral particles / cm³ (right panel). Detailed investigation of Ad-p53 responders revealed that the majority of responders (7 / 9 patients) received Ad-p53 doses around or exceeding 1 × 10¹¹ vp / cm³ (7.81–333.2 × 10¹⁰ vp / cm³). Therefore, doses for Ad-p53 clinical use may exceed 1 × 10¹¹ vp / cm³ of injected tumor volume.

[0042] [Figure 2] Superior overall survival compared to Ad-p53 biomarker / dose-optimized therapy. Superior 1-year and overall survival in patients with a favorable tumor p53 biomarker profile treated with Ad-p53 doses of >7 × 10¹⁰ viral particles / cm³ compared to patients treated with methotrexate, who had a favorable tumor p53 biomarker profile. The results show a statistically significant increase in overall survival with respect to favorable tumor p53 biomarker and Ad-p53 dose-optimized therapy compared to methotrexate (median survival for Ad-p53 treatment: 11.5 months vs. 4.3 months for methotrexate; p<0.016, HR 1.9767).

[0043] [Figure 3] QUADRA-FUSE® Infusion Device. QUADRA-FUSE® (image from REXMEDICAL®) is a multi-branched infusion device with three branches extending from a trocar shaft (insertion shown in the upper right) around a central needle axis, with an adjustable diameter of 1–5 cm depending on the narrow width (W) diameter of the tumor injury. This horizontal expansion allows for broad distribution of the drug through the injury. Each branch has two delivery through-holes (four fluid outlets). Thus, 12 delivery points are obtained with each injection.

[0044] [Figure 4] Efficacy of Ad-p53 + CD122 / 132 agonist + anti-PD-1: Tumor volume. Graphs showing the volume of primary tumors over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132, anti-PD-1, Ad-p53, or any combination of CD122 / 132 + anti-PD-1, Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, or Ad-p53 + CD122 / 132 + anti-PD-1. Significant tumor progression was observed between CD122 / 132, anti-PD-1, and CD122 / 132 + anti-PD-1 treatments, which were reversed by combining with Ad-p53 treatment. The results also show improved efficacy of Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, and Ad-p53+CD122 / 132+anti-PD-1 treatments compared to any one of the other treatments. By day 21, the mean tumor volume in all groups treated with (PBS), CD122 / 132, anti-PD-1, CD122 / 132+anti-PD-1, and Ad-p53 exceeded 2,000 mm3. In contrast, each of the combination therapies—Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, and Ad-p53+CD122 / 132+anti-PD-1—induced a significant reduction in tumor volume compared to either the non-Ad-p53 treatment or Ad-p53 treatment alone. Statistical analysis of variance (ANOVA) of tumor volume at day 21 measured the synergistic antitumor effects of Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, and Ad-p53+CD122 / 132+anti-PD-1 treatments (p<0.0001). However, by day 30, the mean tumor volume in both the Ad-p53+CD122 / 132 and Ad-p53+anti-PD-1 treatment groups also exceeded 2,000 mm3. Importantly, statistical analysis of variance (ANOVA) comparison of tumor volume at day 30 measured that the synergistic antitumor effect was maintained only in the Ad-p53+CD122 / 132+anti-PD-1 treatment combination (p<0.0001 (overall) and p<0.0001 when compared individually with each of the other treatment groups).

[0045] [Figure 5] Complete tumor response rate. Complete tumor response to a treatment is generally understood to be associated with a significant therapeutic effect and is necessary for a curative outcome. As shown in Figure 5, for the p53 treatment group and their controls, only Ad-p53 + CD122 / 132 + anti-PD-1 treatment resulted in complete tumor remission in both primary and contralateral tumors. Complete tumor response was observed in 60% of the Ad-p53+CD122 / 132+anti-PD-1 treatment group (6 out of 10 animals), while no complete tumor response was observed in any of the other treatment groups (Fisher's two-sided exact test comparing Ad-p53+CD122 / 132+anti-PD-1 versus all other treatment groups, p-value < 0.0001; Fisher's two-sided exact test comparing Ad-p53+CD122 / 132+anti-PD-1 versus all other treatment groups, p-value < 0.011). Unexpectedly, the complete tumor response was persistent and was maintained at 40 days in 50% of the Ad-p53+CD122 / 132+anti-PD-1 treatment group, which likely cured these animals with these tumors.

[0046] [Figure 6A]Figures 6A and 6B: Systemic / abscopal treatment effects on contralateral tumor growth. The systemic / abscopal effects of primary tumor treatment on contralaterally transplanted tumors were evaluated in rodents whose primary tumors received one of the Ad-p53 intratumor therapies. Consistent with the unexpectedly substantially increased synergistic effect of Ad-p53+CD122 / 132+anti-PD-1 treatment on primary tumor growth and complete remission rates, we also observed a remarkably potent and statistically significant abscopal effect of Ad-p53+CD122 / 132+anti-PD-1 treatment compared to other Ad-p53 treatment groups. As shown in Figure 6A, contralateral tumor growth disappeared in 90% (9 out of 10 animals) of animals treated with Ad-p53+CD122 / 132+anti-PD-1 for primary tumor growth. In contrast, contralateral tumor growth was observed in 62.5–100% of animals in the other Ad-p53 treatment groups. This difference in contralateral tumor growth was statistically significant (p-value by chi-square test for all treatment groups = 0.0004; p-value by Fisher's two-sided exact test comparing Ad-p53 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group < 0.0430). Figure 6B shows a graph of contralateral tumor volume over time in rodents receiving one of the three most effective primary tumor treatments: Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, or any combination of Ad-p53 + CD122 / 132 + anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes on day 22 measured the synergistic antitumor effect of Ad-p53 + CD122 / 132 + anti-PD-1 therapy (p-value = 0.0435 overall). Only the Ad-p53 + CD122 / 132 + anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the Ad-p53 + anti-PD-1 group (p-value = 0.0360). Taken together, these findings indicate that of all Ad-p53 treatments, only the three combinations of Ad-p53 + CD122 / 132 + anti-PD-1 therapy resulted in curative efficacy by inducing potent local and systemic antitumor immunity mediated by a substantial abscopal effect. [Figure 6B]Figures 6A and 6B: Systemic / abscopal treatment effects on contralateral tumor growth. The systemic / abscopal effects of primary tumor treatment on contralaterally transplanted tumors were evaluated in rodents whose primary tumors received one of the Ad-p53 intratumor therapies. Consistent with the unexpectedly substantially increased synergistic effect of Ad-p53+CD122 / 132+anti-PD-1 treatment on primary tumor growth and complete remission rates, we also observed a remarkably potent and statistically significant abscopal effect of Ad-p53+CD122 / 132+anti-PD-1 treatment compared to other Ad-p53 treatment groups. As shown in Figure 6A, contralateral tumor growth disappeared in 90% (9 out of 10 animals) of animals treated with Ad-p53+CD122 / 132+anti-PD-1 for primary tumor growth. In contrast, contralateral tumor growth was observed in 62.5–100% of animals in the other Ad-p53 treatment groups. This difference in contralateral tumor growth was statistically significant (p-value by chi-square test for all treatment groups = 0.0004; p-value by Fisher's two-sided exact test comparing Ad-p53 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group < 0.0430). Figure 6B shows a graph of contralateral tumor volume over time in rodents receiving one of the three most effective primary tumor treatments: Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, or any combination of Ad-p53 + CD122 / 132 + anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes on day 22 measured the synergistic antitumor effect of Ad-p53 + CD122 / 132 + anti-PD-1 therapy (p-value = 0.0435 overall). Only the Ad-p53 + CD122 / 132 + anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the Ad-p53 + anti-PD-1 group (p-value = 0.0360). Taken together, these findings indicate that of all Ad-p53 treatments, only the three combinations of Ad-p53 + CD122 / 132 + anti-PD-1 therapy resulted in curative efficacy by inducing potent local and systemic antitumor immunity mediated by a substantial abscopal effect.

[0047] [Figure 7]Efficacy of Ad-p53+CD122 / 132+anti-PD-1: Extended survival. Kaplan-Meier survival curves for mice treated with PBS, CD122 / 132+anti-PD-1, Ad-p53, or Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, and Ad-p53+CD122 / 132+anti-PD-1. Log-rank tests showed statistically significant differences in these survival curves (p<0.0001 overall; p<0.0003 for comparisons between the Ad-p53+CD122 / 132+anti-PD-1 treatment group and any other treatment group). These results also demonstrate an unexpected substantial synergistic effect of Ad-p53+CD122 / 132+anti-PD-1 therapy. The median survival in the Ad-p53 + CD122 / 132 + anti-PD-1 treatment group did not reach 40 days, with 80% of this group still alive and showing no evidence of residual tumors. In stark contrast, 98% (49 out of 50) of the animals in the other treatment groups died by day 30, with a median survival ranging from 10 to 28 days.

[0048] [Figure 8]Efficacy of VirRx007 + CD122 / 132 agonist + anti-PD-1: Tumor volume. Graphs showing the volume of primary tumors over time in rodents treated with phosphate-buffered saline (PBS), CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, VirRx007, or a combination of VirRx007 + CD122 / 132, VirRx007 + anti-PD-1, or VirRx007 + CD122 / 132 + anti-PD-1. Significant tumor progression was observed in the groups treated with CD122 / 132 + anti-PD-1 and CD122 / 132 + anti-PD-1, and these trends were reversed by combining them with VirRx007 treatment. The results also demonstrate the improved efficacy of VirRx007 + anti-PD-1 and VirRx007 + CD122 / 132 + anti-PD-1 treatments compared to any of the monotherapy methods. In contrast to the findings with Ad-p53, VirRx007 did not show synergistic effects with CD122 / CD132 treatment. By day 30, the mean tumor volume in all groups treated with (PBS), CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, VirRx007, and VirRx007 + CD122 / CD132 exceeded 2,000 mm3. In contrast, both combination therapies of VirRx007 + anti-PD-1 and VirRx007 + CD122 / 132 + anti-PD-1 induced a significant reduction in tumor volume compared to either non-VirRx007 therapy or VirRx007 therapy alone. Statistical analysis of variance (ANOVA) of tumor volume at day 30 measured the synergistic antitumor effect of VirRx007 + anti-PD-1 and VirRx007 + CD122 / 132 + anti-PD-1 therapy (p < 0.0001, overall and for each of these therapies versus VirRx007). VirRx007 + CD122 / 132 + anti-PD-1 therapy was superior to VirRx007 + anti-PD-1 (p = 0.0002). Surprisingly, while the combination therapy of VirRx007 + CD122 / 132 did not show any clear advantage compared to VirRx007 monotherapy, a synergistic effect was demonstrated with the triple therapy of VirRx007 + CD122 / 132 + anti-PD-1.

[0049] [Figure 9] Complete tumor response rate. Complete tumor response to a treatment is generally understood to be associated with a significant therapeutic effect and is necessary for a curative outcome. As shown in Figure 9, for the VirRx007 treatment group and their controls, only VirRx007 + CD122 / 132 + anti-PD-1 treatment resulted in complete tumor remission in both primary and contralateral tumors. Complete response to both primary and contralateral tumors was observed in 60% of the VirRx007+CD122 / 132+anti-PD-1 treatment group, while no complete response was observed in any of the other treatment groups (Fisher's two-sided exact test comparing the VirRx007+CD122 / 132+anti-PD-1 group versus all other treatment groups, p-value < 0.0001; Fisher's two-sided exact test comparing VirRx007+CD122 / 132+anti-PD-1 versus all other treatment groups, p-value < 0.011). Unexpectedly, the complete response to the tumors was persistent and likely cured these animals with these tumors, and was maintained at 40 days in 50% of the animals treated with VirRx007+CD122 / 132+anti-PD-1.

[0050] [Figure 10A]Figures 10A and 10B: Systemic / abscopal treatment effects on contralateral tumor growth. The systemic / abscopal effects of primary tumor treatment on contralaterally transplanted tumors were evaluated in rodents whose primary tumors received one of the VirRx007 intratumor therapies. Consistent with the unexpectedly substantially increased synergistic effect of VirRx007 + CD122 / 132 + anti-PD-1 treatment on primary tumor growth and complete remission rates, we also observed a remarkably potent and highly statistically significant abscopal effect of VirRx007 + CD122 / 132 + anti-PD-1 treatment compared to other VirRx007 treatment groups. As shown in Figure 10A, contralateral tumor growth disappeared in 80% of animals whose primary tumors were treated with VirRx007 + CD122 / 132 + anti-PD-1. In contrast, contralateral tumor growth was observed in 80–100% of animals in the other VirRx007 treatment groups. This difference in contralateral tumor growth was statistically significant (p-value = 0.0002 by chi-square test comparing all treatment groups; p-value < 0.0230 by Fisher's two-sided exact test comparing VirRx007 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group). These findings suggest that the VirRx007 + CD122 / 132 + anti-PD-1 treatment combination induced potent systemic antitumor immunity and mediated a substantial abscopal effect with potential curative efficacy. Figure 10B shows a graph of contralateral tumor volume over time in rodents receiving one of three most effective combination therapies for primary tumors: VirRx007+CD122 / 132, VirRx007+anti-PD-1, or VirRx007+CD122 / 132+anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes at day 22 measured the synergistic antitumor effect of VirRx007+CD122 / 132+anti-PD-1 treatment (p-value = 0.0171 overall). Only the VirRx007+CD122 / 132+anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the VirRx007+anti-PD-1 group (p-value = 0.0115).These findings, taken together, indicate that of all VirRx007 treatments, only the three combinations of VirRx007 + CD122 / 132 + anti-PD-1 therapy resulted in curative efficacy by inducing potent local and systemic antitumor immunity that mediated a substantial abscopal effect. [Figure 10B]Figures 10A and 10B: Systemic / abscopal treatment effects on contralateral tumor growth. The systemic / abscopal effects of primary tumor treatment on contralaterally transplanted tumors were evaluated in rodents whose primary tumors received one of the VirRx007 intratumor therapies. Consistent with the unexpectedly substantially increased synergistic effect of VirRx007 + CD122 / 132 + anti-PD-1 treatment on primary tumor growth and complete remission rates, we also observed a remarkably potent and highly statistically significant abscopal effect of VirRx007 + CD122 / 132 + anti-PD-1 treatment compared to other VirRx007 treatment groups. As shown in Figure 10A, contralateral tumor growth disappeared in 80% of animals whose primary tumors were treated with VirRx007 + CD122 / 132 + anti-PD-1. In contrast, contralateral tumor growth was observed in 80–100% of animals in the other VirRx007 treatment groups. This difference in contralateral tumor growth was statistically significant (p-value = 0.0002 by chi-square test comparing all treatment groups; p-value < 0.0230 by Fisher's two-sided exact test comparing VirRx007 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group). These findings suggest that the VirRx007 + CD122 / 132 + anti-PD-1 treatment combination induced potent systemic antitumor immunity and mediated a substantial abscopal effect with potential curative efficacy. Figure 10B shows a graph of contralateral tumor volume over time in rodents receiving one of three most effective combination therapies for primary tumors: VirRx007+CD122 / 132, VirRx007+anti-PD-1, or VirRx007+CD122 / 132+anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes at day 22 measured the synergistic antitumor effect of VirRx007+CD122 / 132+anti-PD-1 treatment (p-value = 0.0171 overall). Only the VirRx007+CD122 / 132+anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the VirRx007+anti-PD-1 group (p-value = 0.0115).These findings, taken together, indicate that of all VirRx007 treatments, only the three combinations of VirRx007 + CD122 / 132 + anti-PD-1 therapy resulted in curative efficacy by inducing potent local and systemic antitumor immunity that mediated a substantial abscopal effect.

[0051] [Figure 11] Efficacy of VirRx007 + CD122 / 132 + anti-PD-1: Extended survival. Kaplan-Meier survival curves for mice treated with PBS, CD122 / 132 + anti-PD-1, VirRx007, or any combination of VirRx007 + CD122 / 132, VirRx007 + anti-PD-1, and VirRx007 + CD122 / 132 + anti-PD-1. Log-rank tests showed statistically significant differences in these survival curves (p<0.0001 overall; p<0.0005 when comparing the VirRx007 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group). The unexpected substantial synergistic effect of the VirRx007 + CD122 / 132 + anti-PD-1 treatment also indicates this result. The median survival in the VirRx007 + CD122 / 132 + anti-PD-1 treatment group did not reach 40 days, with 90% of animals still alive. In striking contrast, 98% (49 / 50 animals) in the other treatment groups died by day 40, with median survival ranging from 10 to 33 days. Surprisingly, while there was no clear survival advantage for the VirRx007 + CD122 / 132 combination therapy compared to VirRx007 monotherapy, a synergistic effect was demonstrated for the VirRx007 + CD122 / 132 + anti-PD-1 triple therapy.

[0052] [Figure 12]Efficacy of Ad-IL24 + CD122 / 132 agonist + anti-PD-1: Tumor volume. Graphs showing the volume of primary tumors over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, Ad-IL24, or a combination of Ad-IL24 + CD122 / 132 or Ad-IL24 + CD122 / 132 + anti-PD-1. Significant tumor progression was observed between CD122 / 132, anti-PD-1, and CD122 / 132 + anti-PD-1 treatments, which were reversed by combination with Ad-IL24 treatment. Compared to any of the monotherapys, Ad-IL24 + CD122 / 132 + anti-PD-1 treatment demonstrated improved efficacy. By day 16, the mean tumor volume in all groups treated with (PBS), CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, and Ad-IL24 exceeded 2,000 mm3. In contrast, combination therapy with Ad-IL24 + CD122 / 132 + anti-PD-1 induced a substantial reduction in tumor volume compared to either non-Ad-IL24 therapy or Ad-IL24 therapy alone. Statistical analysis of variance (ANOVA) of tumor volume at day 16 measured the synergistic antitumor effect of Ad-IL24 + CD122 / 132 + anti-PD-1 therapy (p < 0.0001). Compared to either Ad-IL24 (p=0.0025) or CD122 / 132 + anti-PD-1 therapy (p<0.0001), Ad-IL24 + CD122 / 132 + anti-PD-1 therapy resulted in a statistically significant reduction in tumor volume.

[0053] [Figure 13]Efficacy of Ad-IL24 + CD122 / 132 + anti-PD-1: Extended survival. Kaplan-Meier survival curves for mice treated with either PBS, CD122 / 132 + anti-PD-1, Ad-IL24, or a combination of Ad-IL24 + CD122 / 132 + anti-PD-1. Log-rank tests showed statistically significant differences in these survival curves (p<0.0001). This result demonstrates an unexpected, substantial synergistic effect of the Ad-IL24 + CD122 / 132 + anti-PD-1 treatment. Median survival was synergistically improved in the Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group. All animals in the PBS, CD122 / 132 + anti-PD-1, and IL24 treatment groups died by day 16, while 50% of animals in the Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group were alive at day 19. The Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group showed statistically significantly improved survival compared to either Ad-IL24 alone (p=0.0003) or CD122 / 132 + anti-PD-1 treatment group (p<0.0001). Interestingly, the Ad-IL24 + CD122 / 132 pair showed remarkably superior efficacy compared to the CD122 / 132 + anti-PD-1 pair (log-rank test p=0.0002, data not shown).

[0054] [Figure 14]Efficacy of Ad-luciferase (Ad-Luc) negative control + CD122 / 132 agonist + anti-PD-1: Tumor volume. Graphs showing the volume of primary tumors over time in rodents receiving one of the following combinations: phosphate-buffered saline (PBS) control, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, Ad-Luc control, or Ad-Luc control + CD122 / 132, Ad-Luc control + anti-PD-1, or Ad-Luc control + CD122 / 132 + anti-PD-1. In contrast to treatment with Ad-p53, VirRx007, and Ad-IL24, there was no significant increase in treatment efficacy when Ad-Luc was combined with anti-PD-1, CD122 / 132, or CD122 / 132 + anti-PD-1 treatment. By day 16, the mean tumor volume in all groups exceeded 2,000 mm3. Statistical analysis of variance (ANOVA) of tumor volume on day 16 was not statistically significant (p-value = 0.1212; the mean tumor volume among all these treatment groups was not statistically significant).

[0055] [Figure 15] Compared to Ad-Luc control + CD122 / 132 + anti-PD-1, the "triple treatment" regimens using Ad-p53, VirRx007, and Ad-IL24, each combined with CD122 / 132 + anti-PD-1, extended survival. Kaplan-Meier survival curves for mice treated with Ad-p53, VirRx007, Ad-IL24, or CD122 / 132 + anti-PD-1 combined with Ad-Luc control. Log-rank tests showed statistically significant differences in these survival curves (p<0.0001). Ad-p53, VirRx007, and Ad-IL24, when combined with CD122 / 132+anti-PD-1 triple therapy, each showed a statistically significant increase in survival compared to the Ad-Luc+CD122 / 132+anti-PD-1 triple therapy control group (by log-rank test, both Ad-p53 and VirRx007 combined with CD122 / 132+anti-PD-1 triple therapy had p-values ​​< 0.0001; Ad-IL24 combined with CD122 / 132+anti-PD-1 had p-values ​​< 0.015).

[0056] [Figure 16] Efficacy of Ad-p53 + CD122 / 132 (IL15) agonist + anti-PD-1: Tumor volume. Selective CD122 / CD132 agonists combined with tumor suppressor therapy were immune complexes consisting of recombinant IL15 and IL-15-R α-Fc. Graphs show the volume of primary tumors over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132 + anti-PD-1, Ad-p53 alone, or the Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 combination. Significant tumor progression was observed between PBS, CD122 / 132 + anti-PD-1, and Ad-p53 treatments. Consistent with the results of the initial Ad-p53 combination therapy described above, Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy showed substantially improved efficacy compared to any of the treatments. By day 30, the mean tumor volume in the groups treated with PBS, CD122 / 132 + anti-PD-1, and Ad-p53 all exceeded 2,000 mm3. In contrast, the combination therapy using Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 induced a significant reduction in tumor volume. Statistical analysis of variance (ANOVA) of tumor volume measured the synergistic antitumor effect of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 treatment (p < 0.0001 (overall) and p < 0.0001 when compared individually with each of the other treatment groups). [Figure 17]The systemic / abscopal therapeutic effect of Ad-p53+CD122 / 132(IL15)+anti-PD-1 on contralateral tumor growth. The selective CD122 / CD132 agonist, combined with tumor suppressor therapy, was an immune complex consisting of recombinant IL15 and IL-15-R α-Fc. The systemic / abscopal effect in treating primary tumors of contralaterally transplanted tumors was evaluated in rodents whose primary tumors received Ad-p53+CD122 / 132(IL15)+anti-PD-1. Consistent with the unexpected, substantially increased synergistic effect of Ad-p53+CD122 / 132(IL15)+anti-PD-1 treatment on primary tumor growth shown in Figure 16, we also observed a remarkably potent and statistically significant abscopal effect of Ad-p53+CD122 / 132(IL15)+anti-PD-1 treatment compared to other Ad-p53 treatment groups. Figure 17 shows a graph of contralateral tumor volume over time in rodents receiving primary tumor treatment with any of the following combinations: Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, or Ad-p53+CD122 / 132(IL15)+anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes on day 22 measured the synergistic antitumor effect of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 treatment (p-value = 0.0433 overall). Only the Ad-p53 + CD122 / 132 + anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the Ad-p53 + anti-PD-1 group (p-value = 0.0359). Taken together, these findings indicate that of all Ad-p53 treatments, only the three combinations of Ad-p53 + CD122 / 132 + anti-PD-1 treatment resulted in curative efficacy by inducing potent local and systemic antitumor immunity mediated by a substantial abscopal effect.

[0057] [Figure 18]Efficacy of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1: Extended survival. The selective CD122 / CD132 agonist combined with tumor suppressor therapy was an immune complex consisting of recombinant IL15 and IL-15-R α-Fc. Figure 18 shows Kaplan-Meier survival curves for mice treated with either PBS, CD122 / 132 + anti-PD-1, Ad-Luc + CD122 / 132 + anti-PD-1 control, Ad-p53, or Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 combination. Log-rank tests showed statistically significant differences in these survival curves (overall p<0.0001, p-value <0.0001 when comparing the Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 treatment group versus any other treatment group). The results further demonstrate the unexpected substantial synergistic effect of the Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy. In the Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy group, 50% of animals were alive on day 36. In stark contrast, all animals in the other treatment groups died by day 22, with a median survival range of 10–18 days. [Modes for carrying out the invention]

[0058] Tumors are well known to evolve in their early and advanced stages, evading destruction by the immune system. While recent studies have shown some success in reversing this resistance using immune checkpoint inhibitors, the majority of patients do not respond to these treatments. Therefore, in certain embodiments, this disclosure relates to methods and compositions for altering the tumor microenvironment to overcome resistance and enhance the anti-tumor immune response. In one embodiment, a method is provided for treating cancer by expressing p53 and / or MDA-7 in combination with at least one CD122 and CD132 agonist. Specifically, the tumor suppressor gene is administered as a non-replicating adenovirus. In one method, the p53 and / or MDA7 gene therapy is administered in combination with a CD122 / CD132 agonist. CD122 / CD132 agonists can be IL-2 / anti-IL-2 immune complexes, IL-15 / anti-IL-15 immune complexes, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complexes, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein. CD122 / CD132 agonists can also be IL-15 variants (e.g., IL-15N72D) bound to IL-15 receptor α / IgG1 Fc fusion proteins, such as ALT-803 (Rhode et al., 2016).

[0059] Furthermore, the inventors measured that administering additional therapies, such as immune checkpoint inhibitors like anti-PD1 antibodies, enhances antitumor immunity before, during, or after administration of p53 and / or MDA-7 gene therapy in combination with a selective CD122 / CD132 agonist.

[0060] Furthermore, the inventors have measured that administering additional therapies to degrade the extracellular matrix of tumor cells can improve tumor penetration of the combination therapy. In particular, the extracellular matrix degradation therapy is administered before the combination therapy. In one method, the extracellular matrix degradation therapy is a relaxin gene therapy, such as adenovirus relaxin. In particular, adenovirus relaxin is administered intratumorally or intra-arterially.

[0061] Furthermore, the treatment method may include additional anti-cancer therapies, such as cytokines or chemotherapy, to enhance the antitumor effect of the combination therapy provided herein. For example, the cytokine may be granulocyte-macrophage colony-stimulating factor (GM-CSF), and the chemotherapy may be 5-fluorouracil (5FU), or capecitabine, or cyclophosphamide, or a PI3K inhibitor.

[0062] I. Definition As used herein, "essentially absent" in terms of specific components means that a specific component is either not intentionally included in the composition and / or is present as a contaminant or in trace amounts. Therefore, the total amount of a specific component resulting from the unintentional inclusion of a particular component in a composition is well below 0.05%, preferably below 0.01%. Most preferably, the amount of a specific component cannot be analyzed using standard analytical methods.

[0063] As used herein, “a” or “an” may mean one or more. As used within the claims herein, when used with the word “comprising,” the word “a” or “an” may mean one or more.

[0064] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated to mean only substitutes or that the substitutes are mutually exclusive; however, this disclosure supports the definitions of only substitutes and “and / or.” As used herein, “another” may mean at least a second or more.

[0065] Throughout this specification, the term “approximately” is used to indicate that a value includes an inherent deviation of error in the device, method, or test subject used to determine the value. As used herein, “wild type” means a sequence of nucleic acid that is naturally present in a locus within the genome of an organism, and sequences that have been transcribed or translated from such nucleic acid.

[0066] Therefore, the term “wild type” can also mean the amino acid sequence encoded by the nucleic acid. Since a locus can have two or more sequences or alleles in a population of individuals, the term “wild type” encompasses all such naturally occurring alleles. As used herein, the term “polymorphism” means that there is a variation (i.e., the presence of two or more alleles) in a locus of individuals in a population. As used herein, “variant” means a variation in the sequence of a nucleic acid, or a protein, polypeptide, or peptide encoded by it, as a result of recombinant DNA technology.

[0067] When used in relation to proteins, genes, nucleic acids, or polynucleotides within a cell or organism, the term “exogenous” means a protein, gene, nucleic acid, or polynucleotide that has been introduced into that cell or organism by artificial or natural means. Or, in relation to a cell, the term means a cell that has been isolated by artificial or natural means and subsequently introduced into another cell or organism. Exogenous nucleic acids may originate from a different organism or cell. Or, exogenous nucleic acids may be one or more additional copies of nucleic acids that are naturally present within that organism or cell. Exogenous cells may originate from a different organism or from the same organism. As a non-limiting example, exogenous nucleic acids are nucleic acids that are located at a different chromosomal location than where they are found in a natural cell, or that are adjacent to nucleic acid sequences that are different from those otherwise found naturally.

[0068] An expression construct or expression cassette means a nucleic acid molecule capable of directing transcription. An expression construct comprises at least one transcriptional regulator (e.g., a promoter, enhancer, or a construct functionally equivalent thereto) that directs gene expression in one or more desired cell types, tissues, or organs. Additional elements, such as transcription termination signals, may also be included.

[0069] A "vector" or "construct" (sometimes called a delivery system or gene transfer "vehicle") refers to a macromolecule or complex of molecules containing polynucleotides that are delivered to a host cell either in vitro or in vivo.

[0070] A common type of vector, a "plasmid," is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA. In certain cases, plasmids are circular and double-stranded.

[0071] A “origin of replication” ("ori"), or “origin of replication,” is a DNA sequence within a lymphoproliferative herpesvirus, for example, that, if present in a plasmid within a cell, is capable of maintaining the bound sequence within the plasmid and / or at or near the site where DNA synthesis begins. For example, the origin of replication for EBV includes the FR sequence (20 incomplete copies of a 30 bp repeat) and preferably the DS sequence. However, other sites within EBV-binding EBNA-1, such as the Rep* sequence, can be replaced with the DS as the origin of replication (Kirshmaier and Sugden, 1998). Therefore, the origin of replication for EBV includes any functionally equivalent sequence, such as the FR, DS, or Rep* sequence, or a combination of nucleic acid modifications or synthesis derived therefrom. For example, the present invention also uses genetically modified origins of replication for EBV, such as those resulting from the insertion or mutation of individual elements, as specifically described in Lindner, et al., 2008.

[0072] A gene, polynucleotide, coding region, sequence, segment, fragment, or transgene that "codes" a specific protein is a nucleic acid molecule that, when controlled by an appropriate regulatory sequence, is transcribed in vitro or in vivo and optionally translated into a gene product (e.g., polypeptide). The coding region may exist in cDNA, genomic DNA, or RNA form. If in DNA form, the nucleic acid molecule can be single-stranded (i.e., sense strand) or double-stranded. The coding region boundary is determined by a start codon at the 5' (amino) end and a translation termination codon at the 3' (carboxy) end. Examples of genes include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is typically located 3' to the gene sequence.

[0073] The term "regulatory elements" collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRESs), enhancers, splice junctions, etc., that provide for the replication, transcription, post-translational modification, and translation of coding sequences within recipient cells. Not all of these regulatory elements are necessary if the selected coding sequence can be replicated, transcribed, and translated within a suitable host cell.

[0074] The term “promoter” is used herein in its ordinary sense and means a nucleotide region containing a DNA regulatory sequence, where the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating the transcription of a downstream (3' direction) coding sequence. Promoters may contain regulatory proteins and molecules, such as RNA polymerase and other transcription factors, to which specific transcription of a nucleic acid sequence can be initiated. The terms “operatably positioned,” “operatably bound,” “under control,” and “transcriptionally regulated” mean that the promoter is in the correct functional position and / or direction relative to the nucleic acid sequence and controls the transcription initiation and / or expression of that sequence.

[0075] An "enhancer" refers to a nucleic acid sequence that, when placed adjacent to a promoter, confers increased transcriptional activity compared to the transcriptional activity produced by the promoter without an enhancer domain.

[0076] With respect to nucleic acid molecules, "operably bound" or "co-expressed" means that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are linked in a manner that enables the transcription of the nucleic acid molecule. With respect to peptide and / or polypeptide molecules, "operably bound" or "co-expressed" means that two or more peptide and / or polypeptide molecules are linked in a manner that yields a single polypeptide chain, i.e., a fusion polypeptide having at least one property of each peptide and / or polypeptide component of the fusion. Preferably, the fusion polypeptide is a chimera, i.e., composed of heterogeneous molecules.

[0077] "Homologousity" refers to the degree of identity between two polynucleotides or two polypeptides. The agreement between one sequence and another can be measured by techniques known in the art. For example, homology can be measured by directly comparing the sequence information between two polypeptide molecules using a readily available computer program that aligns the sequence information. Alternatively, homology can be measured by hybridizing polynucleotides under conditions that promote the formation of a stable duplex between homologous regions, then degrading them using a single-strand specific nuclease, and measuring the size of the degraded fragments. Two DNA sequences or two polypeptide sequences are "substantially homologous" to each other when, as measured by the above methods, at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match in molecules of a defined length.

[0078] The term “nucleic acid” generally means at least one molecule or chain of DNA, RNA, or its derivatives or mimics containing at least one nucleobase, such as adenine “A”, guanine “G”, thymine “T”, and cytosine “C”, or RNA (such as A, G, uracil “U”, and C), which are naturally occurring purines or pyrimidine bases. The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide.” The term “oligonucleotide” means at least one molecule with a length of about 3 to about 100 nucleobases. The term “polynucleotide” means at least one molecule with a length of more than about 100 nucleobases. While these definitions generally mean at least one single-stranded molecule, in certain embodiments they also include at least one additional chain that is partially, substantially, or completely complementary to at least one single-stranded molecule. Therefore, nucleic acids can comprise at least one double-stranded molecule or at least one triple-stranded molecule, each containing one or more complementary strands or "complement" of a specific sequence including the molecular chain in question.

[0079] As used throughout this application, the term “therapeutic effect” means anything that promotes or improves the health of a patient with respect to the medical treatment of cancer. A list of non-exclusive examples of this includes: extension of the patient’s lifespan over any period of time; reduction or delay of neoplastic progression of the disease; reduction of overgrowth; reduction of tumor growth; delay of metastasis; reduction of the growth rate of cancer cells or tumor cells; induction of apoptosis in any treated cells or any cells affected by treated cells; and reduction of pain in the patient that may result from the patient’s condition.

[0080] An “effective dose” is the minimum amount necessary to produce a measurable improvement or prevention of a particular disease. The effective dose as used herein may vary depending on factors such as the patient’s disease state, age, sex, and weight, as well as the antibody’s ability to induce the desired response in the individual. An effective dose is also the amount at which the therapeutically beneficial effects outweigh any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired outcomes include the elimination or reduction of the risk of disease, reduction of disease severity, or delay of disease onset, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during disease progression. In the case of therapeutic use, beneficial or desired outcomes include clinical outcomes such as a reduction in one or more symptoms caused by the disease, an improvement in the quality of life of the person affected, a reduction in the dose of other drugs required to treat the disease, an enhancement of the effect of another drug (e.g., by targeting), a delay in disease progression, and / or an extension of survival. In the case of cancer or tumors, an effective dose of a drug may have the effect of reducing the number of cancer cells, decreasing tumor size, inhibiting (i.e., delaying to some extent, or preferably stopping) the invasion of cancer cells into peripheral organs, inhibiting (i.e., delaying to some extent, or preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more of the symptoms associated with the disease. An effective dose may be administered in one or more doses. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical field, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, “effective dose” may be considered in relation to the administration of one or more therapeutic agents, and a single active agent may be considered to be given in an effective dose if, when combined with one or more other active agents, a desired result can or does not occur.

[0081] As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating agent, diluent, antimicrobial and antifungal agent, isotonic and absorption retardant, buffer, carrier solution, suspension, colloid, etc. The use of such media and active substances for pharmaceutically active substances is known in the art. Unless any conventional media or active substance is incompatible with the active ingredient, it is conceivable to use conventional media or active substances in therapeutic compositions. Auxiliary active ingredients may also be incorporated into the composition.

[0082] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the target organism to which the preparation is to be administered. Such preparations are sterile. A "pharmaceutically acceptable" excipient (vehicle) is an excipient that can be reasonably administered to the target mammal in order to provide an effective amount of the active ingredient to be used.

[0083] As used herein, the term “treatment” means a clinical intervention designed to alter the natural course of the treated individual or cells during the course of a disease. Desired effects of treatment include a reduction in disease progression, remission or mitigation of the disease, and remission or improved prognosis. For example, an individual is “well treated” if one or more cancer-related symptoms are mitigated or eliminated, including but not limited to a reduction (or destruction) of cancerous cell proliferation, a decrease in disease-related symptoms, an increase in the quality of life of the diseased individual, a reduction in the dosage of other medications required to treat the disease, and / or sustained survival of the individual.

[0084] "Anticancer" drugs can negatively affect cancer cells / tumors in a subject by, for example, promoting the killing of cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the occurrence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting the immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of the subject with cancer.

[0085] The term "antibody" is used herein in its broadest sense and includes, in particular, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity.

[0086] When used in the present invention, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. For example, individual antibodies within that population are identical except for possible mutations, such as naturally occurring mutations that may exist in small amounts. Thus, the modifier “monoclonal” indicates the characteristic of an antibody that is not a mixture of individual antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody containing a target-binding polypeptide sequence obtained by a process involving the selection of a single target polypeptide sequence derived from multiple polypeptide sequences. For example, the selection process may be the selection of a unique clone from multiple pools, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. The selected target-binding sequence can be further modified, for example, to improve affinity for the target, humanize the target-binding sequence, improve production in cell culture, reduce immunogenicity in vivo, or create a multispecific antibody, and antibodies containing the modified target-binding sequence should also be understood to be monoclonal antibodies of the present invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant of the antigen. In addition to its specificity, monoclonal antibody preparations are typically advantageous in that they are not contaminated by other immunoglobulins.

[0087] The term "CD122 / CD132 agonist" or "selective CD122 / CD132 agonist" refers to an activator that preferentially binds to the CD122 / CD132 receptor complex and has low affinity binding to the IL-2α receptor (CD25) or IL-15α receptor. Known selective CD122 / CD132 agonists include: IL2 / anti-IL2 monoclonal antibody immune complexes (e.g., see U.S. Patent Publication No. 20170183403A1, which is incorporated herein by reference in its entirety); recombinant IL-2 mutein having a modified amino acid sequence compared to wild-type IL-2 (e.g., see U.S. Patent Publication No. 2017 / 0044229A1, which is incorporated herein by reference in its entirety); recombinant IL-2 mutein having a modified amino acid sequence compared to wild-type IL-2 in combination with anti-IL2 monoclonal antibody immune complexes (e.g., see International Publication No. 2014100014A1, which is incorporated herein by reference in its entirety); and PEGylated forms of IL-2 such as NKTR-214 (e.g., see Charych et al., which are incorporated herein by reference in its entirety IL-15 / anti-IL-15 monoclonal antibody immune complex (see al., 2016); IL15 / IL15 receptor α-IgG1-Fc (IL15 / IL15Rα-IgG1-Fc) immune complex (see, for example, U.S. Patent Application Publication 20060257361A1, European Patent Application Publication 2724728A1, and Dubois et al., 2008, all of which are incorporated herein by reference); recombinant IL-15 mutein having a modified amino acid sequence compared to wild-type IL-15 in combination with the IL15Rα-IgG1-Fc immune complex (see, for example, U.S. Patent Application Publication 20070160578, the entirety of which is incorporated herein by reference); or a PEGylated form of IL-15 that preferentially binds to CD122 / CD132.

[0088] The term "immune checkpoint" refers to molecules, such as proteins, within the immune system that provide inhibitory signals to components of the immune response in order to balance it. Known immune checkpoint proteins include CTLA-4, PD-1, and their ligands PD-L1 and PD-L2, as well as LAG-3, BTLA, B7H3, B7H4, TIM3, and KIR. It is recognized in the art that pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR constitute immune checkpoint pathways similar to the CTLA-4 and PD-1-dependent pathway (see, for example, Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489).

[0089] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction between a PD-1 axis-binding partner and one or more of its binding partners, thereby restoring or improving T cell function (e.g., proliferation, cytokine production, target cell killing) while eliminating T cell dysfunction resulting from signal transduction along the PD-1 signaling axis. As used herein, PD-1 axis-binding antagonists may include PD-1 binding antagonists, PD-L1 binding antagonists, or PD-L2 binding antagonists.

[0090] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signal transduction resulting from the interaction of PD-1 with one or more binding partners, such as PD-L1 and / or PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more binding partners. In certain embodiments, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. Examples of PD-1 binding antagonists include anti-PD-1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1-binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed in T lymphocyte-mediated signaling via PD-1, thereby reducing the dysfunction of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-1-binding antagonist is an anti-PD-1 antibody. In certain embodiments, the PD-1-binding antagonist is MDX-1106 (nivolumab). In certain embodiments, the PD-1-binding antagonist is MK-3475 (pembrolizumab). In another specific embodiment, the PD-1-binding antagonist is CT-011 (pidilizumab). In yet another specific embodiment, the PD-1-binding antagonist is AMP-224.

[0091] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with the signal transduction resulting from the interaction of PD-L1 with any one or more of its binding partners, such as PD-1 or B7-1. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific embodiment, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, examples of PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with the signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed in T lymphocyte-mediated signal transduction via PD-L1 such that it reduces the dysfunctionality of dysfunctional T cells (e.g., enhances the effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular embodiment, the anti-PD-L1 antibody is YW243.55.S70. In another particular embodiment, the anti-PD-L1 antibody is MDX-1105. In yet another particular embodiment, the anti-PD-L1 antibody is MPDL3280A. In yet another particular embodiment, the anti-PD-L1 antibody is MEDI4736.

[0092] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with the signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific embodiment, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, the PD-L2 antagonist includes anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with the signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed in T lymphocyte-mediated signal transduction via PD-L2 so as to reduce the dysfunctionality of dysfunctional T cells (e.g., enhance the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0093] The term "immune checkpoint inhibitor" means any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduction of function and complete blockade. Specifically, the immune checkpoint protein is a human immune checkpoint protein. Thus, an immune checkpoint protein inhibitor is specifically an inhibitor of a human immune checkpoint protein.

[0094] The term "extracellular matrix-degrading protein" or "extracellular matrix-degrading protein" means any protein that acts on the integrity of the cell matrix, particularly exerting an overall or partial degradation or destabilizing effect on at least one of the components of the matrix or on the binding that integrates these various components.

[0095] In this specification, "abscopal effect" refers to the shrinkage of a tumor outside the scope of localized treatment. For example, local treatment with p53 and / or IL-24, combined with systemic treatment with immune checkpoint therapy, can induce an abscopal effect in distant, untreated tumors.

[0096] II. Cancer suppressors In some embodiments, subjects are administered cancer suppression therapies, such as p53 and / or MDA-7 therapies. Nucleic acids encoding p53 and / or MDA-7 can be provided in various ways known in the art.

[0097] In some embodiments, p53 and MDA-7 tumor suppressor therapies incorporate nucleic acid variants to enhance their activity. In certain embodiments, the variant tumor suppressor nucleic acid is a p53 variant resistant to negative regulation (Yun et al., 2012; the whole is incorporated herein by reference).

[0098] A.p53 In certain embodiments, this disclosure provides combination therapies for the treatment of cancer. Some of the combination therapies provided herein include p53 gene therapies, which involve administering the wild-type p53 gene. Wild-type p53 is recognized as an important growth regulator in many cell types. The p53 gene encodes a 375-amino acid phosphoprotein capable of forming complexes with host proteins such as the large T antigen and E1B. The protein is found in normal tissues and cells, but at much lower concentrations compared to transformed cells or tumor tissue.

[0099] Missense mutations are common for the p53 gene and are essential for its ability to transform as an oncogene. A single gene change promoted by a point mutation can create the oncogenic p53. However, unlike other oncogenes, p53 point mutations are known to occur at at least 30 different codons and often create dominant alleles that produce a shift in cellular phenotype without reduction for homozygotes. Furthermore, many of these dominant-negative alleles appear to be resistant in organisms and pass through the germline. A variety of mutant alleles appear to range from those with minimal dysfunction to potent, pervasive dominant-negative alleles (Weinberg, 1991). Large amounts of mutant p53 have been found in many cells transformed by chemical carcinogens, ultraviolet light, and several viruses.

[0100] In some embodiments, p53 biomarkers are used to select patients for p53 therapy. In certain embodiments, a preferred tumor p53 biomarker profile is defined by either a wild-type p53 gene composition or less than 20% p53-positive cells by immunohistochemistry (U.S. Patent No. 9,746,471 and Nemunaitis et al., 2009; both incorporated in whole by reference).

[0101] B. MDA-7 The combination therapies provided herein may further include MDA-7 gene therapies, which may involve administering a full-length or truncated MDA-7 gene. Interleukin (IL)-24, a protein product of the mda-7 gene, is a cytokine belonging to the IL-10 family of cytokines and is also a tumor suppressor. The cDNA encoding the MDA-7 protein was described by Jiang et al., 1995 (International Publication No. 1995011986). The MDA-7 cDNA encodes an evolutionarily conserved protein of 206 amino acids with a predicted size of 23.8 kDa.

[0102] The nucleic acids encoding MDA-7 provided herein may encode full-length or cleaved human IL-24 protein or polypeptide. A cleaved MDA-7 may contain some or more portions of a contiguous amino acid region of the full-length sequence, but not the entire sequence. A cleaved version can be cleaved at any number of consecutive amino acids at any site in the polypeptide. For example, a cleaved MDA-7 may encode approximately 49–206; approximately 75–206; approximately 100–206; approximately 125–206; approximately 150–206; approximately 175–206; or approximately 182–206 amino acids of human wild-type MDA-7. It is also conceivable that MDA-7 polypeptides containing at least approximately 85%, 90%, and 95% of human wild-type MDA-7 are within the scope of the present invention.

[0103] C. Other tumor suppressors Additional tumor suppressor factors may be used in this disclosure. Examples of gene therapy vectors for use in this disclosure to incorporate other tumor suppressor genes are listed in Table 1, but are not limited to these. [Table 1]

[0104] III. Extracellular Matrix Degradation Methods for enhancing the antitumor effects of tumor suppressor gene therapies and / or immune checkpoint inhibitors are also provided herein. In one embodiment, the delivery and tumor penetration of a gene therapy (e.g., viral distribution) are enhanced by a protein or activator that degrades the extracellular matrix (ECM) or complement of tumor cells.

[0105] The extracellular matrix (ECM) is a collection of extracellular molecules secreted by cells that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of the ECM differs among multicellular constructs. However, cell adhesion, cell-to-cell communication, and differentiation are common functions of the ECM. Components of the ECM that can be targeted by extracellular matrix-degrading proteins include collagen, elastin, hyaluronic acid, fibronectin, and laminin.

[0106] A. Relaxin One extracellular matrix-degrading protein usable in the methods provided herein is relaxin. Relaxin is a 6 kDa peptide hormone structurally related to insulin and insulin-like growth factor. Relaxin is mainly produced in the corpus luteum and endometrium, and its serum levels increase significantly during pregnancy (Sherwood et al., 1984). Relaxin is a potent inhibitor of collagen expression when collagen is overexpressed, but unlike other collagens, it does not significantly alter the basal level of collagen expression. Relaxin degrades collagen by promoting the expression of various MMPs such as MMP2, MMP3, and MMP9, leading to the degradation of connective tissue and basement membranes and the disruption of the extracellular matrix of the birth canal. In addition, the promotion of MMP1 and MMP3 expression by relaxin has also been observed in the lungs, heart, skin, intestines, mammary glands, blood vessels, and vas deferens, where relaxin acts as an inhibitor, preventing collagen overexpression (Qin, X., et al., 1997a; Qin, X., et al., 1997b).

[0107] Administering relaxin protein, or nucleic acids encoding relaxin protein, can induce the degradation of collagen, a major component of the extracellular matrix surrounding tumor cells, thereby disrupting connective tissue and the basement membrane, and leading to the degradation of the extracellular matrix. In particular, when administered to tumor tissue tightly surrounded by connective tissue, the administration of tumor suppressor gene therapy in combination with relaxin shows improved antitumor efficacy.

[0108] Relaxin proteins can be full-length relaxin or a portion of a relaxin molecule that retains biological activity, as described in U.S. Patent No. 5,023,321. Specifically, relaxin is recombinant human relaxin (H2), such as an agent that competitively replaces bound relaxin from a receptor, or other activators having relaxin-like activity. Relaxin can preferably be prepared by any method known to those skilled in the art, as described in U.S. Patent No. 4,835,251. Relaxin analogs or derivatives thereof are described in U.S. Patent No. 5,811,395, and peptide synthesis is described in U.S. Patent Application Publication No. 20110039778.

[0109] An exemplary adenovirus relaxin available for use by the method provided herein is described by Kim et al. (2006). Briefly, by inserting the relaxin gene into the E3 adenovirus region, a relaxin-expressing and reproducible (Ad-ΔE1B-RLX) adenovirus is produced.

[0110] B. Hyaluronidase In some embodiments, any substance capable of hydrolyzing polysaccharides present in the extracellular matrix, such as hyaluronic acid, can be administered. Specifically, the extracellular matrix degrading protein used in the present invention can be hyaluronidase. Hyaluronan (or hyaluronic acid) is a constituent component present in a wide range of the extracellular matrix of vertebrates. This linear polysaccharide, based on glucuronic acid and glucosamine [D-glucuronic acid (1-β-3) N-acetyl-D-glucosamine (1-b-4)], can affect the physicochemical properties of the matrix due to its property of forming a very viscous solution. Hyaluronic acid also interacts with various receptors and binding proteins located on the cell surface. Hyaluronic acid is involved in a number of biological processes such as fertilization, embryonic development, cell migration and differentiation, wound healing, inflammation, tumor growth, and metastasis formation.

[0111] Hyaluronic acid is hydrolyzed by hyaluronidase, and hydrolysis results in the disassembly of the extracellular matrix. Thus, it is contemplated that any substance having hyaluronidase activity, such as hyaluronidase described in Kreil (Protein Sci., 1995, 4:1666-1669), is suitable for use in this method. Hyaluronidase can be a glycoside hydrolase that is a hyaluronate of mammals, reptiles, or Hymenoptera, a glycoside hydrolase that is a hyaluronate from the salivary gland of a hill, or a hyaluronidase derived from a lyase that is a hyaluronate of bacteria, particularly streptococci, pneumococci, and Clostridium perfringens. The enzyme activity of hyaluronidase can be evaluated by conventional techniques as described in Hynes and Ferretti (Methods Enzymol., 1994, 235:606-616) or Bailey and Levine (J.Pharm.Biomed.Anal., 1993, 11:285-292).

[0112] C. Decorin Decorin, a small, leucine-rich proteoglycan, is a ubiquitous component of the extracellular matrix and is primarily found in association with collagen fibrils. Decorin binds to collagen fibrils, delaying the horizontal assembly of individual triple-helix collagen molecules and resulting in a reduction in fibril diameter. Furthermore, decorin can modulate the interactions between cells and other extracellular matrix components, such as fibronectin and thrombospondin. In addition, decorin can influence the remodeling of the extracellular matrix by inducing collagenase, a matrix metalloproteinase. These observations suggest that, at several levels, decorin controls the production and assembly of the extracellular matrix and therefore plays a major role in the remodeling of connective tissue, as described by Choi et al. (Gene Therapy, 17:190-201, 2010) and Xu et al. (Gene Therapy, 22(3):31-40, 2015).

[0113] An exemplary adenovirus-type decorin available for use in the methods provided herein is described by Choi et al. (Gene Therapy, 17:190-201, 2010). Briefly, a replicable (Ad-ΔE1B-DCNG) adenovirus expressing decorin is produced by inserting the decorin gene into the E3 adenovirus region. Another adenovirus-type decorin available for use in the methods provided herein is described by Xu et al. (Gene Therapy, 22(3):31-40, 2015). Similarly, a replicable (Ad.dcn) adenovirus expressing decorin is produced by inserting the decorin gene into the E3 adenovirus region.

[0114] IV. Nucleic acids Nucleic acids can be produced by any technique known to those skilled in the art. Non-limited examples of synthetic nucleic acids, particularly synthetic oligonucleotides, include those produced by in vitro chemical synthesis using phosphotriester, phosphite, or phosphoramidite chemical phenomena and solid-phase techniques, as described in European Patent No. 266,032, or by deoxynucleoside H-phosphonate intermediates, as described in Froehler et al., 1986 and U.S. Patent No. 5,705,629. Non-limited examples of enzymatically produced nucleic acids include those produced by enzymes in amplification reactions such as PCR (see, for example, U.S. Patents No. 4,683,202 and 4,682,195), or by the synthesis of oligonucleotides, as described in U.S. Patent No. 5,645,897. Non-limited examples of biologically produced nucleic acids include recombinant nucleic acid production in living cells, such as recombinant DNA vector production in bacteria (see, for example, Sambrook et al., 1989).

[0115] Nucleic acids, regardless of their own sequence length, can be combined with other nucleic acid sequences, including but not limited to promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, and coding segments, to create one or more nucleic acid constructs. The total length may vary significantly between nucleic acid constructs. Therefore, while nucleic acid segments of almost any length can be used, the total length is preferably limited for ease of preparation or use in the intended recombinant nucleic acid protocol.

[0116] A. Nucleic acid delivery by expression vectors The vectors provided herein are primarily designed to express therapeutic tumor suppressor genes (e.g., p53 and / or MDA-7) and / or extracellular matrix-degrading genes (e.g., relaxin) under the control of regulated eukaryotic cell promoters (i.e., constitutional, stimulative, repressive, and tissue-specific). In some embodiments, p53 and MDA-7 can be expressed simultaneously within the vector. In other embodiments, p53 and / or MDA-7 can be expressed simultaneously with extracellular matrix-degrading genes. Where there is no other reason, the vectors may also contain a selection marker to facilitate in vitro manipulation.

[0117] Those skilled in the art will have ample equipment for constructing vectors using standard recombination techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Examples of vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), including retroviral vectors (e.g., derived from Moloney's mouse leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors including replicable, non-replicable, and gutless forms, adeno-associated virus (AAV) vectors, Simian virus 40 (SV-40) vectors, bovine papillomavirus, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey mouse sarcoma virus vectors, mouse mammary cancer virus vectors, and Rous sarcoma virus vectors.

[0118] 1. Viral vector Viral vectors encoding tumor suppressor factors and / or extracellular matrix-degrading genes can be provided in certain aspects of the present invention. In the production of recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences for heterologous (or non-natural) proteins. A viral vector is a type of expression construct that uses a viral sequence to introduce nucleic acids, and optionally proteins, into cells. Due to the ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and integrate into the host cell genome to express viral genes, these viruses are stably and efficiently made attractive candidates for transporting foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain aspects of the present invention are described below.

[0119] Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patent Nos. 6,013,516 and 5,994,136).

[0120] Recombinant lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses capable of infecting non-dividing cells (where a suitable host cell is transfected with two or more vectors having packaging function, namely gag, pol, and env, as well as rev and tat) are described in U.S. Patent No. 5,994,136, incorporated herein by reference.

[0121] a. Adenovirus vectors One method for delivering tumor suppressor factors and / or extracellular matrix-degrading genes involves the use of adenovirus expression vectors. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this characteristic is offset by the highly efficient gene transfer achieved by these vectors. Adenovirus expression vectors include constructs that (a) support the packaging of constructs and (b) contain sufficient adenovirus sequences to ultimately express the recombinant gene construct cloned therein.

[0122] Adenovirus replication and manipulation are known to those skilled in the art, and it exhibits a broad host range both in vitro and in vivo. This group of viruses can be obtained with high titers, e.g., 10⁹–10¹¹ plaque-forming units per mL, and these are highly infectious. The adenovirus life cycle does not require integration into the host cell genome. The exogenous gene delivered by the adenovirus vector is episomal and therefore has low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), which demonstrates the safety and therapeutic potential of this in vivo gene transfer vector.

[0123] Knowing the genetic makeup of adenoviruses, which are 36kb linear double-stranded DNA viruses, makes it possible to replace large adenovirus DNA with foreign sequences of up to 7kb (Grunhaus and Horwitz, 1992). In contrast to retroviruses, adenovirus DNA can replicate like an episome without potential genotoxicity, so infection of host cells with adenovirus does not result in chromosome alteration. Adenoviruses are also structurally stable, and no genomic transposition has been detected after large-scale amplification.

[0124] Adenoviruses are particularly suitable as gene transfer vectors due to their medium-sized genomes, ease of manipulation, high titer, broad range of target cells, and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units, separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for transcriptional regulation of the viral genome and a small number of cellular genes. Expression of the E2 region (E2A and E2B) leads to the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell blockade (Renan, 1990). The late gene products, which contain most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript derived from the major late promoter (MLP). The MLP (located at 16.8mu) is particularly effective during the later stages of infection, and all mRNA derived from this promoter has a 5' triplicate leader (TPL) sequence, which allows specific mRNAs to be translated.

[0125] The recombinant adenovirus provided herein can be generated by homologous recombination of a shuttle vector and a proviral vector. Wild-type adenovirus can be generated from this process by possible recombination of the two proviral vectors. Therefore, a single clone of the virus is isolated from individual plaques and its genomic structure is investigated.

[0126] Adenovirus vectors can be replicable, non-replicable, or conditionally non-replicable, and the properties of the adenovirus vector are not considered important for the successful implementation of the present invention. The adenovirus can be any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is a specific starting material for obtaining a conditionally non-replicable adenovirus vector for use in the present invention. This is because adenovirus type 5 is a human adenovirus for which much biochemical and genetic information is known, and it has historically been used in the most numerous constructs that use adenovirus as a vector.

[0127] Nucleic acids can be introduced into adenovirus vectors as a replacement for the coding sequence. For example, in a non-replicating adenovirus vector, the sequence encoding E1 may be removed. Polynucleotides encoding the target gene can also be inserted into E3 replacement vectors, either in place of a deleted E3 region or in the E4 region where a helper cell line or helper virus complements the E4 deletion, as described by Karlsson et al. (1986).

[0128] The generation and propagation of non-replicating adenovirus vectors can be carried out using helper cell lines. A unique helper cell line called 293, when transformed from human embryonic kidney cells with an Ad5 DNA fragment, structurally expresses the E1 protein (Graham et al., 1977). Since the E3 region is segregable from the adenovirus genome (Jones and Shenk, 1978), the adenovirus vector, with the assistance of 293 cells, delivers foreign DNA to either the E1 or E3 region, or both (Graham and Prevec, 1991).

[0129] Helper cell lines can be derived from human cells, such as human fetal kidney cells, muscle cells, hematopoietic cells, or other human embryonic mesenchymal or epithelial cells. Alternatively, helper cells can be derived from cells of other mammalian species that are tolerant to human adenovirus. Examples of such cells include Vero cells or other monkey embryonic mesenchymal or epithelial cells. As mentioned above, there are 293 specific helper cell lines.

[0130] Methods for producing recombinant adenoviruses, such as U.S. Patent No. 6740320, incorporated herein by reference, are known in the art. Racher et al. (1995) also disclose an improved method for culturing cells and growing adenoviruses. In one embodiment, an aggregate of native cells is grown by seeding individual cells in a silicone-coated spinner flask (Techne, Cambridge, UK) containing 1 liter of 100-200 mL of culture medium. After stirring at 40 rpm, cell viability is estimated using trypan blue. In another embodiment, Fibra-Cel microcarrier (Bibby Sterlin, Stone, UK) (5 g / L) is used as follows: Cell seed cells resuspended in 5 mL of medium are added to the carrier (50 mL) in a 250 mL Erlenmeyer flask and allowed to stand for 1-4 hours with appropriate stirring. The medium is then replaced with 50 mL of fresh medium, and shaking is initiated. To produce the virus, cells were grown to approximately 80% confluence, then the culture medium was changed (final volume 25%), and adenovirus was added at an MOI of 0.05. The culture medium was left to stand overnight, then the volume was increased to 100%, and shaking was started for another 72 hours.

[0131] b. Retroviral vectors Furthermore, tumor suppressor factors and / or extracellular matrix-degrading genes can be encoded by retroviral vectors. Retroviruses are a group of single-stranded RNA viruses characterized by their ability to convert cellular RNA into double-stranded DNA within infected cells through a reverse transcription process (Coffin, 1990). The resulting DNA is then stably integrated into the cell chromosome as a provirus, instructing the synthesis of viral proteins. Integration preserves the gene sequence in the recipient cell and its offspring. The retroviral genome contains three genes, gag, pol, and env, which encode capsid proteins, polymerase enzymes, and envelope components, respectively. The sequence found upstream of the gag gene contains a signal for packaging the genome into virions. Two long-terminal repeat (LTR) sequences are located at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also necessary for integration into the host cell genome (Coffin, 1990). To construct a retroviral vector, the nucleic acid encoding the target gene is inserted into the viral genome in place of a specific viral sequence, creating a virus that cannot replicate.

[0132] To create virions, packaging cells containing the gag, pol, and env genes but without the LTR and packaging components are constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is incorporated into this cell line along with the retroviral LTR and packaging cells (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged within the viral particle, which is then secreted into the cell medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retrovirus is then collected and optionally concentrated for use in gene transfer. Retroviral vectors can infect a wide range of cell types. However, host cell division is required for transfer and stable expression (Paskind et al., 1975).

[0133] A concern with using deficient retroviral vectors is the potential emergence of wild-type, replicable viruses within the packaging cells. This can occur due to a recombination event in which intact sequences from the recombinant virus are inserted upstream of gag, pol, and env sequences integrated into the host cell genome. However, packaging cell lines are available that should significantly reduce the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

[0134] c. Adeno-associated virus vectors Adeno-associated viruses (AAVs) are an attractive vector system for use in this disclosure due to their high inclusion rate and their ability to infect non-dividing cells, making them useful for delivering genes to mammalian cells (Muzyczka, 1992). AAVs are infectable to a wide range of hosts (Tratschin, et al., 1984; Laughlin, et al., 1986; Lebkowski, et al., 1988; McLaughlin, et al., 1988), which means that AAVs are suitable for use in the present invention. Details regarding the production and use of rAAV vectors are described in U.S. Patents No. 5,139,941 and No. 4,797,368. AAV is a dependent parvovirus in that it requires co-infection with another virus (either an adenovirus or an element of the herpesvirus family) and subsequent proliferation in cultured cells (Muzyczka, 1992).

[0135] In the absence of co-infection with a helper virus, the wild-type AAV genome is integrated into human chromosome 19, where it exists potentially as a provirus, through its terminals (Kotin et al., 1990; Samulski et al., 1991). However, unless the AAV Rep protein is also expressed, rAAV is not limited to chromosome 19 in terms of integration (Shelling and Smith, 1994). When cells carrying the AAV provirus are co-infected with a helper virus, the AAV genome is "rescued" from the chromosome or recombinant plasmid, and normal replication and infection are established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990; Muzyczka, 1992).

[0136] Typically, recombinant AAV (rAAV) viruses are produced by co-transfection of a plasmid containing a target gene with two AAV terminal repeats adjacent to each other (McLaughlin et al., 1988; Samulski et al., 1989; each incorporated herein by reference) with an expression plasmid containing a wild-type AAV coding sequence without terminal repeats, such as pIM45 (McCarty et al., 1991). Cells are also infected with or transfected with an adenovirus or plasmid containing the adenovirus genes necessary for AAV helper function. The stock of rAAV viruses thus produced is then contaminated with an adenovirus that must be physically isolated from rAAV particles (e.g., by a cesium chloride density gradient). Alternatively, an adenovirus vector containing the AAV coding region, or a cell line containing the AAV coding region and some or all of the adenovirus helper genes, can be used (Yang et al., 1994; Clark et al., 1995). Cell lines containing rAAV DNA as an incorporated provirus can also be used (Flotte et al., 1995).

[0137] d. Other viral vectors In this disclosure, other viral vectors can be used as constructs. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) and herpesvirus can be used. They offer several attractive properties for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0138] Molecularly cloned strains of the Venezuelan encephalitis (VEE) virus have been genetically refined as replicable vaccine vectors for the expression of heterologous viral proteins (Davis et al., 1996). Studies have shown that VEE infection stimulates a potential CTL response, suggesting that VEE may be very useful as a vector for vaccination (Caley et al., 1997).

[0139] In further embodiments, the nucleic acid encoding chimeric CD154 is contained within an infectious virus modified to express a specific binding ligand. Thus, the viral particle specifically binds to an allogeneic receptor on a target cell, delivering its contents to the cell. A novel approach has recently been developed that is designed to specifically target retroviral vectors based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification allows for specific infection of hepatocytes via the sialycoprotein receptor.

[0140] For example, targeting of recombinant retroviruses was designed using biotinylated antibodies against retroviral envelope proteins and specific cell receptors. Antibodies were conjugated via biotin components using streptavidin (Roux et al., 1989). Antibodies against major histocompatibility complex class I and class II antigens demonstrated in vitro infection of various human cells possessing these surface antigens with allotropic viruses (Roux et al., 1989).

[0141] 2. Regulatory factors The expression cassettes contained in the vectors useful in this disclosure specifically include a eukaryotic cell transcription promoter responsively bound to a protein-coding sequence (5' to 3' direction), a splice signal including an intervening sequence, and a transcription termination / polyadenylation sequence. Promoter and enhancer that regulate the transcription of gene-coding proteins in eukaryotic cells are composed of multiple genetic elements. Cellular mechanisms can collect and integrate the regulatory information carried by each element, thereby enabling different genes to evolve different, often complex, transcriptional regulatory patterns. Promoter types used in the context of the present invention include constitutional, inducible, and tissue-specific promoters.

[0142] a. Promoter / Enhancer The expression constructs provided herein include promoters that drive the expression of tumor suppressor and / or extracellular matrix-degrading genes. Promoters generally contain sequences that function to locate the start site of RNA synthesis. The most well-known example is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the SV40 late gene, separate factors overlapping their own start site help to fix the start site. Additional promoter factors control the frequency of transcription initiation. Typically, these are located in a region 30-110 bp upstream of the start site, although many promoters have been shown to also contain functional factors in the granules of the start site. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcription reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoded RNA. Because the spacing between promoter factors is often flexible, promoter function is preserved when factors invert or move relative to one another.

[0143] In the TK promoter, the spacing between promoter factors can be increased to 50 bp before activity begins to decline. Depending on the promoter, the individual factors appear to function either cooperatively or independently to activate transcription. Promoters may or may not be used with "enhancers," which implies the cis action of regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0144] A promoter may be naturally associated with the nucleic acid sequence, obtainable by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be called “endogenous.” Similarly, an enhancer may be naturally associated with the nucleic acid sequence, located either upstream or downstream of that sequence. Alternatively, certain advantages can be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which means a promoter that is not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancer also means an enhancer that is not normally associated with the nucleic acid sequence in its natural environment. Examples of such promoters or enhancers include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are not “naturally present,” i.e., those containing different factors and / or mutations that alter the expression of different transcription factor regions. For example, the most commonly used promoters in recombinant DNA constructs include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthesizing promoter and enhancer nucleic acid sequences, the sequences can also be prepared using recombinant cloning and / or nucleic acid amplification techniques, including PCR®, in conjunction with the disclosed compositions (see, for example, U.S. Patents 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, it has been conceivable that regulatory sequences directing the direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria and chloroplasts can also be used.

[0145] Ideally, it is important to use promoters and / or enhancers that efficiently direct the expression of a DNA segment within a selected organelle, cell type, tissue, organelle, or organism for expression. Those skilled in molecular biology generally understand the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter used can be constitutional, tissue-specific, and inducible, and / or useful, under conditions suitable for directing the high expression of the introduced DNA segment, which is advantageous, for example, in large-scale recombinant protein and / or peptide production. Promoter can be heterogeneous or endogenous.

[0146] Furthermore, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Database EPDB, via the World Wide Web at epd.isb-sib.ch / ) can also be used to drive expression. Using T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from specific bacterial promoters if the appropriate bacterial polymerase is provided either as part of a delivery complex or as an additional gene expression construct.

[0147] Non-limiting examples of promoters include early or late viral promoters, e.g., SV40 early or late promoter, cytomegalovirus (CMV) pre-early promoter, Rous sarcoma virus (RSV) early promoter; eukaryotic cell promoters, e.g., β-actin promoter (Ng, 1989; Quitsche et al., 1989), GADPH promoter (Alexander et al., 1988, Ercolani et al., 1988), metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and linkage response factor promoters near the smallest TATA box, e.g., cyclic AMP response factor promoter (cre), serum response factor promoter (sre), phorbol ester promoter (TPA), and response factor promoter (tre). It is also possible to use a human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank deposit number X05244, nucleotide 283-341) or a mouse mammary cancer promoter (available from ATCC, catalog number ATCC 45007). In certain embodiments, the promoter is CMV IE, Dectin 1, Dectin 2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I or MHC class II promoter, but any other promoter useful for driving the expression of p53, MDA-7, and / or relaxin genes is applicable to carrying out the present invention.

[0148] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the potential to act over relatively long distances (up to several kilobases from the target promoter), regardless of cis and direction. However, the function of the enhancer is not necessarily limited to long distances such that it can function even when very close to a given promoter.

[0149] b. Initiation signals and related expression Specific start signals can also be used in the expression constructs provided in this disclosure for efficient translation of coding sequences. These signals include an ATG start codon or an adjacent sequence. It may be necessary to provide an exogenous translational regulatory signal, including an ATG start codon. Those skilled in the art will be able to quickly measure this and provide the required signal. It is well known that the start codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational regulatory signal and start codon can be either natural or synthetic. Expression efficiency can be improved by including appropriate transcriptional enhancers.

[0150] In certain embodiments, multiple genetic or polycistronic signaling codes can be constructed using internal ribosome entry site (IRES) factors. IRES factors can bypass the ribosome scanning model of 5'-methylated cap-dependent translation and initiate translation at an internal location (Pelletier and Sonenberg, 1988). IRES factors derived from two elements of the picornavirus family (polio and encephalomyocarditis) (Pelletier and Sonenberg, 1988) and IRESs derived from mammalian signaling codes (Macejak and Sarnow, 1991) have been described. IRES factors can be ligated to heterologous open reading frames. Multiple open reading frames, each separated by an IRES, can be transcribed to each other to create polycistronic signaling information. Due to the IRES factors, each open reading frame can approach the ribosome for efficient translation. A single promoter / enhancer can be used to efficiently express multiple genes and transcribe a single signaling signal (see U.S. Patents 5,925,565 and 5,935,819, respectively, incorporated herein by reference).

[0151] Furthermore, specific 2A sequence factors can be used to generate associated or co-expression of genes in the constructs provided in this disclosure. For example, co-expression of genes can be achieved by using cleavage sequences to ligate open reading frames to form a single cistron. Exemplary cleavage sequences include F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A) (Minskaia and Ryan, 2013).

[0152] c. Origin of replication To propagate the vector within a host cell, the host cell may contain one or more origin sites (often called "ori"), such as the oriP of EBV described above, which is a nucleic acid sequence specific to the site where replication is initiated, or a recombinant oriP corresponding to a similar or improved programming function. Alternatively, the origins of replication or autonomous replication sequences (ARS) of other extrachromosomal replicating viruses described above may be used.

[0153] 3. Selectable and screenable markers In some embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers confer a identifiable change to the cells, facilitating the identification of cells containing the expression vector. Generally, selection markers are markers that confer a property that enables selection. Positive selection markers are markers whose presence enables selection, while negative selection markers are markers whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0154] Typically, including drug selection markers assists in the cloning and identification of transformed cells, e.g., genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, and zeosin, with histidinol being a useful selection marker. Depending on the introduced conditions, in addition to markers that confer phenotypes enabling the identification of transformed cells, other types of screenable markers, such as GFP, which are colorimetric, can also be conceived. Alternatively, enzymes that can be screened as positive selection markers, such as herpes simplex virus-derived thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), can be utilized. Those skilled in the art will also be familiar with the know-how of using immunomarkers, sometimes in conjunction with FACS analysis. The marker used is not considered important as long as it is possible to simultaneously express the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0155] B. Other methods of nucleic acid delivery In addition to viral delivery of nucleic acids encoding tumor suppressor factors and / or extracellular matrix-degrading genes, the following are further methods for delivering recombinant genes to a given host cell and are therefore considered to be within the scope of this disclosure.

[0156] For the introduction of nucleic acids such as DNA or RNA, any suitable method for delivering nucleic acids to transform cells, as described herein or known to those skilled in the art, may be used. Such methods include direct delivery of DNA by ex vivotransfection (Wilson et al., 1989, Nabel et al., 1989); injection (US Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each incorporated herein by reference), including microinjection (US Patent No. 5,384,253, Tur-Kaspa et al., incorporated herein by reference); and electroporation (US Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al.). al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using polyethylene glycol after DEAE-dextran (Gopal, 1985); direct ultrasonic loading (Fechheimer et al., 1987); liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al.By means of transfection (Wu and Wu, 1987; Wu and Wu, 1988); by means of micro-jet bombardment (PCT International Publication Nos. 94 / 09699 and 95 / 06128; U.S. Patents Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, each incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al. Examples include, but are not limited to, methods such as those by (al., 1990; U.S. Patents No. 5,302,523 and No. 5,464,765, respectively, incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Patents No. 5,591,616 and No. 5,563,055, respectively, incorporated herein by reference); by desiccation / inhibition-mediated DNA incorporation (Potrykus et al., 1985); and any combination of such methods. By applying these techniques, organelles, cells, tissues, or living organisms can be transformed stably or transiently.

[0157] 1. Electroporation In certain specific embodiments of this disclosure, a gene construct is introduced into target overgrowth cells by electroporation. Electroporation involves exposing cells (or tissues) and DNA (or DNA complexes) to a high-voltage discharge.

[0158] Transfection of eukaryotic cells using electroporation has been extremely successful. Using this method, mouse pre-B lymphocytes have been transfected with the human κ-immunoglobulin gene (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0159] It has been conceived that electroporation conditions can be optimized for overgrowth cells from different sources. One might particularly desire to optimize parameters such as voltage, capacitance, time, and the composition of the electroporation medium. Other routine adjustments will be known to those skilled in the art; see, for example, Hoffman, 1999; Heller et al., 1996.

[0160] 2. Lipid-mediated transformation In further embodiments, tumor suppressor factors and / or extracellular matrix-degrading genes can be trapped in liposomes or lipid formulations. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an aqueous medium inside. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. These form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo auto-reorganization before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Gene constructs complexed with lipofectamine (Gibco BRL) have also been conceived.

[0161] Lipid-mediated delivery of nucleic acids and in vitro expression of foreign DNA have proven very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated the potential of lipid-mediated delivery and expression of foreign DNA in cultured chicken embryos, Healer cells, and liver cancer cells.

[0162] Lipid-based nonviral formulations offer an alternative to adenovirus gene therapy. While numerous cell culture studies document lipid-based nonviral gene delivery, systemic gene delivery with lipid-based formulations remains limited. The primary limitation in nonviral lipid-based gene delivery is the toxicity of cationic lipids, including the nonviral delivery vehicle. The in vivo toxicity of liposomes, in particular, explains the discrepancy in in vitro and in vivo gene delivery outcomes. Another factor contributing to this conflicting data is the difference in the stability of the lipid vehicle with and without serum proteins. The interaction between the lipid vehicle and serum proteins has a dramatic effect on the stability properties of the lipid vehicle (Yang and Huang, 1997). Cationic lipids attract and bind to negatively charged serum proteins. Lipid vehicles associated with serum proteins are either lysed by macrophages or taken up by macrophages, thereby being removed from the bloodstream. Current in vivo lipid delivery methods utilize subcutaneous, intradermal, intratumor, or intracranial injections to avoid toxicity and stability issues associated with cationic lipids in the bloodstream. Interactions between lipid vehicles and plasma proteins are the cause of the discrepancy between in vitro efficiency (Felgner et al., 1987) and in vivo gene transfer (Zhu el al., 1993; Philip et al., 1993; Solodin et al., 1995; Liu et al., 1995; Thierry et al., 1995; Tsukamoto et al., 1995; Aksentijevich et al., 1996).

[0163] Advances in lipid formulations have improved the efficiency of in vivo gene transfer (Templeton et al. 1997; International Publication No. 98 / 07408). A novel lipid formulation composed of equimolar ratios of 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP) and cholesterol significantly improves in vivo systemic gene transfer by approximately 150 times. DOTAP: The cholesterol lipid formulation forms a unique structure called a "sandwich liposome." This formulation has been reported to "sandwich" DNA between invaginated bilayers, or "cup" structures. Beneficial features of these lipid structures include positive ρ, colloidal stabilization by cholesterol, two-dimensional DNA packing, and increased serum stability. Patent applications No. 60 / 135,818 and 60 / 133,116 discuss formulations that can be used in conjunction with the present invention.

[0164] The production of lipid formulations is often achieved by (I) reverse-phase evaporation, (II) dehydration and rehydration, (III) dialysis with detergent, and (IV) thin-film hydration, followed by ultrasonic treatment or continuous extrusion of the liposome mixture. Once produced, the lipid structure can be used to encapsulate toxic (chemotherapeutic) or unstable (nucleic acid) compounds when they are in blood circulation. Lipid encapsulation results in low toxicity and a long serum half-life for such compounds (Gabizon et al., 1990). Lipid-based gene transfer methods are used in the treatment of numerous diseases, particularly in the treatment of hyperproliferative disorders, to improve conventional therapies or establish novel therapies.

[0165] V. Selective CD123 / CD132 agonists In certain embodiments, subjects are administered at least one CD122 / CD132 agonist, such as a CD122 / CD132 agonist that selectively binds to the CD122 / CD132 receptor complex and has low affinity binding to the CD25 or IL15α receptor. CD122 / CD132 can be selected from recombinant IL-22 mutein having a modified amino acid sequence compared to wild-type IL2 (U.S. Patent Application Publication 2017 / 0044229; the whole is incorporated by reference). In certain embodiments, selective CD122 / CD132 agonists have modified amino acid sequences compared to IL-2 / anti-IL-2 monoclonal antibody immune complexes (US Patent Publication No. 20170183403A1; the whole is incorporated by reference), or recombinant IL-2 mutein (International Publication No. 2014100014A1; the whole is incorporated by reference), such as NKTR-214, PEGylated forms of IL2 (Charych et al., 2016), IL15 / anti-IL15 monoclonal antibody immune complexes, IL15 / IL15 receptor α-IgG1-Fc (IL15 / IL15Rα-IgG1-Fc) immune complexes (US Patent Publication No. 20060257361A1, European Patent Publication No. 2724728A1, and Dubois et al. (al., 2008), recombinant IL-15 mutein having a modified amino acid sequence compared to wild-type IL-15 combined with the IL15Rα-IgG1-Fc immune complex (U.S. Patent Application Publication No. 20070160578; the entire application is incorporated herein), or a PEGylated form of IL-15 that selectively binds to CD122 / CD132. In some embodiments, two or more CD122 / CD132 agonists are utilized.

[0166] VI. Tumor-lysing viruses In some embodiments, the disclosure includes administering at least one tumor lysis virus. In some embodiments, the tumor lysis virus is modified to express p53, MDA-7, IL-12, a TGF-β inhibitor, ADP, and / or an IL-10 inhibitor. In certain embodiments, the tumor lysis virus is a single-stranded or double-stranded DNA virus, an RNA virus, an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus, a herpesvirus, a poxvirus, a vaccinia virus, a vesicular stomatitis virus, a poliovirus, a Newcastle disease virus, an Epstein-Barr virus, an influenza virus, a reovirus, a myxoma virus, a marabavirus, a rhabdovirus, an enadenochusileb, or a coxsackievirus. In some embodiments, the tumor lysis virus is modified to express granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-12. In some embodiments, the tumor lysis virus is further defined as talimogene laharpalebeck (T-VEC). In some embodiments, the tumor lysis adenovirus vector is derived from a modified TERT promoter tumor lysis adenovirus (U.S. Patent No. 8,067,567; the whole is incorporated herein by reference), and / or an HRE-E2F-TERT hybrid promoter tumor lysis adenovirus (PCT / KR2011 / 004693; the whole is incorporated herein by reference), and / or an adenovirus having a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is deficient by an Elb-19K clone insertion site (European Patent Application Publication No. 2403951A2; the whole is incorporated herein by reference), all of which can be modified to express therapeutic genes. In some embodiments, tumor-lysis adenovirus vectors are derived from E1b-deficient tumor-lysis adenovirus (Yu and Fang, 2007; Li, 2009; both of these are incorporated by reference).

[0167] Examples of tumor lysis viruses include, but are not limited to, adenoviruses that overexpress adenovirus death protein (ADP), as described in U.S. Patent No. 7,589,069,B1 (which is incorporated in its entirety), such as Ad5-yCD / mutTKSR39rep-hIL12, Cavatak®, CG0070, DNX-2401, G207, HF10, IMLYGIC®, JX-594, MG1-MA3, MV-NIS, OBP-301, Reolysin®, Toca 511, Oncorine, RIGVIR, and VirRx007, and N1L-deficient vaccinia viruses expressing IL12, as described in PCT / GB2015 / 051023 (which is incorporated in its entirety). Other exemplary tumor lysis viruses are described, for example, in International Publication Nos. 2015 / 027163, 2014 / 138314, 2014 / 047350, and 2016 / 009017, all of which are incorporated herein by reference.

[0168] In a particular embodiment, the tumor lysis virus agent is talimogene laharpalebeck (T-VEC), a tumor lysis herpes simplex virus genetically modified to express GM-CSF. T-VEC is HSV-1[strain JS1]ICP34.5- / ICP47- / hGM-CSF(OncoVEX GM CSFPreviously known as) is an oncolytic immunotherapy delivered intratumorally that includes an immunostimulatory HSV-1 that selectively replicates in solid tumors. (Lui et al., 2003; U.S. Patent Nos. 7,223,593 and 7,537,924; incorporated herein by reference.) In October 2015, the U.S. FDA approved T-VEC under the trade name IMLYGIC™ for the treatment of melanoma in patients with inoperable tumors. The characteristics and administration methods of T-VEC are described, for example, in the package insert of IMLYGIC™ (Amgen, 2015) and U.S. Patent Application Publication No. 2015 / 0202290 (both incorporated herein by reference). For example, talimogene laherparepvec is typically administered on Day 1 of Week 1 at a dose of up to 4.0 mL of 10 6 plaque-forming units / mL (PFU / mL), followed by Day 1 of Week 4 and then every other week (±3 days) at a dose of up to 4.0 mL of 10 8 PFU / mL by intratumoral injection into injectable skin, subcutaneous, and nodular tumors. The recommended volume of talimogene laherparepvec injected into the tumor depends on the tumor size and must be determined according to the injection volume guidelines. Although T-VEC has shown clinical activity in melanoma patients, many cancer patients either do not respond to T-VEC treatment or end their response to the treatment. In one embodiment, p53 and / or MDA-7 nucleic acids, and at least one CD122 / CD132 agonist can be administered after, during, or before T-VEC treatment to, for example, reverse treatment resistance.

[0169] In some embodiments, an E1b-deficient tumor-lysis adenovirus is combined with at least one selective CD122 / CD132 agonist and at least one immune checkpoint inhibitor. Exemplary E1b-deficient tumor-lysis adenoviruses include H101 (Oncorine), Onyx 015, or H103, which express heat shock protein 70 (HSP70), or tumor-lysis adenovirus H102, in which Ad E1a gene expression is driven by an α-fetoprotein (AFP) promoter, resulting in selective replication in hepatocellular carcinoma and other AFP-overexpressing cancers compared to normal cells (Yu and Fang, 2007; Li, 2009; both of these are incorporated by reference).

[0170] Further exemplary CD122 / CD132 agonists for use in the present invention include, but are not limited to, the active substances listed in Table 2 below. [Table 2-1] [Table 2-2]

[0171] VII. Immune checkpoint inhibitors In certain embodiments, the disclosure provides a method for combining immune checkpoint blockade with tumor suppressor gene therapy, such as p53 and / or MDA-7 gene therapy. Immune checkpoints are molecules in the immune system (e.g., co-stimulatory molecules) that either increase or decrease signaling. Inhibitory immune checkpoints that may be targeted by blockade of checkpoint molecules include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activator gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and the V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0172] Immune checkpoint inhibitors may be drugs such as small recombinant forms of ligands or receptors, or in particular antibodies, such as human antibodies (e.g., International Publication No. 2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogues thereof may be used, and in particular, chimeric, humanized, or human forms of antibodies may be used. As those skilled in the art will know, alternative and / or equivalent names may be used for certain antibodies described herein. Such alternative and / or equivalent names are interchangeable within the scope of the invention. For example, lambrolizumab is known to be also known by alternative and equivalent names MK-3475 and pembrolizumab.

[0173] It has been conceived that any of the immune checkpoint inhibitors known in the art that stimulate an immune response may be used. These include inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. These immune checkpoint inhibitors include, but are not limited to, agents that target immune checkpoint proteins and pathways involved in PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, known LAG3 inhibitors in the art include soluble LAG3 (IMP321 or LAG3-Ig, disclosed in International Publication No. 2009044273), as well as mouse or humanized antibodies that block human LAG3 (e.g., IMP701, disclosed in International Publication No. 2008132601), or fully human antibodies that block human LAG3 (such as those disclosed in European Patent No. 2320940). Another example is provided by using an inhibitor against BTLA, including, but not limited to, an antibody that blocks the interaction of human BTLA with its ligand (such as 4C7 disclosed in International Publication No. 2011014438). Yet another example is provided by using an agent that neutralizes B7H4, including, but not limited to, an antibody against human B7H4 (disclosed in International Publication Nos. 2013025779 and 2013067492), or a soluble recombinant form of B7H4 (such as those disclosed in U.S. Patent Application Publication No. 20120177645). Yet another example is provided by an agent that neutralizes human B7-H3, including, but not limited to, an antibody that neutralizes human B7-H3 (for example, MGA271, disclosed as BRCA84D in U.S. Patent Application Publication No. 20120294796, and its derivatives). Another example is a TIM3-targeting agent, including but not limited to antibodies that target human TIM3 (e.g., those disclosed in International Publication No. 2013006490A2, or anti-human TIM3 blocking antibody F38~2E2 disclosed by Jones et al., J Exp Med. 2008;205(12):2763-79).

[0174] Furthermore, two or more immune checkpoint inhibitors (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody) can be used in combination with tumor suppressor genes. For example, p53 gene therapy and immune checkpoint inhibitors (e.g., anti-KIR antibody and / or anti-PD-1 antibody) can be administered to enhance innate antitumor immunity, and then IL24 gene therapy and immune checkpoint inhibitors (e.g., anti-PD-1 antibody) can be administered to induce an adaptive antitumor immune response.

[0175] A. PD-1 axis antagonist T cell dysfunction or anergy occurs simultaneously with the induction and maintenance of the inhibitory receptor programmed cell death 1 polypeptide (PD-1). Therefore, therapeutic targeting of PD-1 and other molecules that signal through interaction with PD-1, such as programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2), is provided herein. PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern.Immun.2007 19(7):813). Therefore, for example, in tumor CD8 + This specification provides inhibition of PD-L1 / PD-1 interactions in combination with p53, ADP, and / or MDA-7 gene therapies to enhance T cell-mediated killing.

[0176] This specification provides a method for treating cancer or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist in combination with p53, ADP(VirRx007), and / or an MDA-7 gene therapy. Also provided herein is a method for enhancing immune function in an individual requiring enhanced immune function, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist and p53, ADP(VirRx007), and / or an MDA-7 gene therapy.

[0177] For example, PD-1 axis-coupled antagonists include PD-1-binding antagonists, PDL1-binding antagonists, and PDL2-binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0178] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In specific embodiments, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In specific embodiments, the PDL1 binding partner is PD-1 and / or B7-1. In yet another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, the PDL2 binding partner is PD-1. The antagonist can be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Publication Nos. 20140294898, 2014022021, and 20110008369 (all incorporated herein by reference).

[0179] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing an extracellular or PD-1 binding moiety of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in International Publication No. 2006 / 121168. Pembrolizumab is MK-3475, Merck 3475, also known as lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in International Publication No. 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in International Publication No. 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in International Publication Nos. 2010 / 027827 and 2011 / 066342. Further PD-1 binding antagonists include pidilizumab, also known as CT-011, MEDI0680, also known as AMP-514, and REGN2810.

[0180] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, or avelumab, also known as MSB00010118C. In certain embodiments, the immune checkpoint inhibitor is a PD-L2 antagonist such as rHIgM12B7. In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist such as IMP321 and BMS-986016, but is not limited thereto. The immune checkpoint inhibitor may be an adenosine A2a receptor (A2aR) antagonist such as PBF-509.

[0181] In some embodiments, the antibodies described herein (e.g., anti-PD-1 antibody, anti-PDL1 antibody, or anti-PDL2 antibody) further comprise a human or mouse constant region. In further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In even more specific embodiments, the human constant region is IgG1. In even more specific embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In even more specific embodiments, the antibody has reduced or minimal effector function. In even more specific embodiments, minimal effector function results from production in prokaryotic cells. In even more specific embodiments, minimal effector function results from an "effector-free Fc mutation," i.e., non-glycosylation.

[0182] Therefore, antibodies used herein can be deglycosylated. Antibody glycosylation is typically either N-linked or O-linked. N-linking means attaching a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences within a polypeptide creates a potential glycosylation site. O-linked glycosylation means attaching one of the sugars N-acetylgalactoseamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from antibodies is conveniently achieved by modifying the amino acid sequence so that one of the aforementioned tripeptide sequences (for the N-linked glycosylation site) is removed. Modifications can be made by substituting an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0183] For example, an antibody or its antigen-binding fragment can be produced using methods known in the art by a process comprising culturing host cells containing nucleic acids encoding any of the aforementioned anti-PDL1, anti-PD-1, or anti-PDL2 antibodies or antigen-binding fragments in a form suitable for expression, under conditions suitable for producing such antibodies or fragments, and recovering the antibody or fragment.

[0184] B. CTLA-4 Another immune checkpoint that can be targeted by the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank deposit number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein, CD28, both molecules bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulating signals. Intracellular CTLA4 is found on regulatory T cells and may be important for its function. T cell activation by T cell receptors and CD28 increases the expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0185] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), its antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide.

[0186] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the method of the present invention can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, International Publication Nos. 01 / 14424, 98 / 42752; 00 / 37504 (CP675,206, tremelimumab; also formerly known as tichilimumab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (Antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the manner disclosed herein. The teachings of each of the aforementioned publications are incorporated herein by reference. Regarding binding to CTLA-4, antibodies that compete with any of these antibodies recognized in the art may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Publication No. 2001014424, Publication No. 2000037504 and U.S. Patent No. 8017114, all of which are incorporated herein by reference.

[0187] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, International Publication No. 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for and / or binds to the same epitope on CTLA-4, similar to the antibodies described above. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibodies described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0188] Other molecules that modulate CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent No. 5,844,905, 5,885,796, and International Patent Application Publications No. 1,995,001,994, and 1,998,042,752 (all incorporated herein by reference), as well as immunoadhesins, such as those described in U.S. Patent No. 8,329,867 (all incorporated herein by reference).

[0189] C. Killer immunoglobulin-like receptor (KIR) Another immune checkpoint inhibitor for use in the present invention is an anti-KIR antibody. Suitable anti-human KIR antibodies (or VH / VL domains derived therefrom) for use in the present invention can be produced using methods well known in the art.

[0190] Alternatively, anti-KIR antibodies recognized in the art can be used. Anti-KIR antibodies can cross-reactive with multiple inhibitory KIR receptors and enhance the cytotoxicity of NK cells having one or more of these receptors. For example, anti-KIR antibodies can bind to KIR2D2DL1, KIR2DL2, and KIR2DL3, respectively, and can enhance NK cell activity by reducing, neutralizing, and / or reversing the inhibition of NK cell cytotoxicity mediated by any of these KIRs. In some embodiments, anti-KIR antibodies do not bind to KIR2DS4 and / or KIR2DS3. For example, monoclonal antibodies 1-7F9 (also known as IPH2101), 14F1, 1-6F1, and 1-6F5, described in International Publication No. 2006 / 003179 (the teachings of which are incorporated herein by reference), can be used. Antibodies that compete with any of these art-recognized antibodies for binding to KIRs can also be used. Further available anti-KIR antibodies used in the art include, for example, those described in International Publication Nos. 2005 / 003168, 2005 / 009465, 2006 / 072625, 2006 / 072626, 2007 / 042573, 2008 / 084106, 2010 / 065939, 2012 / 071411, and 2012 / 160448.

[0191] An exemplary anti-KIR antibody is lirirumab (also known as BMS-986015 or IPH2102). In another embodiment, the anti-KIR antibody comprises the heavy-chain and light-chain complementarity-determining regions (CDRs) or variable regions (VRs) of lirirumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy-chain variable (VH) region of lirirumab, as well as the CDR1, CDR2, and CDR3 domains of the light-chain variable (VL) region of lirirumab. In another embodiment, the antibody has at least about 90% amino acid sequence identity of the variable region with lirirumab.

[0192] VIII. Treatment method This specification provides a method for treating cancer or delaying the progression of cancer in an individual, comprising administering to the individual an effective amount of at least one CD122 / CD132 agonist and at least one tumor suppressor gene therapy (e.g., p53 and / or MDA-7 gene therapy, or viral tumor disintegration therapy - VirRx007). The therapy may further comprise at least one immune checkpoint inhibitor (e.g., a PD-1 axis-binding antagonist and / or a CTLA-4 antibody).

[0193] In some embodiments, the treatment results in the maintenance of the response in the individual after the completion of treatment. The methods described herein may be found to be used in therapeutic conditions where enhanced immunogenicity is desired, such as increased oncoimmunogenicity for cancer treatment. Also provided herein are methods for enhancing immune function in an individual having cancer, the such individual comprising administering to the individual an effective amount of a CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein and / or IL-15 mutein), as well as a p53 and / or MDA-7 tumor suppressor gene therapy, or a viral tumor disintegration therapy such as VirRx007. The CD122 / CD132 agonist can be an IL-15 variant (e.g., IL-15N72D) bound to an IL-15 receptor α / IgG1 Fc fusion protein, such as ALT-803. In some embodiments, the individual is human.

[0194] In some embodiments, the subject is further administered a tumor suppressor immunogene therapy (see PCT / US2016 / 060833, the whole of which is incorporated herein by reference). In some embodiments, the subject is further administered additional viral and nonviral gene therapies (see PCT / US2017 / 065861, the whole of which is incorporated herein by reference). In some embodiments, the replicable and / or nonreplicable viral and / or nonviral gene therapies can deliver one or more therapeutic genes, which may be tumor suppressor genes or immunostimulatory genes.

[0195] Examples of cancers for which treatment is conceivable include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, osteosarcoma, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, pre-neoplastic injury in the lungs, colorectal cancer, melanoma, and bladder cancer.

[0196] In some embodiments, the individual has cancer that is resistant to (or has been shown to be resistant to) one or more anticancer therapies. In some embodiments, resistance to anticancer therapies may include cancer recurrence or resistant cancer. Recurrence may mean the reappearance of cancer in the original location or a new location after treatment. In some embodiments, resistance to anticancer therapies may include cancer progression during treatment with the anticancer therapy. In some embodiments, the cancer is in the early or late stages.

[0197] In some embodiments, the subject is also treated with PD-1 axis-coupled antagonists and / or immune checkpoint inhibitors such as anti-CTLA-4 antibodies. The individual may have cancer that expresses PD-L1 biomarkers (e.g., shown to express in diagnostic tests) or have a high tumor mutagram. In some embodiments, the patient's cancer expresses insufficient PD-L1 biomarkers. In some embodiments, the patient's cancer expresses many PD-L1 biomarkers. PD-L1 biomarkers can be detected in a sample using methods selected from the group consisting of FACS, Western blotting, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof. The tumor burden of high mutations can be determined by genome sequencing (e.g., Foundation One CDx assay).

[0198] In some embodiments, the subject may also be treated with a histone deacetylase (HDAC) inhibitor (e.g., tractinostat, an orally administered class 1 histone deacetylase selective inhibitor, formerly known as CHR-3996 or VRx-3996).

[0199] The efficacy of any of the methods described herein (for example, a combination of therapies including administering an effective amount of a combination of at least one CD122 / CD132 agonist, p53, ADP, and / or MDA-7, gene therapy, at least one immune checkpoint inhibitor, and / or at least one HDAC inhibitor) can be tested in various models known in the art, such as clinical or preclinical models. Suitable preclinical models are exemplified herein and may further include, but are not limited to, ID8 ovarian cancer, GEM models, B16 melanoma, RENCA renal cell carcinoma, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma models of cancer.

[0200] In some embodiments of the methods of this disclosure, the cancer has a small amount of T cell infiltration. In some embodiments, the cancer does not have detectable T cell infiltration. In some embodiments, the cancer is a non-immunogenic cancer (e.g., non-immunogenic colorectal cancer and / or ovarian cancer). Although not bound by theory, combination therapy may have T cell (e.g., CD4) levels compared to before administration of the combination. + T cells, CD8 + This can increase the priming, activation, and / or proliferation of T cells (memory T cells).

[0201] In some embodiments of the methods of this disclosure, activated CD4 and / or CD8 T cells in an organism are characterized by enhanced cytolytic activity compared to γ-IFN-producing CD4 and / or CD8 T cells and / or combinations before administration. γ-IFN can be measured by any means known in the art, such as cell fixation, saturation, and staining with an antibody against γ-IFN, with intracellular cytokine staining (ICS). Cytolytic activity can be measured by any means known in the art, such as a cell-killing assay using mixed effector and target cells.

[0202] This disclosure is useful for any human cells involved in an immune response, either as a target of the immune system or as part of the immune system's response to an exogenous target. The methods include ex vivo, in vivo, and various other methods involving the injection of polynucleotides or vectors into host cells. The methods also include direct injection into tumors or tumor beds, and local or local injection into tumors.

[0203] A. Administration The combination therapies provided herein include administering a selective CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein), as well as p53, ADP, and / or MDA-7 gene therapy. The combination therapies can be administered by any preferred method known in the art. For example, CD122 / CD132 agonists (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein), as well as p53 and / or MDA-7 gene therapies, can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, one or more CD122 / CD132 agonists are present in separate compositions as p53, ADP, and / or MDA-7 gene therapies, or their expression constructs. In some embodiments, the CD122 / CD132 agonists are present in the same composition as the p53 and / or MDA-7 gene therapy. In certain embodiments, subjects are administered a nucleic acid encoding p53, ADP, and / or a nucleic acid encoding MDA-7 before, concurrently with, or after at least one CD122 / CD132 agonist.

[0204] One or more CD122 / CD132 agonists, as well as p53, ADP, and / or MDA-7 gene therapies, may be administered via the same or different routes of administration. In some embodiments, CD122 / CD132 agonists are administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, p53, ADP, and / or MDA-7 gene therapies are administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Effective doses of CD122 / CD132 agonists, as well as p53, ADP, and / or MDA-7 gene therapies, may be administered for the prevention or treatment of disease. The appropriate dose of CD122 / CD132 agonists, and / or p53, ADP, and / or MDA-7 gene therapies, can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician. In some embodiments, combination therapy with at least one selective CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein) and p53, ADP, and / or MDA-7 gene therapies is synergistic, thereby... Compared to treatment as individual agents, there is an effect that surpasses the additive effect of individual doses of p53, ADP, and / or MDA-7 gene therapies when combined with at least one CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein).

[0205] For example, a therapeutically effective dose of a CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein) is administered subcutaneously or intravenously at intervals ranging from weekly, bi-weekly, every three weeks, or every four weeks, in a dose range of 5 to 100 μg / kg.

[0206] For example, when a therapeutically effective dose of one or more CD122 / CD132 agonists, as well as p53, ADP, and / or MDA-7 gene therapies, is administered in combination with an immune checkpoint inhibitor such as an antibody, the therapeutically effective dose is in the range of approximately 0.01 to approximately 50 mg / kg of the patient's body weight, regardless of whether it is a single or multiple dose. In some embodiments, the antibody used is, for example, approximately 0.01 to approximately 45 mg / kg, approximately 0.01 to approximately 40 mg / kg, approximately 0.01 to approximately 35 mg / kg, approximately 0.01 to approximately 30 mg / kg, approximately 0.01 to approximately 25 mg / kg, approximately 0.01 to approximately 20 mg / kg, approximately 0.01 to approximately 15 mg / kg, approximately 0.01 to approximately 10 mg / kg, approximately 0.01 to approximately 5 mg / kg, or approximately 0.01 to approximately 1 mg / kg per day. In some embodiments, the antibody is administered at 15 mg / kg. However, other drug regimens may be useful. In one embodiment, the anti-PD-L1 antibody described herein is administered to humans in doses of approximately 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, or 1400 mg in cycles of 1 to 21 days. The dose may be administered as a single dose, such as an injectable, or as multiple doses (e.g., two or three doses). Progress of this treatment can be easily monitored by conventional techniques.

[0207] Intratumoral injection, or injection into tumor vascular structures, is specifically conceivable for the p53, ADP, and / or MDA-7 gene therapy components of combination therapy. Local, local, or systemic administration may also be preferred. For tumors larger than 4 cm, the administered volume is approximately 4–10 mL (particularly 10 mL), while for tumors smaller than 4 cm, a volume of approximately 1–3 mL (particularly 3 mL) is used. Multiple injections delivered as a single dose occupy a volume of approximately 0.1–0.5 mL. For example, adenovirus particles can be advantageously contacted by administering multiple injections to the tumor.

[0208] Treatment regimens can vary considerably, often depending on the type of tumor, its location, the progression of the disease, and the patient's health and age. Clearly, some tumors require more aggressive treatment, while at the same time, some patients cannot tolerate more demanding protocols. Clinicians are best suited to making such decisions based on the known efficacy and (if any) toxicity of therapeutic agents.

[0209] In certain embodiments, the tumor being treated may not be resectable, at least initially. Combination therapy can increase the resectableness of the tumor by shrinking the surrounding tissue or by removing certain invasive portions. Resection is performed after combination therapy. Further treatment after resection will serve to eliminate any remaining disease.

[0210] Therapies can include various "unit doses." A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount administered, as well as specific routes and formulations, are within the scope of the art of those skilled in the clinical field. A unit dose does not necessarily have to be administered as a single injection, but can include a series of infusions over a period of time. For convenience, the unit doses of the present invention can be described in terms of plaque-forming units (PFUs) against viral constructs. A unit dose is 10 3 , 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 The range is pfu or higher. Alternatively, depending on the type of virus and the achievable titer, 1-100, 10-50, 100-1000, or up to approximately 1 x 10¹⁶ to the patient or patient's cells. 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , or 1 × 10 15 It delivers, or more infectious viral particles (VPs).

[0211] B. Injectable compositions and formulations One method for delivering one or more expression constructs encoding human p53, ADP, and MDA-7 to the overgrowth cells of the present invention is by intratumoral injection, while CD122 / CD132 agonists, immune checkpoint inhibitors, and HDAC inhibitors are administered systemically. However, all pharmaceutical compositions disclosed herein can be administered intratumoral, parenterally, intravenously, intradermally, intra-arterially, intramuscularly, percutaneously, or even intraperitoneally, as described in U.S. Patents 5,543,158, 5,641,515, and 5,399,363, which are all incorporated herein by reference.

[0212] Nucleic acid constructs can be delivered by syringe or any other method used for injecting solutions, provided that the expression construct can pass through a needle of the specific gauge required for injection. A novel needle-free injection system is described (U.S. Patent No. 5,846,233) having a nozzle defining an ampoule chamber for holding a solution and an energy device for pushing the solution from the nozzle to the delivery site. A syringe system for use in gene therapy that allows for the precise injection of a predetermined amount of solution multiple times at any depth is also described (U.S. Patent No. 5,846,225). Another available injection system is the QuadraFuse device, which has a branched needle adjustable to different depths via an attached syringe.

[0213] Solutions of the active compound as a free base or a pharmacokinetically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. Suitable drug forms for injectable use include sterile aqueous solutions or dispersions, and sterile powders for immediate preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468). In all cases, the form must be sterile and fluid enough to allow for easy injection. The form must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. For example, by using coating agents such as lecithin, the required particle size can be maintained in the case of a dispersion, and by using surfactants, appropriate fluidity can be maintained. Microbial activity can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugar chains or sodium chloride. Sustained absorption of the injectable composition can be achieved by using absorption retarders in the composition, such as aluminum monostearate and gelatin.

[0214] For parenteral administration in aqueous solutions, for example, the solution must be appropriately buffered as needed, and the liquid diluent is first isotonicized with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this regard, available sterile aqueous media will be known to those skilled in the art from the perspective of this disclosure. For example, one dose may be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous injection fluid, or injected at the intended injection site (see, for example, Remington's Pharmaceutical Sciences 22md Edition). Some dose variations may occur as needed depending on the condition of the subject being treated. In any case, the person administering the drug will determine the appropriate dose for each individual subject. Furthermore, for administration to humans, the preparation must meet the sterility, pyrogenicity, general safety, and purity requirements of the FDA Office's Biologics Standards.

[0215] Sterile injectable solutions are prepared by combining the required amount of active compound in a suitable solvent, along with other components as needed, and then sterilizing by filtration. Generally, dispersions are prepared by mixing various sterilized active ingredients with a sterile vehicle containing a basic dispersion medium and other components as needed. For sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which involve obtaining the active ingredient powder plus any additional desired components from a pre-sterilized and filtered solution.

[0216] The compositions disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) and those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine. During formulation, the solution is administered in a therapeutically effective dose in a manner compatible with the dosage form. The formulations are readily administered in various dosage forms, such as injectable solutions and drug-release capsules.

[0217] C. Additional anti-cancer treatments To enhance the efficacy of p53, ADP, and / or MDA-7 nucleic acids, and at least one CD122 / CD132 agonist, these can be combined with at least one additional active agent effective in the treatment of cancer. More generally, these other compositions are provided in combination doses effective in killing or inhibiting cell proliferation. This process may involve contacting cells with the expression construct and the active agent or multiple factors simultaneously. This can be achieved by contacting cells with a single composition or pharmacological formulation containing both active agents, or by contacting cells with two different compositions or formulations, one of which contains the expression construct and the other contains the second active agent. Alternatively, the expression construct may come into contact with proliferating cells, and the additional treatment may affect other cells in the immune system or tumor microenvironment, thereby improving the anti-tumor immune response and therapeutic effect. At least one additional anticancer treatment may be, but is not limited to, surgery, chemotherapy (e.g., administration of protein kinase inhibitors or EGFR-targeted therapy), radiotherapy, cryotherapy, hyperthermia therapy, phototherapy, radioresection therapy, hormone therapy, immunotherapy including but not limited to immune checkpoint inhibitors, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, or biological therapy (e.g., monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes, or gene therapy). Biological therapies may be, but are not limited to, gene therapies such as tumor suppressor gene therapy, cell death protein gene therapy, cell cycle regulator gene therapy, cytokine gene therapy, toxic gene therapy, immunogen therapy, suicide gene therapy, prodrug gene therapy, anti-proliferation gene therapy, enzyme gene therapy, or anti-angiogenic factor gene therapy.

[0218] Gene therapy can precede or follow other activator therapies with intervals ranging from a few minutes to several weeks. In embodiments where other activators and expression constructs are applied to cells individually, it is generally ensured that the time between each delivery is not significantly extended so that the activators and expression constructs can still exert a combined beneficial effect. In such examples, it is conceivable that cells can be brought into contact with each other in both ways, and preferably in about 12 to 24 hours, and preferably in about 6 to 12 hours. However, in some cases, if the interval between each administration takes several days (e.g., 2, 3, 4, 5, 6, or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks), it may be desirable to significantly extend the duration of treatment. In certain embodiments, one or more therapies can be continued with or without other therapies as maintenance therapy.

[0219] Various combinations can be used, with gene therapy and CD122 / CD132 agonists being "A", and the second active agent, i.e., immune checkpoint inhibitors, being "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0220] 1. Chemotherapy Cancer treatment generally includes various combination therapies, both chemo-based and radio-based. Examples of combination chemotherapy include cisplatin (CDDP), carboplatin, procarbazine, mechloretamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, precomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor conjugates, taxol, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans-platinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, temozolomide (aqueous form of DTIC), or any analogues or derivatives of the aforementioned drugs. Combining chemotherapy with biological therapies is known as biochemotherapy. Chemotherapy can also be administered in small, continuous doses, a practice known as metronome chemotherapy.

[0221] Further combination chemotherapy options include, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocon, metsuredopa and uredopa; ethyleneimine and methylamelamelamine, altoretamine, triethylenemelamelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamelamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); briostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin ( Includes synthetic analogs KW-2189 and CB1-TM1); elucerobin; pancratistatin; sarcodicin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembitin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, e.g., engine antibiotics (e.g., calichemycin, in particular calichemycin gamma I and calichemycin omega I1); dynemycin, including dynemycin A; bisphosphonates, e.g., clondronate; esperamicin;and neocardinostatin chromophore and related pigment protein enediin antibiotics chromophore, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epil Zolubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, pteropterin, trimethrexate; purine analogs, e.g., fludara Vinn, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., mitotane, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofsphamide glycoside; aminolevulinic acid; enyluracil; am Sacrin; Bestrabusil; Bisanthren; Edatraxate; Defofamine; Demecoltin; Diadiquan; Eflornithine; Erliptinium acetate; Eposilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Maytansinoids, e.g., Maytansine and Ansamitosine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Loxoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Lazoxane; Schizophyllan;Spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, loridine A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinate complexes, e.g., cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP -16); ifosfamide; mitoxantrone; vincristine; vinblastine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; capecitabine; carboplatin, procarbazine, precomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, trans platinum, and any pharmaceutically acceptable salts, acids, or derivatives of the above. In certain embodiments, the compositions provided herein can be combined with histone deacetylase inhibitors. In certain embodiments, the compositions provided herein can be combined with gefitinib. In another embodiment, this embodiment can be practiced in combination with Gleevec (for example, a patient may be administered about 400 to about 800 mg / day of Gleevec). In a particular embodiment, one or more chemotherapy drugs may be used in combination with the compositions provided herein.

[0222] 2. Radiation therapy Other widely used factors that cause DNA damage include those commonly known as Y rays, X-rays, and / or the delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also known, such as microwaves and ultraviolet light. All of these factors are most likely to have a wide-ranging impact on DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The range of X-ray irradiation varies from 50-200 roentgens per day over a long period (3-4 weeks) to 2000-6000 roentgens per single irradiation. The range of radioisotope irradiation varies widely, depending on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by newly formed cells.

[0223] 3. Immunotherapy Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors may be, for example, antibodies specific to certain markers on the surface of tumor cells. Only antibodies can serve as therapeutic effectors, or can mobilize other cells to actually influence cell killing. Antibodies may also be conjugated to drugs or toxins (chemotherapy, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.), or they may simply function as targeting agents. Alternatively, effectors may be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells, as well as genetically modified variants of these cell types that express chimeric antigen receptors. Mda-7 gene introduction into tumor cells induces tumor cell death and apoptosis. Apoptotic tumor cells are captured by dendritic cells and reticuloendothelial cells, including macrophages, and presented to the immune system to generate antitumor immunity (Rovere et al., 1999; Steinman et al., 1999).

[0224] Those skilled in the art of cancer immunotherapy will understand that other adjunctive immunotherapies, including but not limited to GM-CSF, can be added to the regimens described above to further enhance efficacy and increase the number of myeloid-derived innate immune cells, the number of low-dose cyclophosphamide or PI3K inhibitors (e.g., PI3Kδ inhibitors) to eliminate innate and adaptive immune and 5FU-inhibiting T regulatory cells (e.g., capecitabine), and the number of PI3K inhibitors or histone deacetylase inhibitors to eliminate inhibitory myeloid-derived immunosuppressive cells. For example, PI3K inhibitors include, but are not limited to, LY294002, perifosine, BKM120, duvelisib, PX-866, BAY80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, paromide 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136. In some embodiments, the PI3K inhibitor is a PI3Kδ inhibitor such as idelalisib RP6530, TGR1202, and RP6503, but is not limited to these. Further PI3K inhibitors are disclosed in U.S. Patent Applications Publications 20150291595, 20110190319, and International Patent Applications Publications 2012146667, 2014164942, 2012062748, and 2015082376. Immunotherapy may also include the administration of interleukins such as IL-2 or interferons such as INFα.

[0225] Examples of immunotherapies that can be combined with p53, ADP, and / or MDA-7 gene therapies, as well as CD122 / CD132 agonists, include: immune adjuvants (e.g., Mycobacterium bovine, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (U.S. Patent Nos. 5,801,005, 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies (e.g., interferon α, β, and γ; interleukins (IL-1, IL-2), GM-CSF, and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and These include Villaseca (1998; U.S. Patent Nos. 5,830,880 and 5,846,945), and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. Herceptin has antitumor activity and is approved for use in the treatment of malignant tumors (Dillman, 1999). Combination therapy of cancer with Herceptin and chemotherapy has been shown to be more effective than individual therapies. Therefore, it is conceivable that one or more anticancer therapies may be used in conjunction with the p53, ADP, and / or MDA-7 gene therapies described herein.

[0226] Additional immunotherapies that can be used in combination with p53, ADP, and / or MDA-7 gene therapies and CD122 / CD132 agonists include immune checkpoint inhibitors, costimulatory receptor agonists, innate immune cell stimulants, or innate immunity activators. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some embodiments, at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is tremelimumab or ipilimumab. In certain embodiments, at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. In some embodiments, the anti-KIR antibody is lirirumab. In some embodiments, the PD-L1 inhibitor is durvalumab, atezolizumab, or avelumab. In some embodiments, the PD-L2 inhibitor is rHIgM12B7. In some embodiments, the LAG3 inhibitor is IMP321 or BMS-986016. In some embodiments, the A2aR inhibitor is PBF-509.

[0227] In some embodiments, at least one immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) axis-binding antagonist. In certain embodiments, the PD-1 axis-binding antagonist is selected from the group consisting of PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. In some embodiments, the PD-1 axis-binding antagonist is a PD-1 binding antagonist. In certain embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PDL1 and / or PDL2. In particular, the PD-1 binding antagonist is a monoclonal antibody or its antigen-binding fragment. In some embodiments, the PD-1 binding antagonist is nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224.

[0228] In certain embodiments, at least one checkpoint inhibitor is selected from inhibitors of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some embodiments, at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is tremelimumab or ipilimumab. In certain embodiments, at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. In some embodiments, the anti-KIR antibody is lirirumab. In some embodiments, the PD-L1 inhibitor is durvalumab, atezolizumab, or avelumab. In some embodiments, the PD-L2 inhibitor is rHIgM12B7. In some embodiments, the LAG3 inhibitor is IMP321 or BMS-986016. In some embodiments, the A2aR inhibitor is PBF-509.

[0229] Co-stimulatory receptor agonists can be anti-OX40 antibodies (e.g., MEDI6469, MEDI6383, MEDI0562, and MOXR0916), anti-GITR antibodies (e.g., TRX518, and MK-4166), anti-CD137 antibodies (e.g., urelumab, and PF-05082566), anti-CD40 antibodies (e.g., CP-870, 893, Chi Lob 7 / 4), or anti-CD27 antibodies (e.g., valrirumab, also known as CDX-1127). Stimulants for innate immune cells include, but are not limited to, KIR monoclonal antibodies (e.g., lirirumab), inhibitors of cytotoxic inhibitory receptors (e.g., KLRC and NKG2A, also known as CD94, e.g., the monoclonal antibody monalizumab, and anti-CD96, also known as TACTILE), and Toll-like receptor (TLR) agonists. TLR agonists can be BCG, TLR7 agonists (e.g., poly0ICLC, and imiquimod), TLR8 agonists (e.g., reximod), or TLR9 agonists (e.g., CPG7909). Activators of innate immune cells such as natural killer (NK) cells, macrophages, and dendritic cells include IDO inhibitors, TGFβ inhibitors, and IL-10 inhibitors. An exemplary activator of innate immunity is indoximod. In some embodiments, immunotherapy involves interferon gene-stimulating (STING) agonists (Corrales et al., 2015).

[0230] Other immunotherapies conceivable for use in the methods of this disclosure include those described by Tchekmedyian et al., 2015, which are incorporated herein by reference. Immunotherapy may include the suppression of T regulatory cells (Tregs), myeloid-derived immunosuppressive cells (MDSCs), and cancer-associated fibroblasts (CAFs). In some embodiments, the immunotherapy is a tumor vaccine (e.g., whole tumor cell vaccines, dendritic cell vaccines, DNA and / or RNA expression vaccines, peptides, and recombinant tumor-associated antigen vaccines) or adoptive cell therapy (ACT) (e.g., T cells, natural killer cells, and LAK cells). T cells and / or natural killer cells may be modified using chimeric antigen receptors (CARs) or T cell receptors (TCRs) for specific tumor antigens. As used herein, a chimeric antigen receptor (or CAR) may mean any modified receptor that is specific to the antigen of interest and, when expressed on a T cell or natural killer cell, confers the specificity of the CAR to that T cell or natural killer cell. Once prepared using standard molecular techniques, T cells or natural killer cells expressing chimeric antigen receptors can be introduced into patients using techniques such as adoptive cell transfer. In some embodiments, the T cells are CD4 and / or CD8 T cells activated in the organism, characterized by γ-IFN''-producing CD4 and / or CD8 T cells and / or enhanced cytolytic activity compared to before administration of the combination. CD4 and / or CD8 T cells may show increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins. CD4 and / or CD8 T cells can be effector memory T cells. In certain embodiments, CD4 and / or CD8 effector memory T cells are CD44 high CD62L low It is characterized by having the expression of [something].

[0231] In certain embodiments, two or more immunotherapies can be combined with additional immune checkpoint inhibitors, including p53, ADP, and / or MDA-7 gene therapies, as well as CD122 / CD132 agonists, in combination with T cell costimulatory receptor agonists or TIL ACT. Other combinations include T cell checkpoint blockade + costimulatory receptor agonist, T cell checkpoint blockade to improve innate immune cell function, checkpoint blockade + IDO inhibition, or checkpoint blockade + adoptive T cell introduction. In certain embodiments, the immunotherapy includes a combination of an anti-PD-L1 immune checkpoint inhibitor (e.g., avelumab), a 4-1BB (CD-137) agonist (e.g., utomirumab), and an OX40 (TNFRS4) agonist. The immunotherapy can be combined with histone deacetylase (HDAC) inhibitors such as 5-azacitidine and entinostat.

[0232] Immunotherapy may be a cancer vaccine comprising one or more cancer antigens, in particular a protein or its immunogenic fragment, DNA or RNA encoding the above cancer antigen, in particular a protein or its immunogenic fragment, cell solubilized products, and / or protein preparations derived from tumor cells. As used herein, a cancer antigen is an antigenic substance present in cancer cells. In principle, any protein produced in cancer cells that is upregulated in cancer cells or has an abnormal structure due to mutation compared to normal cells can act as a cancer antigen. In principle, cancer antigens may be products of mutated or overexpressed oncogenes and tumor suppressor genes; products of other mutated genes, overexpressed or abnormally expressed cellular proteins; cancer antigens produced by oncogenic viruses; carcinoembryonic antigens; altered cell surface glycolipids and glycoproteins; or cell type-specific differentiation antigens. Examples of cancer antigens include products of abnormally or overexpressed ras and p53 genes. Other examples include tissue differentiation antigens, mutant protein antigens, oncogenic viral antigens, carcinogenic testis antigens, and vascular or stromal specific antigens. Tissue differentiation antigens are antigens specific to certain types of tissue. Mutant proteins can be far more specific to cancer cells because normal cells do not contain these proteins. Normal cells present normal protein antigens to MHC molecules, while cancer cells present mutant versions. Several viral proteins have been suggested to form cancer, and some viral antigens are also cancer antigens. Carcinogenic testis antigens are antigens that are primarily expressed in testicular germ cells, but are also expressed in fetal ovarian and trophoblast cells. Some cancer cells abnormally express these proteins, thus presenting these antigens and enabling attack by T cells specific to these antigens. Exemplary antigens of this type include CTAG1 B and MAGEA1, as well as rindopeptim, a 14-mer intradermal injectable peptide vaccine targeted at epidermal growth factor receptor (EGFR) VIII variants. Lindopeptide is particularly suitable for treating gliablastoma when used in combination with the CD95 / CD95L signaling pathway inhibitors described herein.Furthermore, proteins that are normally produced in very small amounts but whose production dramatically increases in cancer cells can trigger an immune response. An example of such a protein is the enzyme tyrosinase, which is necessary for melanin production. Normally, tyrosinase is produced in trace amounts, but its quantity increases dramatically in melanoma cells. Carcinoembryonic antigens are another important category of cancer antigens. Examples include alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). These proteins are usually produced in the early stages of embryonic development and disappear by the time the immune system is fully developed. Therefore, self-tolerance does not develop against these antigens. Abnormal proteins are also produced by cells infected with oncoviruses, such as EBV and HPV. Cells infected with these viruses contain potential viral DNA that is transcribed, and the resulting proteins produce an immune response. Cancer vaccines can include peptide cancer vaccines, which, in some embodiments, are peptide vaccines designed for individual use. In some embodiments, the peptide cancer vaccine is a polyvalent long peptide vaccine, a polypeptide vaccine, a peptide cocktail vaccine, a hybrid peptide vaccine, or a peptide-pulsed dendritic cell vaccine.

[0233] Immunotherapy may be an antibody, such as one as part of a polyclonal antibody preparation, or a monoclonal antibody. The antibody may be a humanized antibody, a chimeric antibody, an antibody fragment, a bispecific antibody, or a single-chain antibody. Antibodies disclosed herein include, but are not limited to, Fab, Fab', and F(ab')2, Fd, single-chain Fvs(scFv), single-chain antibodies, disulfide-linked Fvs(sdfv), and fragments containing either a VL or VH domain. In some embodiments, the antibody or fragment thereof specifically binds to epidermal growth factor receptor (EGFR1, Erb-B1), HER2 / neu(Erb-B2), CD20, vascular endothelial growth factor (VEGF), insulin-like growth factor receptor (IGF-1R), TRAIL receptor, epithelial cell adhesion molecules, carcinoembryonic antigen, prostate-specific membrane antigen, mucin-1, CD30, CD33, or CD40.

[0234] Examples of monoclonal antibodies that can be used in combination with the compositions provided herein include trastuzumab (anti-HER2 / neu antibody); pertuzumab (anti-HER2 mAb); cetuximab (chimeric monoclonal antibody against epidermal growth factor receptor EGFR); panitumumab (anti-EGFR antibody); nimotuzumab (anti-EGFR antibody); zaltumumab (anti-EGFR mAb); nesitumumab (anti-EGFR mAb); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); rituximab (chimeric mouse / human anti-CD20 mAb); obinutuzumab (anti-CD20 mAb); ofatumumab (anti-CD20 mAb); tositumomab-I131 (anti-CD20 mAb); Ibritumomab tiuxetan (anti-CD20 mAb); Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); Ranibizumab (anti-VEGF mAb); Aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgG1 Fc); AMG386 (angiopoietin-1 and 2 linked peptides fused to IgG1 Fc); Darotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti-Campus-1 / CD52 mAb); Brentuximab vedotin (anti-CD30 mAb); Catumakisomab (bispecific mAb targeting epithelial cell adhesion molecules and CD3); Naptumomab (anti-5T4 Examples include, but are not limited to, mAbs; dilentuximab (anti-carbonic anhydrase ix); or faretzumab (anti-folate receptor).Other examples include Panorex(trademark)(17-1A)(mouse monoclonal antibody); Panorex(@(17-1A)(chimeric mouse monoclonal antibody)); BEC2(anti-idiotype mAb, mimic of the GD epitope)(with BCG); Oncolym(Lym-1 monoclonal antibody); SMART M195 Ab(humanized 13'1 LYM-1(Oncolym), Ovarex(B43.13, anti-idiotype mouse mAb)); 3622W94 mAb that binds to the pancarcinoma antigen EGP40(17-1A) in adenocarcinoma; Zenapax(SMART Anti-Tac(IL-2 receptor)); SMART M195 Examples of antibodies include Ab (humanized Ab); NovoMAb-G2 (general-purpose cancer-specific Ab); TNT (chimeric mAb against histone antigens); TNT (chimeric mAb against histone antigens); Gliomab-H (monoclonal-humanized Ab); GNI-250 Mab; EMD-72000 (chimeric EGF antagonist); LymphoCide (humanized IL.L.2 antibody); and bispecific antibodies such as MDX-260 that target GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab, or ImmuRAIT-CEA. Examples of antibodies are disclosed in U.S. Patents 5,736,167, 7,060,808, and 5,821,337.

[0235] Further examples of antibodies include zanulimumab (anti-CD4 mAb), keriximab (anti-CD4 mAb); ipilimumab (MDX-101; anti-CTLA-4 mAb); remelimumab (anti-CTLA-4 mAb); daclizumab (anti-CD25 / IL-2R mAb); basiliximab (anti-CD25 / IL-2R mAb); MDX-1106 (anti-PD1 mAb); antibody against GITR; GC1008 (anti-TGF-β antibody); meterimumab / CAT-192 (anti-TGF-β antibody); reldelimumab / CAT-152 (anti-TGF-β antibody); ID11 (anti-TGF-β antibody); denosumab (anti-RANKL mAb); BMS-663513 (humanized anti-4-1BB mAb); SGN-40 (humanized anti-CD40 mAb); CP870,893 (human anti-CD40 mAb); infliximab (chimeric anti-TNF mAb); adalimumab (human anti-TNF mAb); certolizumab (humanized Fab anti-TNF); golimumab (anti-TNF); etanercept (extracellular domain of TNFR fused to IgG1 Fc); beratacept (extracellular domain of CTLA-4 fused to Fc); abatacept (extracellular domain of CTLA-4 fused to Fc); belimumab (anti-B lymphocyte stimulator); muromonab-CD3 (anti-CD3 mAb); otelixizumab (anti-CD3 mAb); teprizumab (anti-CD3 mAb); tocilizumab (anti-IL6R mAb); REGN88 (anti-IL6R mAb); ustekinumab (anti-IL-12 / 23 Examples include mAb); ustekinumab (anti-IL-12 / 23 mAb); natalizumab (anti-α4 integrin); vedolizumab (anti-α4 β7 integrin mAb); T1 h (anti-CD6 mAb); epratuzumab (anti-CD22 mAb); efalizumab (anti-CD11a mAb); and atacicept (extracellular domains of transmembrane activators and calcium regulatory ligand interactors fused to Fc).

[0236] a. Passive immunotherapy Numerous different approaches exist to passive immunotherapy for cancer. These can be broadly categorized as follows: injection of antibodies alone; injection of antibodies conjugated to toxins or chemotherapeutic agents; injection of antibodies conjugated to radioisotopes; injection of anti-idiotype antibodies; and finally, purging of tumor cells into the bone marrow.

[0237] Human monoclonal antibodies are preferred for use in passive immunotherapy because they cause little to no side effects in patients. Human monoclonal antibodies against ganglioside antigens have been administered intrafocally to patients suffering from recurrent melanoma (Irie & Morton, 1986). Degeneration was observed in 6 out of 10 patients after daily or weekly intrafocal injections. In another study, moderate success was achieved by intrafocal injection of two types of human monoclonal antibodies (Irie et al., 1989).

[0238] It may be preferable to administer two or more monoclonal antibodies directed against two different antigens, or even more specifically, antibodies with multiple antigen specificities. The treatment protocol may also include the administration of lymphokines or other immunostimulants, as described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in further detail elsewhere in this specification.

[0239] b. Active immunotherapy In active immunotherapy, antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions, i.e., "vaccines," are generally administered along with different bacterial adjuvants (Ravindranath & Morton, 1991; Morton & Ravindranath, 1996; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993). In melanoma immunotherapy, patients who elicit a high IgM response often have a better survival rate than patients who do not elicit any or very little IgM antibodies (Morton et al., 1992). IgM antibodies are often transient antibodies, with exceptions to this rule appearing to be anti-ganglioside or anti-carbohydrate antibodies.

[0240] c. Adoptive immunotherapy In adoptive immunotherapy, lymphocytes circulating in the patient's blood or lymphocytes infiltrated by a tumor are isolated in vitro, activated with lymphokines such as IL-2, or transduced with genes for tumor necrosis, and then re-administered (Rosenberg et al., 1988; 1989). To achieve this, an immunologically effective amount of activated lymphocytes is administered to an animal or human patient together with an antigenic peptide composition incorporating an adjuvant as described herein. The activated lymphocytes are most preferably the patient's own cells, previously isolated from the blood or tumor and activated (or "swollen") in vitro. This form of immunotherapy has produced several cases of regression in melanoma and renal cell carcinoma, but the proportion of responsive subjects was lower compared to those that did not respond. More recently, higher response rates have been observed when such adoptive immunotherapy incorporates recombinant T cells expressing chimeric antigen receptors (CARs), known as CAR T-cell therapy. Similarly, both autologous and allogeneic natural killer cells are isolated, swollen, and genetically modified to express receptors or ligands that facilitate the binding and killing of tumor cells.

[0241] 4. Other active substances To improve the therapeutic effect of the treatment, it has been conceived that other active ingredients may be used in combination with the compositions provided herein. These additional active ingredients include immunostimulants, ingredients that affect the upregulation of cell surface receptors and gap junctions, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, or ingredients that increase the sensitivity of hyperproliferating cells to apoptosis-inducing factors. Examples of immunostimulants include tumor necrosis factor; interferon α, β, and γ; IL-2 and other cytokines; or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. It has also been conceived that the apoptosis-inducing ability of the compositions provided herein can be enhanced by establishing autocrine or paracrine effects in hyperproliferating cells by upregulating cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL. The anti-hyperproliferative effect on adjacent hyperproliferating cell populations is increased by increasing intracellular signaling through an increase in the number of gap junctions. In another embodiment, cell proliferation inhibitors or differentiation agents can be used in combination with the compositions provided herein to improve the anti-hyperproliferative effect of the treatment. Cell adhesion inhibitors have been conceived to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It has further been conceived that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with the compositions provided herein to improve the efficacy of the treatment.

[0242] In further embodiments, the other active ingredient may be one or more tumor lysis viruses. These tumor lysis viruses may be modified to express p53 and / or IL24, and / or other genes such as cytokines, ADP, or heat shock proteins. Examples of tumor lysis viruses include single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia viruses, vesicular stomatitis viruses, polioviruses, Newcastle disease viruses, Epstein-Barr viruses, influenza viruses and reoviruses, myxoma viruses, maraba viruses, rhabdoviruses, enadenochusileb, or coxsackieviruses. In certain embodiments, the other active ingredient may be talimogene laharpalebeck (T-VEC), a tumor lysis herpes simplex virus genetically modified to express GM-CSF. Talimogene Laharpalebeck, HSV-1 [JS1 strain] ICP34.5- / ICP47- / hGM-CSF (OncoVEX GM CSF T-VEC (formerly known as ) is an intratumor-delivered tumor-disintegrating immunotherapy containing an immune-enhancing HSV-1 that selectively replicates in solid tumors. (Lui et al., Gene Therapy, 10:292-303, 2003; U.S. Patent Nos. 7,223,593 and 7,537,924; incorporated herein by reference.) In October 2015, the U.S. FDA approved T-VEC under the trade name IMLYGIC® for the treatment of melanoma in patients with inoperable tumors. The characteristics and administration of T-VEC are described, for example, in the IMLYGIC® package insert (Amgen, 2015) and U.S. Patent Application Publication No. 2015 / 0202290 (both incorporated herein by reference). For example, T-VEC is typically administered at a maximum dose of 4.0 mL, 10 times, on day 1 of week 1. 6 The dose is plaque-forming units / mL (PFU / mL), followed by a maximum of 4.0 mL on day 1 of week 4, and then every other week thereafter (±3 days), for 108 It is administered by intratumoral injection into injectable cutaneous, subcutaneous, and nodular tumors at doses of PFU / mL. The recommended volume of tarimozine laharpalepbek injected into the tumor depends on the tumor size and should be determined according to injection volume guidelines. Although T-VEC has shown clinical activity in melanoma patients, many cancer patients either do not respond to T-VEC treatment or terminate their response to treatment. In one embodiment, p53, ADP, and / or MDA-7 nucleic acids, along with at least one CD122 / CD132 agonist, can be administered after, during, or before T-VEC treatment to reverse treatment resistance, for example. Exemplary tumor lysis viruses include, but are not limited to, Ad5-yCD / mutTKSR39rep-hIL12, Cavatak®, CG0070, DNX-2401, G207, HF10, IMLYGIC®, JX-594, MG1-MA3, MV-NIS, OBP-301, Reolysin®, Toca 511, Oncorine (H101), Onyx-015, H102, H103, and RIGVIR. Other exemplary tumor lysis viruses are described, for example, in International Publications 2015 / 027163, 2014 / 138314, 2014 / 047350, and 2016 / 009017, all of which are incorporated herein by reference.

[0243] In certain embodiments, hormone therapy may also be used in combination with these embodiments or with any other cancer treatments described above. The use of hormones can be used in the treatment of certain cancers, such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer, to reduce or block the effects of specific hormones, such as testosterone or estrogen. This treatment is often used as a treatment option, in combination with at least one other cancer treatment, or to reduce the risk of metastasis.

[0244] In some embodiments, further anticancer agents are protein kinase inhibitors or monoclonal antibodies that inhibit protein kinases or receptors involved in growth factor signaling pathways, such as EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor, or BRAF inhibitors. Non-limiting examples of protein kinase or growth factor signaling pathway inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, vandetanib, and AP2. Examples include 3451, vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridafololimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0245] In some embodiments, PI3K inhibitors include buparisib, idelalisib, BYL-719, dactrisib, PF-05212384, pictilisib, copanlisib, copanlisib dihydrochloride, ZSTK-474, GSK-2636771, duvelisib, GS-9820, PF-04691502, SAR-245408, SAR-245409, sonolisib, alhexin, GDC-0032, GDC-0980, apitricib, piraralisib, DLBS-1425, PX-866, voxtalisib, AZD-8186, BGT-226, DS-7423, GDC-0084, GSK-21-26458, and INK-1-1. 17, SAR-260301, SF-1 1 26, AMG-319, BAY-1082439, CH-51 32799, GSK-2269557, P-71 70, PWT-33597, CAL-263, RG-7603, LY-3023414, RP-5264, RV-1729, Taselicib, TGR-1 202, GSK-418, INCB-040093, Panulisib, GSK-105961 5, CNX-1351, AMG-51 1, PQR-309, 17β-hydroxywartmannine, AEZS-129, AEZS-136, HM-5016699, IPI-443, ONC-201, PF-4989216, RP-6503, SF-2626, X-339, XL-499, PQR-401, AEZS-132, CZC-24832, KAR-4141, PQR-31 1, PQR-316, RP-5090, VS-5584, X-480, AEZS-126, AS-604850, BAG-956, CAL-130, CZC-24758, ETP-46321, ETP-471 87, GNE-317, GS-548202, HM-032, KAR-1 139, LY-294002, PF-04979064, PI-620, PKI-402, PWT-143, RP-6530, 3-HOI-BA-01, AEZS-134, AS-041 164, AS-252424, AS-605240, AS-605858, AS-606839, BCCA-621 C, CAY-10505, CH-5033855, CH-51 08134, CUDC-908, CZC-19945, D-106669, D-87503, DPT-NX7, ETP-46444, ETP-46992, GE-21, GNE-123, GNE -151, GNE-293, GNE-380, GNE-390, GNE-477, GNE-490, GNE-493, GNE-614, HMPL-51 8, HS-104, HS-1 06, HS-1 16, HS-173, HS-196, IC-486068, INK-055, KAR 1 141, KY-1 2420, Wortmannin, Lin-05, NPT-520-34, PF-04691503, PF-06465603, PGNX-01, PGNX-02, PI 620, PI-103, PI-509, PI-516, PI-540, PIK-75, PWT-458, RO-2492, RP-5152, RP-5237, SB-201 5, SB-2312, SB-2343, SHBM-1009, SN 32976, SR-13179, SRX-2523, SRX-2558, SRX-2626, SRX-3636, SRX-5000, TGR-5237, TGX- 221, UCB-5857, WAY-266175, WAY-266176, EI-201, AEZS-131, AQX-MN100, KCC-TGX, OXY-1 1 1 The PI3K inhibitor is selected from the group consisting of A, PI-708, PX-2000, and WJD-008.

[0246] Further cancer treatments include, for example, the following: epidermal growth factor receptors (EGFR, EGFR1, ErbB-1, HER1), ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family, fibroblast growth factor (FGFR) family, FGFR ligand family. - Vascular endothelial growth factor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; Ephrin (EPH) receptor family; AXL receptor family; Leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; Receptor-type tyrosine kinase-like orphan receptor (ROR) receptor family; Discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; Transforming growth factor α (TGF-α), TGF-α receptor; transforming growth factor β (TGF-β), TGF-β receptor; interleukin-13 receptor α2 chain (1L13Ralpha2), interleukin-6 (IL-6), IL-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (hematopoietin family) and class II (interferon / IL-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), Death Receptor Family, TRAIL Receptor; Oncological Testis (CT) Antigen, Lineage-Specific Antigen, Differentiation Antigen, α-Actinin-4, ARTC1, Breakpoint Cluster Region Abelson (Bcr-abl) Fusion Product, B-RAF, Caspase-5 (CASP-5), Caspase-8 (CASP-8), β-Catenin (CTNNB1), Cell Division Cycle 27 (CDC27), Cyclin-Dependent Kinase 4 (CDK4), CDKN2A, COA-1, Dek-Can Fusion Protein, EFTUD-2, Prolongation Factor 2 (ELF2),Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low-density lipid receptor / GDP-L fucose:β-D-galactose 2α-L fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, arginine to isoleucine substitution at residue 170 of the α-helix of the α2 domain in the HLA-A2 gene (HLA-A*201-R170I), MLA-A11, heat shock protein 70-2 mutation (HSP70-2M), K IAA0205, MART2, Melanoma ubiquitous mutations 1, 2, 3 (MUM-1, 2, 3), Prostatic acid phosphorylation enzyme (PAP), NeoPAP, Myosin class 1, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, Triose phosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6 7, 8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL recognition antigen in melanoma (CAMEL), MAGE-A1 (MAGE-1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MA GE-C1, MAGE-C2, Mucin 1 (MUC1), MART-1 / / Melan-A (MLANA), gp100, gp100 / Pme117 (S1LV), Tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17, SSX-1, 2, 3, 4, TRP2-1NT2, Carcinoembryonic Antigen (CEA), Kallikfein 4, Mammaglobin-A, OA1, Prostate-Specific Antigen (PSA), Prostate-Specific Membrane Antigen, TRP-1 / gp75, TRP-2, Adipophyllin,Interferon-inducible protein (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250 intestinal carboxylesterase (iCE)), α-fetoprotein (AFP), M-CSF, mdm-2 (e.g., HDM201, also known as MDM2, HDM2 and / or HDM4, to reverse the inhibition of p53 activity, etc.), small molecule inhibitors, cis-imidazoline (e.g., Nutrin), benzodiazepine (BDP), Spirooxyindole), MUCI, p53 (TP53), PBF, FRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, Sulbibin (BIRCS), Human telomerase reverse transcriptase (hTERT), Telomerase, Wilms tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2 TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotype, Bence Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA125), cancer antigen 19-9 (CA 19-9), β-human chorionic gonadotropin, 1-2 microglycurin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, salglamostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cell 4 (ART-4), carcinoembryonic antigen peptide 1 (CAP-1), calcium-activated chloride ion channel 2 (CLCA2), cyclophyllin B (Cyp-B), human signet ring tumor 2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7,It has been conceivable that antibodies, peptides, polypeptides, small molecule inhibitors, siRNAs, miRNAs, or gene therapies may target major and minor capsid antigens, Epstein-Barr virus (EBV) proteins (EBV latent membrane proteins - LMP1, LMP2, etc.), hepatitis B or C virus proteins, and HIV proteins.

[0247] IX. Product or Kit Also provided herein are manufactured articles or kits comprising at least one CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein), as well as nucleic acids encoding p53, ADP, and / or MDA-7 (e.g., ad-p53 and / or ad-MDA-7). The product or kit may further include a package insert containing instructions for using at least one CD122 / CD132 agonist in combination with tumor suppressor gene therapy to treat cancer in an individual, delay the progression of cancer, or enhance the immune function of an individual with cancer. Any of the CD122 / CD132 agonists and the nucleic acids encoding p53, ADP, and / or MDA-7 described herein may be included in the product or kit. The kit may further include an extracellular matrix-degrading protein or an expression construct encoding an extracellular matrix-degrading protein.

[0248] In some embodiments, at least one selective CD122 / CD132 agonist (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutain, and / or IL-15 mutain), as well as the nucleic acid encoding p53, ADP, and / or the nucleic acid encoding MDA-7, are present in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or alloy (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and labels on or attached to the container may indicate instructions for use. The product or kit may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. In some embodiments, the product further includes one or more other active ingredients (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for one or more active ingredients include, for example, bottles, vials, bags, and syringes. [Examples]

[0249] X. Examples The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques developed by the inventors to function well in the implementation of the present invention and can therefore be considered to constitute preferred embodiments for its implementation. However, those skilled in the art will understand that, in terms of the present disclosure, many modifications can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention, while still obtaining the same or similar results.

[0250] Example 1 - Ad-p53 and Ad-IL24 tumor suppressor factors, and tumor lysis virus (VirRx007) immunogene therapy combined with selective CD122 / 132 agonists and immune checkpoint inhibitors for enhanced local and systemic efficacy and reversal of resistance to previous immunotherapy. The efficacy of combining CD122 / CD132 agonists with tumor suppressors and viral oncolysis immunotherapy to enhance local and systemic antitumor effects, including in tumors resistant to previous immunotherapy, was demonstrated in immune-responsive animal tumor models. The following treatment methods, doses, and schedules were used:

[0251] Animals, tumor dissemination, and measurement: Pathogen-free C57BL / 6 (B6) male mice (6-8 weeks old, obtained from Charles River Labs) were used. B16F10 melanoma cells (ATCC, 5 × 10⁶) were collected in the right flank of the animals. 5 Cells (suspended in a serum-free medium) were subcutaneously injected to form a "primary tumor." The tumor size was approximately 50 mm. 3 Treatment is initiated when this is reached, and this is called day 1 of treatment. Tumor growth is monitored by measuring the length (L) and width (w) of the tumor, and the following formula is used: Volume = 0.523 L(w) 2 The tumor volume was calculated using [a specific method / tool]. Animals were monitored for up to 40 days, and the tumor was approximately 2000 mm². 3 They sacrificed it when they reached that point.

[0252] Viral vectors used in these experiments were non-replicable human adenovirus type 5 (Ad5) encoding either the p53 or IL24 tumor suppressor gene, and replicable tumor-disintegrating adenovirus modified to overexpress ADP (VirRx007). The construction, properties, and purification of the vectors are reported elsewhere regarding the Ad5 / CMV p53, IL24, and VirRx007 vectors (Zhang 1994; Mahashilkar et al., 2001; U.S. Patent No. 7589069B1). Three of the four doses of the viral vector were administered intratumorally. For Ad-p53 and / or ADP (VRX-007), the viral vector was administered on days 2, 5, and 8. For Ad-IL24, the vector was administered on days 3, 5, 7, and 9 (at 48-hour intervals). In the group evaluating viral therapy combining CD122 / CD132 agonists and immune checkpoint inhibitors, an additional intratumoral viral injection was administered on day 21. Each viral dose was 5 × 10¹⁶ units in a volume of 50 μL. 9 It contained virus particles.

[0253] CD122 / CD132 agonist therapy: For the B16F10 model, mouse IL-2 (eBioscience or R&D Systems Minneapolis, MN) was mixed with S4B6-1 anti-mouse IL-2 antibody (Bioxcell, West Lebanon, NH or BD Biosciences) in a 2:1 molar ratio to create selective CD122 / CD132 agonist immune complexes. For studies involving human T cells, human IL-2 was mixed with MAB602 anti-human IL-2 antibody (R&D Systems). IL-2 / S4B6 or IL-2 / MAB602 mAb immune complexes were administered intraperitoneally (IP) at a dose of 2.5 μg of IL-2 on days 2, 6, and 10. Alternatively, IL-2 / S4B6 mAb immune complexes were injected on days 2-6 (1.0 μg of IL-2). Immune complexes are prepared by incubating anti-IL-2 monoclonal with IL-2 at room temperature for 15 minutes.

[0254] In several mouse experiments, the CD122 / CD132 agonist included recombinant mouse IL-15 (eBiosciences) and IL-15-Rα-Fc (R&D Systems). These were combined and incubated at 37°C for 30 minutes to prepare immune complexes. Once the tumors were palpable, this selective CD122 / CD132 agonist immune complex was administered intravenously for two consecutive days. An alternative schedule involves administering the IL-15 immune complex intraperitoneally on days 3, 5, and 7 after the tumors are palpable. For IL-15 immune complex studies, recombinant mouse IL-15 (Peprotech, Rocky Hill, CT, USA) is used in vivo in a dose of 2 μg of recombinant mouse IL-15 per injection, administered intravenously once a week. Recombinant mouse IL-15Rα Fc chimeric protein was obtained from R&D Systems (Minneapolis, MN) and used in equimolar doses with IL-15 cytokine (12 μg of IL-15-Ra-Fc per injection for every 2 μg of IL-15 protein in the immune complex).

[0255] Immune checkpoint inhibitors: To mimic the general clinical state of tumor progression during immune checkpoint inhibitor therapy, anti-PD-1 therapy was initiated intraperitoneally on day 1 at a dose of 200 μg per mouse and administered every 3 days until day 30. In some experiments, to evaluate the efficacy of tumor suppression and tumor lysis virus VirRx007 therapy in combination with selective CD122 / 132 agonists and immune checkpoint inhibitors, which were resistant to previous immunotherapy, tumor suppression therapy was initiated after tumor progression with anti-PD-1 therapy by administering the first tumor suppression therapy dose for 1-2 days after initiating anti-PD-1 therapy. B16F10 and B16 melanoma models are known to be highly resistant to immunotherapy. In these models, tumors progress similarly with immune checkpoint inhibitors and selective CD122 / 132 therapy to control therapy using phosphate-buffered saline (PBS). We purchased a specially formulated anti-mouse PD-1 antibody (CD279) for in vivo use from BioXcell (catalog number BE0146).

[0256] Reversal of resistance to previous immunotherapy: The ability of selective CD122 / CD132 agonists and tumor suppressor or viral tumor-cleaving therapies combined with immune checkpoint inhibitor therapy to reverse resistance to previous immunotherapy was also demonstrated. To mimic the general clinical state of tumor progression during immune checkpoint inhibitor therapy, anti-PD-1 therapy was initiated intraperitoneally on day 1 at a dose of 10 mg / kg and administered every 3 days until day 30. In some experiments, to evaluate the effect of tumor suppressor or viral tumor-cleaving therapy combined with CD122 / CD132 therapy in tumors resistant to previous immunotherapy, the initial doses of tumor suppressor and CD122 / CD132 therapy were administered for 1-2 days after initiating anti-PD-1 therapy, and combination therapy was initiated after tumor progression with anti-PD-1 therapy. These studies were conducted in B16F10 and B16 melanoma models, which are known to be highly resistant to immunotherapy. In these models, tumors progress similarly with immune checkpoint inhibitor therapy compared to control therapy using phosphate-buffered saline (PBS). An anti-mouse PD-1 antibody (CD279), specifically formulated for in vivo use and acting as an immunomodulatory agent against PD-L1 and anti-LAG-3, was purchased from BioXcell (catalog number BE0146). The anti-mouse PD-L1 antibody (clone 9G2, Biolegend) and / or anti-CTLA-4 antibody (clone UC10-4F10-11;Altor) were administered intraperitoneally at a dose of 100 μg twice weekly for two weeks.

[0257] The effectiveness of treatment and its synergistic interactions were demonstrated by measuring tumor volume in the primary and contralateral tumors, and by statistical analyses of these analyses using t-tests, analysis of variance (ANOVA), and Kruskal-Wallis ANOVA, as well as by comparing survival rates using Kaplan-Meier and log-rank tests.

[0258] Surprisingly, the findings revealed a substantial synergistic effect between the Ad-p53+CD122 / 132+anti-PD-1 and VirRx007+CD122 / 132+anti-PD-1 therapies, resulting in a potential curative treatment associated with complete tumor remission in both the primary and contralateral tumors. This effect demonstrated a remarkably superior abscopal effect in isolated tumors not injected with the tumor suppressor therapy. These effects resulted in exceptionally long overall survival. Statistically significant improvements in reduced tumor growth and increased survival were also observed with the Ad-IL24+CD122 / 132+anti-PD-1 therapy.

[0259] Ad-p53 + CD122 / 132 agonist and checkpoint inhibitor immunotherapy: The efficacy of Ad-p53 treatment in combination with CD122 / 132 agonist and anti-PD-1 therapy was evaluated by confirming tumor volume (in primary and contralateral tumors), complete tumor response, and survival. Regarding primary tumor volume, the graph in Figure 4 shows the tumor volume over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, Ad-p53, or a combination of Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, or Ad-p53 + CD122 / 132 + anti-PD-1. There was significant tumor progression between CD122 / 132, anti-PD-1, and CD122 / 132 + anti-PD-1 therapies, which was reversed by combining them with Ad-p53 therapy. Compared to any of the monotherapys, the efficacy of Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, and Ad-p53 + CD122 / 132 + anti-PD-1 therapies was improved. By day 21, the mean tumor volume in all groups treated with (PBS), CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, and Ad-p53 was 2,000 mm². 3The tumor volume exceeded 2,000 mm³. In contrast, combination therapy with Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, and Ad-p53+CD122 / 132+anti-PD-1 each induced a significant reduction in tumor volume compared to non-Ad-p53 therapy or Ad-p53 therapy alone. Statistical analysis of variance (ANOVA) of tumor volume at day 21 measured the synergistic antitumor effect of Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, and Ad-p53+CD122 / 132+anti-PD-1 therapy (p<0.0001). However, by day 30, the mean tumor volume in the Ad-p53+CD122 / 132 and Ad-p53+anti-PD-1 therapy groups also exceeded 2,000 mm³. 3 It exceeded [value]. Importantly, statistical analysis of variance (ANOVA) comparison of tumor volume at day 30 measured that the synergistic antitumor effect was maintained only in the Ad-p53 + CD122 / 132 + anti-PD-1 treatment combination (p < 0.0001 (overall) and p < 0.0001 (individual) compared to each of the separate treatment groups).

[0260] Ad-p53 treatment group: Evaluation of complete tumor response rate. Complete tumor response to a treatment is generally understood to be associated with important therapeutic efficacy and is necessary for a curative outcome. As shown in Figure 5, for the p53 treatment group and their controls, only Ad-p53 + CD122 / 132 + anti-PD-1 treatment resulted in complete tumor remission in both primary and contralateral tumors. Complete tumor response, for both primary and contralateral tumors, was observed in 60% of the Ad-p53+CD122 / 132+anti-PD-1 treatment group, while complete tumor response for both primary and contralateral tumors was not observed in any of the other treatment groups (Fisher's two-sided exact test comparing Ad-p53+CD122 / 132+anti-PD-1 group versus all other treatment groups, p-value < 0.0001; Fisher's two-sided exact test comparing Ad-p53+CD122 / 132+anti-PD-1 group versus all other treatment groups, p-value < 0.011). Unexpectedly, the complete tumor response was persistent and was maintained at 40 days in 50% of the Ad-p53+CD122 / 132+anti-PD-1 group, likely curing these animals with these tumors.

[0261] Ad-p53 Treatment Group: Systemic / Abscopal Therapy Effect on Contralateral Tumor Growth. The systemic / abscopal effect of primary tumor treatment on contralaterally transplanted tumors was evaluated in rodents whose primary tumors received one of the Ad-p53 treatments, and the results are shown in Figure 6. Consistent with the unexpectedly substantially increased synergistic effect of Ad-p53 + CD122 / 132 + anti-PD-1 treatment on primary tumor growth and complete remission rates, we also observed a remarkably potent and statistically significant abscopal effect of Ad-p53 + CD122 / 132 + anti-PD-1 treatment compared to other Ad-p53 treatment groups. As shown in Figure 6A, contralateral tumor growth disappeared in 90% (9 out of 10 animals) of animals treated with Ad-p53 + CD122 / 132 + anti-PD-1 for primary tumors. In contrast, contralateral tumor growth was observed in 62.5–100% of animals in the other Ad-p53 treatment groups. This difference in contralateral tumor growth was statistically significant (p-value by chi-square test for all treatment groups = 0.0004; p-value by Fisher's two-sided exact test comparing Ad-p53 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group < 0.0430). Figure 6B shows a graph of contralateral tumor volume over time in rodents receiving one of the three most effective primary tumor treatments: Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, or any combination of Ad-p53 + CD122 / 132 + anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes on day 22 measured the synergistic antitumor effect of Ad-p53 + CD122 / 132 + anti-PD-1 therapy (p-value = 0.0435 overall). Only the Ad-p53 + CD122 / 132 + anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the Ad-p53 + anti-PD-1 group (p-value = 0.0360). Taken together, these findings indicate that of all Ad-p53 treatments, only the three combinations of Ad-p53 + CD122 / 132 + anti-PD-1 therapy resulted in curative efficacy by inducing potent local and systemic antitumor immunity mediated by a substantial abscopal effect.

[0262] Ad-p53 treatment group: Efficacy of treatment leading to sustained survival. Kaplan-Meier survival curves for mice treated with PBS, CD122 / 132 + anti-PD-1, Ad-p53, or Ad-p53 + CD122 / 132, Ad-p53 + anti-PD-1, and Ad-p53 + CD122 / 132 + anti-PD-1 are shown in Figure 7. Log-rank tests showed statistically significant differences in these survival curves (p<0.0001 overall; p<0.0003 when comparing the Ad-p53 + CD122 / 132 + anti-PD-1 treatment group to any other treatment group). The unexpected substantial synergistic effect of the Ad-p53 + CD122 / 132 + anti-PD-1 treatment also indicates this result. In the group treated with Ad-p53 + CD122 / 132 + anti-PD-1 therapy, the median survival was not even reached at 40 days, with 80% of animals still alive. In stark contrast, 98% (49 out of 50) of animals in the other treatment groups died by day 30, with median survival ranging from 10 to 28 days.

[0263] VirRx007 + CD122 / 132 agonist and checkpoint inhibitor immunotherapy: The equally impressive and unexpected therapeutic efficacy of VirRx007 in combination with CD122 / 132 agonists and anti-PD-1 therapy was also observed by confirming tumor volume (in primary and contralateral tumors), complete tumor response, and survival. Regarding primary tumor volume, the graph in Figure 8 shows the tumor volume over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132 + anti-PD-1, CD122 / 132 + anti-PD-1, VirRx007, or any combination of VirRx007 + CD122 / 132, VirRx007 + anti-PD-1, or VirRx007 + CD122 / 132 + anti-PD-1. There was significant tumor progression between CD122 / 132, anti-PD-1, and CD122 / 132 + anti-PD-1 therapies, which was reversed by combination with VirRx007 therapy. The results show improved efficacy of VirRx007 + anti-PD-1 and VirRx007 + CD122 / 132 + anti-PD-1 therapies compared to any of the monotherapys. In contrast to findings with Ad-p53, VirRx007 did not show synergy with CD122 / CD132 therapy. By day 30, the mean tumor volume exceeded 2,000 mm3 in all groups treated with PBS, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, VirRx007, and VirRx007 + CD122 / CD132. In contrast, both combination therapies of VirRx007 + anti-PD-1 and VirRx007 + CD122 / 132 + anti-PD-1 induced a significant reduction in tumor volume compared to either non-VirRx007 therapy or VirRx007 therapy alone. Statistical analysis of variance (ANOVA) of tumor volume at day 30 measured the synergistic antitumor effect of VirRx007 + anti-PD-1 and VirRx007 + CD122 / 132 + anti-PD-1 therapy (p < 0.0001, overall and for each of these therapies versus VirRx007). VirRx007 + CD122 / 132 + anti-PD-1 therapy was superior to VirRx007 + anti-PD-1 (p = 0.0002).Surprisingly, while the combination therapy of VirRx007 + CD122 / 132 did not show any clear advantage compared to VirRx007 monotherapy, a synergistic effect was demonstrated with the triple therapy of VirRx007 + CD122 / 132 + anti-PD-1.

[0264] Evaluation of complete tumor response rates in the VirRx007 treatment group. Complete tumor response to a treatment is generally understood to be associated with a significant therapeutic effect and is necessary for a curative outcome. As shown in Figure 9, in the VirRx007 treatment group and their controls, only VirRx007 + CD122 / 132 + anti-PD-1 treatment resulted in complete tumor remission in both primary and contralateral tumors. Complete tumor response, for both primary and contralateral tumors, was observed in 60% of the VirRx007+CD122 / 132+anti-PD-1 treatment group, while complete tumor response for both primary and contralateral tumors was not observed in any of the other treatment groups (Fisher's two-sided exact test comparing the VirRx007+CD122 / 132+anti-PD-1 group versus all other treatment groups, p-value < 0.0001; Fisher's two-sided exact test comparing VirRx007+CD122 / 132+anti-PD-1 versus all other treatment groups, p-value < 0.011). Unexpectedly, the complete tumor response was persistent and likely cured these animals with these tumors, and was maintained at 40 days in 50% of the VirRx007+CD122 / 132+anti-PD-1 treatment group.

[0265] VirRx007 Treatment Group - Effect of Systemic / Abscopal Therapy on Contralateral Tumor Growth. The systemic / abscopal effect of primary tumor treatment on contralaterally transplanted tumors was evaluated in rodents whose primary tumors received one of the VirRx007 treatments, and the results are shown in Figure 10. Consistent with the unexpectedly substantially increased synergistic effect of VirRx007 + CD122 / 132 + anti-PD-1 treatment on primary tumor growth and complete remission rates, we also observed a remarkably potent and very statistically significant abscopal effect of VirRx007 + CD122 / 132 + anti-PD-1 treatment compared to other VirRx007 treatment groups. As shown in Figure 10A, contralateral tumor growth disappeared in 80% of animals receiving VirRx007 + CD122 / 132 + anti-PD-1 primary tumor treatment. In contrast, contralateral tumor growth was observed in 80–100% of animals in the other VirRx007 treatment groups. This difference in contralateral tumor growth was statistically significant (p-value = 0.0002 by chi-square test comparing all treatment groups; p-value < 0.0230 by Fisher's two-sided exact test comparing VirRx007 + CD122 / 132 + anti-PD-1 treatment group versus any other treatment group). These findings suggest that the VirRx007 + CD122 / 132 + anti-PD-1 treatment combination induced potent systemic antitumor immunity and mediated a substantial abscopal effect with potential curative efficacy. Figure 10B shows a graph of contralateral tumor volume over time in rodents receiving one of three most effective combination therapies for primary tumors: VirRx007+CD122 / 132, VirRx007+anti-PD-1, or VirRx007+CD122 / 132+anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes at day 22 measured the synergistic antitumor effect of VirRx007+CD122 / 132+anti-PD-1 treatment (p-value = 0.0171 overall). Only the VirRx007+CD122 / 132+anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the VirRx007+anti-PD-1 group (p-value = 0.0115).These findings, taken together, indicate that of all VirRx007 treatments, only the three combinations of VirRx007 + CD122 / 132 + anti-PD-1 therapy resulted in curative efficacy by inducing potent local and systemic antitumor immunity that mediated a substantial abscopal effect.

[0266] VirRx007 treatment group – efficacy of the treatment resulting in sustained survival. Figure 11 shows Kaplan-Meier survival curves for mice treated with either PBS, CD122 / 132 + anti-PD-1, VirRx007, or any combination of VirRx007 + CD122 / 132, VirRx007 + anti-PD-1, or VirRx007 + CD122 / 132 + anti-PD-1. Log-rank tests showed statistically significant differences in these survival curves (p<0.0001 overall; p<0.0005 for comparisons between the VirRx007 + CD122 / 132 + anti-PD-1 treatment group and any other treatment group). The unexpected substantial synergistic effect of the VirRx007 + CD122 / 132 + anti-PD-1 treatments also demonstrates these results. The median survival in the VirRx007 + CD122 / 132 + anti-PD-1 treatment group did not reach 40 days, with 90% of animals still alive. In striking contrast, 98% (49 / 50 animals) in the other treatment groups died by day 40, with median survival ranging from 10 to 33 days. Surprisingly, while there was no clear survival advantage for the VirRx007 + CD122 / 132 combination therapy compared to VirRx007 monotherapy, a synergistic effect was demonstrated for the VirRx007 + CD122 / 132 + anti-PD-1 triple therapy.

[0267] Ad-IL24 + CD122 / 132 agonist and checkpoint inhibitor immunotherapy: The efficacy of similar excellent therapies, such as Ad-IL24 in combination with CD122 / 132 agonists and anti-PD-1 therapy, was also observed by evaluating primary tumor volume and survival. Regarding primary tumor volume, the graph in Figure 12 shows the tumor volume over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, Ad-IL24, or a combination of Ad-IL24 + CD122 / 132 or Ad-IL24 + CD122 / 132 + anti-PD-1. There was significant tumor progression between CD122 / 132, anti-PD-1, and CD122 / 132 + anti-PD-1 therapies, which were reversed by combination with Ad-IL24 therapy. Compared to any of the monotherapy methods, Ad-IL24 + CD122 / 132 + anti-PD-1 therapy demonstrated improved efficacy. By day 16, the mean tumor volume in all groups treated with PBS, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, and Ad-IL24 exceeded 2,000 mm3. In contrast, the combination therapy of Ad-IL24 + CD122 / 132 + anti-PD-1 induced a substantial reduction in tumor volume compared to either non-Ad-IL24 therapy or Ad-IL24 therapy alone. Statistical analysis of variance (ANOVA) of tumor volume at day 16 measured the synergistic antitumor effect of Ad-IL24 + CD122 / 132 + anti-PD-1 therapy (p < 0.0001). Compared to either Ad-IL24 (p=0.0025) or CD122 / 132 + anti-PD-1 therapy (p<0.0001), Ad-IL24 + CD122 / 132 + anti-PD-1 therapy resulted in a statistically significant reduction in tumor volume.

[0268] Ad-IL24 Treatment Group - Efficacy of Treatment for Sustained Survival. Figure 13 shows Kaplan-Meier survival curves for mice treated with either PBS, CD122 / 132 + anti-PD-1, Ad-IL24, or a combination of Ad-IL24 + CD122 / 132 + anti-PD-1. Log-rank tests showed statistically significant differences between these survival curves (p<0.0001). This result demonstrates an unexpected, substantial synergistic effect of the Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group. Median survival was synergistically improved in the Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group. All animals in the PBS, CD122 / 132 + anti-PD-1, and IL24 treatment groups died by day 16, while 50% of animals in the Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group were alive at day 19. The Ad-IL24 + CD122 / 132 + anti-PD-1 treatment group showed statistically significantly improved survival compared to either Ad-IL24 alone (p=0.0003) or CD122 / 132 + anti-PD-1 treatment group (p<0.0001). Interestingly, the Ad-IL24 + CD122 / 132 pair showed remarkably superior efficacy compared to the CD122 / 132 + anti-PD-1 pair (log-rank test p=0.0002, data not shown).

[0269] Ad-luciferase (Ad-Luc) negative control. Ad-Luc control + CD122 / 132 agonist + anti-PD-1: Tumor volume. The graph in Figure 14 shows the volume of primary tumors over time in rodents receiving one of the following combinations: phosphate-buffered saline (PBS) control, CD122 / 132, anti-PD-1, CD122 / 132 + anti-PD-1, Ad-Luc control, or Ad-Luc control + CD122 / 132, Ad-Luc control + anti-PD-1, and Ad-Luc control + CD122 / 132 + anti-PD-1. In contrast to treatment with Ad-p53, VirRx007, and Ad-IL24, there was no significant increase in treatment efficacy when Ad-Luc was combined with anti-PD-1, CD122 / 132, or CD122 / 132 + anti-PD-1 treatment. By day 16, the mean tumor volume in all groups exceeded 2,000 mm³. Statistical analysis of variance (ANOVA) of tumor volume at day 16 was not statistically significant (p-value = 0.1212; the mean tumor volume across all treatment groups was not statistically significant).

[0270] The superiority of the "triple treatment" regimens of Ad-p53, VirRx007, and Ad-IL24 combined with CD122 / 132 + anti-PD-1 compared to Ad-Luc-negative controls using CD122 / 132 + anti-PD-1. Figure 15 clearly shows that the "triple treatment" regimens of Ad-p53, VirRx007, and Ad-IL24 combined with CD122 / 132 + anti-PD-1 each statistically significantly increased survival compared to treatment with Ad-Luc + CD122 / 132 + anti-PD-1. According to the log-rank test, there was a statistically significant difference in these survival curves (p<0.0001). Ad-p53, VirRx007, and Ad-IL24, when combined with CD122 / 132+anti-PD-1 triple therapy, each showed a statistically significant increase in survival compared to the Ad-Luc+CD122 / 132+anti-PD-1 triple therapy control group (by log-rank test, both Ad-p53 and VirRx007 combined with CD122 / 132+anti-PD-1 triple therapy had p-values ​​< 0.0001; Ad-IL24 combined with CD122 / 132+anti-PD-1 had p-values ​​< 0.015).

[0271] Experiments involving CD122 / CD132 agonists consisting of recombinant mouse IL-15 and IL-15-Rα-Fc. In these studies, selective CD122 / CD132 agonists were prepared by incubation of these reagents together at 37C for 30 minutes, and the resulting immune complexes were intraperitoneally injected on days 3, 5, and 7 after the tumors became palpable.

[0272] Ad-p53 + CD122 / 132 (IL15) agonist and checkpoint inhibitor immunotherapy - Tumor volume: The efficacy of Ad-p53 treatment in combination with IL15-based CD122 / 132 agonists and anti-PD-1 therapy was evaluated by confirming tumor volume (in primary and contralateral tumors) and survival. Regarding primary tumor volume, the graph in Figure 16 shows the tumor volume over time in rodents receiving either phosphate-buffered saline (PBS) control, CD122 / 132 + anti-PD-1, Ad-p53 alone, or a combination of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1. There was significant tumor progression between PBS, CD122 / 132 + anti-PD-1, and Ad-p53 treatments. Consistent with the results of the initial Ad-p53 combination therapy described above, Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy demonstrated substantially improved efficacy compared to all other treatments. By day 30, the mean tumor volume in the groups treated with PBS, CD122 / 132 + anti-PD-1, and Ad-p53 all exceeded 2,000 mm3. In contrast, the combination therapy using Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 induced a significant reduction in tumor volume. Statistical analysis of variance (ANOVA) of tumor volume measured the synergistic antitumor effect of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy (p < 0.0001 (overall) and p < 0.0001 when compared individually with each of the other treatment groups).

[0273] Ad-p53 + CD122 / 132 (IL15) agonist and checkpoint inhibitor immunotherapy - Systemic / abscopal therapeutic effects on contralateral tumor growth. The systemic / abscopal effects in the treatment of primary tumors of contralaterally transplanted tumors were evaluated in rodents whose primary tumors received one of the Ad-p53 therapies, and the results are shown in Figure 17. Consistent with the unexpected, substantially increased synergistic effect of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy on primary tumor growth shown in Figure 16, we also observed a remarkably potent and highly statistically significant abscopal effect of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy compared to other Ad-p53 therapy groups. Figure 17 shows a graph illustrating the time course of contralateral tumor volume in rodents receiving primary tumor treatment with one of the following combinations: Ad-p53+CD122 / 132, Ad-p53+anti-PD-1, or Ad-p53+CD122 / 132(IL15)+anti-PD-1. Statistical analysis of variance (ANOVA) of these contralateral tumor volumes at day 22 measured the synergistic antitumor effect of Ad-p53+CD122 / 132(IL15)+anti-PD-1 treatment (p-value = 0.0433 overall). Only the Ad-p53+CD122 / 132+anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the Ad-p53+anti-PD-1 group (p-value = 0.0359).

[0274] Ad-p53 + CD122 / 132 (IL15) agonist and checkpoint inhibitor immunotherapy – therapeutic effects resulting in sustained survival. Figure 18 shows Kaplan-Meier survival curves for mice treated with either PBS, CD122 / 132 + anti-PD-1, Ad-Luc + CD122 / 132 + anti-PD-1 control, Ad-p53, or a combination of Ad-p53 + CD122 / 132 (IL15) + anti-PD-1. Log-rank tests showed statistically significant differences in these survival curves (overall p<0.0001, p-value <0.0001 comparing the Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 treatment group to any other treatment group). The results further demonstrate an unexpected, substantial synergistic effect of the Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 treatment. In the group treated with Ad-p53 + CD122 / 132 (IL15) + anti-PD-1 therapy, 50% of the animals were alive on day 36. In stark contrast, all animals in the other treatment groups died by day 22, with a median survival of 10–18 days.

[0275] Example 2 - Application using a vaccinia vector modified with N1L deficiency, IL12 expression, and a selective CD122 / CD132 agonist in combination with a PI3Kδ / γ inhibitor, for both topical and systemic administration. In another embodiment of this therapeutic approach, a novel tumor-disintegrating vaccinia virus called VVL 15-N1L-IL12 is used as an additional therapeutic virus to enhance the efficacy of the approach described in Example 1 above. Several strains of tumor-disintegrating vaccinia virus, such as the Western Reserve, Wyeth, and Lister strains, have been reported. Various knockout mutants of each of these strains have been created. Wang et al. (International Publication No. 2015 / 150809A1) have developed a TK-deficient vaccinia virus strain with an inactivated N1L gene that exhibits improved selectivity and antitumor efficacy. N1L is thought to inhibit apoptosis and NF-κB activation in infected cells. In addition to regulating the natural killer (NK) cell response, deletion of the N1L gene has been shown to result in an increase in NF-κB-regulated pro-inflammatory antiviral cytokines. N1L-deficient derivatives are described in Wang et al., 2015 (Patent International Publication No. 2015 / 150809A1). To enhance the antitumor efficacy of VVL 15N1L, GM-CSF, IL-12, and IL-21, tumor suppressor factors and other therapeutic genes are inserted into the N1L region of the VVL 15N1L vector. These therapeutic "armed" VVL 15N1L vectors are used one at a time or in combination with the treatments described in Example 1 above to improve the local and abscopal effects of the treatment.

[0276] In addition to evaluating the approaches described in Examples 1 and 2 above, viral vectors are also combined with PI3K inhibitors. Examples incorporating PI3Kδ or PI3Kγ / δ inhibitors are described to improve the intravenous administration of viral vectors. Animals received 75 mg / kg via the tail vein. -1 At a concentration of IC87114 (PI3Kδ inhibitor), 3 hours later, 1 × 10¹⁶ units were added to 100 μL of PBS. 8PFU / mice receive intravenous VVL 15N1L vector. This treatment is administered at least three times on days 0, 3, and 5. These treatments are combined with the same therapies as described above. Tumor size and animal survival are measured, and the data are analyzed as described above, showing increased efficacy of therapies combined with Ad-p53 and / or Ad-IL24 and / or VVL 15N1L vector, CD122 / CD132 agonists, immune checkpoint inhibitors, and PI3K inhibitors.

[0277] Example 3 - Combination therapy including intratumor Ad-p53, CD122 / CD132 agonist, and anti-PD-1 therapy in patients with progression of prior treatment including both immunotherapy. Each vial is supplied with a volume of 2 mL of Ad-p53, and 1 × 10⁶ units are used per 1 mL. 12 It contains viral particles (vp). Ad-p53 is supplied as a sterile viral suspension in phosphate-buffered saline (PBS) containing 10% (v / v) glycerol as a stabilizer. Before administration, dilute and filter Ad-p53 according to the procedure described. Administer the anti-PD-1 therapy according to the instructions for use in the FDA-approved package insert. CD122 / CD132 agonist therapy (e.g., IL-2 / anti-IL-2 immune complex, IL-15 / anti-IL-15 immune complex, IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, PEGylated IL-2, PEGylated IL-15, IL-2 mutein, and / or IL-15 mutein) should be administered subcutaneously or intravenously at intervals ranging from weekly, bi-weekly, every three weeks, or every four weeks, in doses ranging from 5 to 100 μg / kg. CD122 / CD132 agonists can be IL-15 variants (e.g., IL-15N72D) bound to the IL-15 receptor α / IgG1 Fc fusion protein, such as ALT-803.

[0278] Therapies are designed to improve the prognosis of patients with advanced HNSCC, for example, by using Ad-p53, selective CD122 / CD132 agonists, and anti-PD-1 antibodies. Clinical efficacy of combination therapy includes assessment of overall response rate [ORR = partial response (PR) + complete response (CR)], complete remission rate (CRR), sustained response rate (DRR = PR + CR maintained for at least 6 months); rate and time of metastasis to endovascular organs; progression-free survival (PFS) and overall survival (OS). The effects of the study drugs on lymphocyte phenotype and serum cytokines, disease-related biomarkers, antibody responses to selected antigens, and humoral and cellular responses to tumor antigens are also evaluated. Efficacy endpoints correlate with PD-L1, PD-L2, immune cell infiltrations, and tumor mutational burden biomarkers in diagnostic analyses.

[0279] Patients receive intratumoral injections of Ad-p53 every 28 days on days 1, 2, and 3. Nivolumab infusions are administered every two weeks, starting on day 5, along with selective CD122 / CD132 agonist therapy (e.g., IL2 / anti-IL2 immune complex, and / or IL15 / anti-IL15 immune complex, and / or IL15 / IL15 receptor α-IgG1-Fc (IL15 / IL15Rα-IgG1-Fc) immune complex, and / or PEGylated IL2, and / or PEGylated IL15, and / or IL2 mutein, and / or IL15 mutein) at intervals ranging from weekly, bi-weekly, every three weeks, or every four weeks, either subcutaneously or intravenously, in doses ranging from 5 to 100 μg / kg.

[0280] Determination of Ad-p53 injection dose (mL) and Ad-p53 injection method based on the tumor injury diameter described in Table 3. Table 3 should be used to identify the Ad-p53 injection dose (mL) for each tumor injury corresponding to the two-dimensional injury diameter measured by CT or MRI scan. The Ad-p53 injection dose (mL) should be 1 × 10⁻⁶. 12The amount of virus particles (vp) / mL must be subtracted from the provided 2 mL vial. The injectable dose (mL) of Ad-p53 corresponding to the diameter of the injury described is 1 cm 3 At least 1 × 10⁻¹⁴ per tumor volume 11 Each tumor injury received by the Ad-p53 dose of viral particles (VP) results in a therapeutic dose. This therapeutic dose was determined from tumor response, survival, and safety data from previous Ad-p53 clinical trials.

[0281] All tumor damage should be treated. However, the MTD of Ad-p53 is 2.5 × 10⁶ per treatment day. 13 Since this is a VP (Voluntary Practice) treatment, the total amount of Ad-p53 injected (mL) must be less than 25 mL. The table below lists the corresponding Ad-p53 injection methods used for each injury, based on the amount of Ad-p53 injected (mL). Based on the amount of Ad-p53 injected (mL), use the Ad-p53 injection method listed in the table below for each injury. According to the instructions for use in the package insert, an Ad-p53 injection dose of 2 mL or less must be administered using the fine-needle syringe technique; however, if using the Quadra-Fuse device shown below, an Ad-p53 injection dose of 4 mL or less must be administered.

[0282] [Table 3] a: Diameter of the damage - L is the longer diameter and W is the shorter diameter (cm) (rounded up to the nearest integer). b: 1cm 3 Approximately 1 × 10⁻¹⁶ per tumor volume. 11 Each tumor damage received by the Ad-p53 dose of viral particles (vp) results in 1 × 10⁻¹⁶ 12 Injectable dose (mL) of Ad-p53 from a vial containing vp (viral particles) / mL. The MTD of Ad-p53 is 2.5 × 10⁶ per treatment day. 13 Since this is a VP (Voluntary Patient) procedure, the total amount of Ad-p53 injected (mL) must be less than 25mL. c: Method of injection for Ad-p53.

[0283] Ad-p53 injection method: Based on the amount of Ad-p53 injection dose (mL) used, the Ad-p53 injection method listed in Table 3 should be used for each injury. Using fine-needle syringe techniques, an Ad-p53 injection dose (mL) of 2 mL or less must be administered; however, if using the Quadra-Fuse device described, an Ad-p53 injection dose (mL) greater than 2 mL must be administered.

[0284] Fine-needle syringe for Ad-p53 injection doses (mL) of 2 mL or less: For injuries where the Ad-p53 injection dose (mL) is 2 mL or less, the Ad-p53 injection dose (either 1 or 2 mL) must be delivered using a standard 1 mL syringe equipped with a 27 gauge needle. One-quarter of the total Ad-p53 injection dose (mL) must be injected into each quadrant of the tumor injury where the needle is positioned, maximizing the distribution of the injection within each quadrant.

[0285] Quadra-Fuse device for Ad-p53 injectable doses (mL) exceeding 2 mL: Regarding damage when the injectable dose (mL) of Ad-p53 exceeds 2 mL, the injectable dose of Ad-p53 must be delivered using a Quadra-Fuse delivery device (Rex Medical, PA). The Quadra-Fuse device (FDA Grade 1 medical device) consists of a single central trocar from which three prisms extend radially with an adjustable diameter of 1–5 cm (Figure 3).

[0286] As detailed below, the Quadra-Fuse device enables the precise and simultaneous delivery of drugs to multiple areas of injury through diffusion.

[0287] 1. Dispense the appropriate injection dose (mL) of Ad-p53 from Table 3, corresponding to the two-dimensional diameter of the tumor injury, into a standard syringe and attach it to the expansion tube of the Quadra-Fuse device.

[0288] 2. Treat the lower half of the tumor injury first, using the device's depth marker to position the central trocar end of the Quadra-Fuse at the bottom of the longest length diameter (L) of the injury, and align it with the longest diameter of the injury under CT or ultrasound guidance. Adjust the Quadra-Fuse device's pointed array treatment diameter to less than 1 cm of the shortest width diameter (W) of the tumor injury (pointed array treatment diameter = width tumor diameter - 1 cm). After adjusting the Quadra-Fuse device to the pointed array treatment diameter, open the pointed end and deliver a quarter dose (mL) of Ad-p53 to this location. (Note: Use the same pointed array treatment diameter for all four doses of Ad-p53 for each injury.) Retract the pointed end and rotate the device 60° at the same depth. Then, open the pointed end again and deliver a second quarter dose (mL) of Ad-p53 to this location. These procedures ensure that half of the therapeutic dose of Ad-p53 is efficiently delivered to the lower half of the tumor injury.

[0289] 3. To treat the upper half of the tumor injury, retract the apex and move the central trocar end to the midpoint of the longest L diameter of the tumor injury. Extend the apex again to the treatment diameter of the apex array (= width of tumor diameter - 1 cm) and deliver the third quarter injection dose (mL) of Ad-p53 to this position. Retract the apex and rotate the device 60° at the same midpoint depth of the tumor. Then, open the apex again and deliver the last quarter injection dose (mL) of Ad-p53 to this position.

[0290] In this way, a total of 48 sites in each tumor injury receive Ad-p53. In each apex array deployment, each apex has two through-holes (four fluid outlets) for a total of 12 simultaneous drug injection sites (4 apex array deployments × 12 = 48 Ad-p53 delivery sites).

[0291] Treatment of 5 × 4 cm tumor injury: As shown in Table 3, tumors with injury diameters of L (longest diameter) 5 cm and W (shortest diameter) 4 cm are treated with a 4 mL injection dose of Ad-p53 using the Quadra-Fuse Ad-p53 injection method.

[0292] Under CT or US guidance, the central trocar end of the Quadra-Fuse should first be positioned at the bottom of the longer tumor diameter, L=5cm. The apex should be expanded to a apex array therapeutic diameter of 3cm (width tumor diameter 4cm - 1cm = 3cm), and the first quarter therapeutic dose (1mL) should be injected. (Note: Use the same apex array therapeutic diameter for all four injectable doses of Ad-p53 in this injury.) The apex should be retracted, rotating the device 60° at the same bottom of the tumor at a depth of 5cm. The apex should be expanded again to a 3cm apex array therapeutic diameter, and the second quarter dose of Ad-p53, 1mL, should be injected. These steps ensure that half of the injectable dose of Ad-p53 is delivered to the lower half of the tumor.

[0293] To treat the upper half of the tumor injury, the depth of the Quadra-Fuse central trocar is raised to the midpoint of the longest injury diameter = 2.5 cm. The apex is then expanded again to a therapeutic diameter of 3 cm in the apex array, and the therapeutic dose (1 mL) of the third quarter is injected. Along the longest tumor diameter, while maintaining the same intratumoral depth (2.5 cm), the apex is deflated, the central trocar is rotated 60°, and the apex is expanded again to a therapeutic diameter of 3 cm in the apex array, and the fourth and final quarter, 1 mL of Ad-p53, is injected. Through these steps, half of the injected dose of Ad-p53 is delivered to the upper half of the tumor. In total, these steps deliver 4 mL of Ad-p53 to 48 injection points within the tumor injury.

[0294] Treatment duration: Each treatment cycle lasts 28 days (4 weeks). Day 1 of the treatment cycle is the first day of the study treatment administration. The treatment regimen is as follows:

[0295] Treatment regimen: The scheduled treatment days using Ad-p53 are days 1, 2, and 3 every 28 days. Nivolumab is administered every two weeks, starting on day 5. Anti-PD-1 therapies should be administered according to the instructions for use in the FDA-approved prescribing information. Selective CD122 / CD132 agonist therapies (e.g., IL2 / anti-IL2 immune complex, and / or IL15 / anti-IL15 immune complex, and / or IL15 / IL15 receptor α-IgG1-Fc (IL15 / IL15Rα-IgG1-Fc) immune complex, and / or PEGylated IL2, and / or PEGylated IL15, and / or IL2 mutein, and / or IL15 mutein) should be administered subcutaneously or intravenously at intervals ranging from weekly, bi-weekly, every three weeks, or every four weeks, in doses ranging from 5 to 100 μg / kg.

[0296] On day 28 or 29 of the study, tests to assess the tumor's location and measurements will be completed, after which a new treatment cycle will be initiated.

[0297] Unless there is local disease progression (excluding new treatable damage) or an unacceptable adverse event occurs, the patient will be treated in three or more cycles.

[0298] Criteria for evaluating effectiveness: 1. Monitor tumor size by CT or MRI. Measurements will be taken on day 28 or 29 of the study, after which, if using CT or MRI, an injection will be given on day 1 of the third cycle, followed by scans every 8 weeks. Apply RECIST 1.1 criteria. 2. The duration of response is defined as the time elapsed from the response date to the progression date. 3. Progression-free survival is defined as the time elapsed from the date of randomization to the date on which progression was recorded. 4. Overall survival is defined as the time elapsed from randomization to the day of death. 5. Efficacy endpoints correlate with PD-L1, PD-L2, immune cell infiltration, and tumor mutational burden biomarkers in diagnostic analysis.

[0299] Safety assessment: 1. Reporting of adverse events. 2. Physical examination, vital signs test, and laboratory tests, including CBC, biochemistry, and urinalysis. 3. Distribution of adeno vectors in the body based on antibody testing.

[0300] For therapeutic and research patient inclusion criteria, a preferred biomarker for Ad-p53 efficacy is needed, which may be either the wild-type p53 gene sequence or p53-positive tumor cells (less than 20%), as described in Sobol et al., 2012.

[0301] In light of the recent breakthroughs in the appointment and acceleration of authorization of anti-PD-1 in relapsed HNSCC, we conducted a meta-analysis of Ad-p53 treatment data in patients with relapsed HNSCC to identify potential treatment doses and schedules and improve published anti-PD-1 outcomes. The meta-analysis included patients with relapsed HNSCC (n=54) who had a favorable p53 biomarker profile and the majority had received prior surgery, radiotherapy, and platinum-based chemotherapy. In the meta-analysis, the highest response rates were observed in clinical trials, with Ad-p53 administered intratumorally at a schedule of three times per week, either three consecutive days of treatment for the first week or every other week for the first two weeks of each monthly treatment cycle. All responders (as defined by RECIST 1.1 criteria) had a response rate of 7 × 10⁶ per cm³ of tumor volume. 10 The Ad-p53 dose exceeded the viral particle count.

[0302] As shown in the table below, compared to patients treated with a lower dose of Ad-p53, 7 × 10 10 virus particles / cm 3 There was a statistically significant difference in tumor response among patients who received treatment beyond 7 × 10⁻¹⁰. 10 virus particles / cm 3 For Ad-p53 exceeding 53, 7 × 10¹⁶ doses were administered for a 31% (9 / 29) tumor response. 10 virus particles / cm 3For Ad-p53 less than 53, the percentage is 0% (0 / 25); p = 0.0023.

[0303] [Table 6]

[0304] Figure 1 shows 7 × 10 10 virus particles / cm 3 Compared to Ad-p53 doses of less than 7 × 10⁶ (right panel), 10 virus particles / cm 3 This waterfall plot shows tumor response in the subgroup of patients treated with Ad-p53 exceeding a certain threshold (left panel).

[0305] The majority of those who responded (7 out of 9 patients) received 1 × 10 11 vp / cm 3 Nearby, or exceeding (7.81~333.2×10 10 vp / cm 3 Further detailed investigations of Ad-p53 responders revealed that they had received the Ad-p53 dosage.

[0306] In a previous Phase 3 clinical trial of relapsed HNSCC, patients treated with methotrexate had a favorable Ad-p53 biomarker profile, compared to patients treated with 7 × 10⁶ 10 virus particles / cm 3 1-year and overall survival in patients with a favorable Ad-p53 biomarker profile treated with Ad-p53 doses exceeding [value missing]. Results are shown in Figure 2, showing a statistically significant increase in Ad-p53 biomarkers and overall survival with dose-optimized Ad-p53 treatment compared to methotrexate (median survival with Ad-p53 treatment: 11.5 months vs. 4.5 months with methotrexate; p<0.016, HR 1.9767).

[0307] As shown in Table 4, the optimal Ad-p53 treatment for recurrent HNSCC from meta-analysis data was comparable to standard-of-care (SOC) chemotherapy and anti-PD-1 therapy reported by Ferris et al., 2016, in terms of the efficacy endpoint of tumor response, one-year survival, and overall central survival.

[0308] [Table 4-1]

[0309] Therefore, the dose of Ad-p53 for combination therapy with a selective CD122 / CD132 agonist and anti-PD-1 therapy (tumor volume 1 cm) 3 Winning combination: 1 x 10 11 Select vp) based on this data.

[0310] Example 4 - Combination therapy using Ad-MDA7 (IL24), CD122 / CD132 agonist, and anti-PD1 antibody Anti-PD-1 therapy is an approved treatment for patients with advanced, unresectable melanoma. While anti-PD-1 therapy presents a breakthrough treatment that offers benefits to many patients, clinical data from multiple studies indicate that the majority of patients do not respond to this treatment.

[0311] This treatment is designed to improve the prognosis of patients with advanced melanoma through the use of Ad-MDA-7 (Note: Ad-MDA-7 = Ad-IL24), as well as CD122 / CD132 agonists and anti-PD-1 antibodies. The clinical efficacy of the combination therapy includes evaluation of the overall response rate [ORR = partial response (PR) + complete response (CR)], complete remission rate (CRR), sustained response rate (DRR = PR + CR maintained for at least 6 months); rate and time of metastasis to endovascular organs; progression-free survival (PFS) and overall survival (OS). The effects of the study drugs on lymphocyte phenotype and serum cytokines, disease-related biomarkers, antibody responses to selected antigens, and humoral and cellular responses to tumor antigens are also evaluated.

[0312] Furthermore, tumor samples will be investigated for the amount and characteristics of inflammatory infiltrates (e.g., the expression of CD8 and CD4 cells, and programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1), respectively, in lymphocytes and tumor cells), as well as for the pathological correlation of clinical activity, including (but not limited to) tumor mutation loading.

[0313] If the patient responds at that time, they should be treated for a maximum of 12 months or 18 months. Patients who respond (CR or PR) at 12 months must continue treatment until 18 months or until clinically related progressive disease (PDr), whichever comes first.

[0314] Because immunotherapy can cause a delay in the onset of the tumor response and may be associated with false tumor inflammation related to tumor progression, three types of PD are defined. Clinically unrelated progressive disease (PDn) is defined as PD in patients who do not experience a decline in performance and / or, in the physician's opinion, do not require alternative treatment. Patients showing PDn are permitted to continue treatment. Clinically related progressive disease (PDr) is defined as PD associated with a decline in performance and / or, in the physician's opinion, the patient does not require alternative treatment. Patients with PDr remain on treatment for 24 weeks unless, in the physician's opinion, other treatments are guaranteed. CNS progressive disease (PDcns) is defined as progression in the central nervous system (brain).

[0315] The therapeutic Ad-IL24 is prepared in a neutral buffer containing physiological saline and 10% glycerol, with a concentration of 1 × 10⁶. 12 It is supplied as a frozen vial suspension (2.0 mL per vial) at a concentration of vp / mL. There is no minimum tumor size requirement for injection. The first group of tumors to be treated should include skin lesions to enhance the immunological effect of the treatment mediated by skin antigen-presenting cells.

[0316] Each patient may have up to 20 lesions, provided there are no solitary lesions with a longest diameter exceeding 5 cm. The ultimate goal is to treat all lesions with at least one cycle of Ad-IL24 therapy (intratumoral injections twice weekly for 3 weeks). Each patient's lesions are divided into Ad-IL24 treatment groups, and the number of lesions in each group is determined by tumor diameter and dose escalation cohort, ensuring that the Ad-IL24 delivered in each treatment does not exceed the total volume permitted for each treatment day as specified in the dose escalation scheme outlined in Table 4. The total dose (volume) delivered to the tumor does not exceed the volume specified in Table 4, and the amount injected into each individual tumor in each treatment group depends on the size of the tumor nodule and is determined according to the following algorithm: ● For tumors with a maximum length of 0.5 c...

Claims

1. A combination for use in a method of treating cancer in a subject, wherein the combination comprises (1) a nucleic acid encoding p53 and / or a nucleic acid encoding MDA-7, (2) at least one CD122 / CD132 agonist, and (3) at least one immune checkpoint inhibitor, the method comprising administering the combination to the subject, wherein the at least one CD122 / CD132 agonist selectively binds to CD122 / CD132, the at least one CD122 / CD132 agonist comprises an IL-2 / anti-IL-2 immune complex or an IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, and the at least one immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.

2. The combination according to claim 1, characterized in that the subject is administered nucleic acid encoding p53.

3. The combination according to claim 1, characterized in that the subject is administered nucleic acid encoding MDA7.

4. The combination according to claim 1, characterized in that the subject is administered a nucleic acid encoding p53 and a nucleic acid encoding MDA7.

5. The combination according to claim 1, wherein IL-15 is pre-composited with IL-15Rα and selectively binds to CD122 / CD132.

6. The combination according to claim 1, characterized in that one, two, three, or four types of CD122 / CD132 agonists are administered to the subject.

7. The combination according to claim 1, wherein at least one of the CD122 agonists and / or CD132 agonists is not F42K.

8. The combination according to claim 1, wherein the cancer is metastatic.

9. The combination according to claim 1, wherein the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are located in an expression cassette within a viral vector.

10. The combination according to claim 9, wherein the viral vector is an adenovirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus vector, a herpesvirus vector, a vesicular stomatitis virus vector, or a polyomavirus vector.

11. The combination according to claim 10, wherein the vaccinia virus vector is further defined as an NIL-deficient vaccinia virus vector.

12. The combination according to claim 10, wherein the adenovirus vector is further defined as an adenovirus vector in which ADP expression is increased.

13. The combination according to claim 1, characterized in that the nucleic acid encoding p53 and / or the nucleic acid encoding MDA-7 are administered intratumorally to the subject.

14. The combination according to claim 1, wherein the cancer is melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, hepatocellular carcinoma, retinoblastoma, astrocytoma, gliablastoma, leukemia, neuroblastoma, head cancer, neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, genitourinary cancer, airway cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, carcinoma, sarcoma, central nervous system cancer, peripheral nervous system cancer, lymphoma, brain cancer, colon cancer, or bladder cancer.

15. The combination according to claim 1, further comprising administering at least one additional anti-cancer treatment.

16. The combination according to claim 15, wherein the at least one additional anticancer treatment is surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, radioresection, or biological therapy.

17. The combination according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, and AMP-224.

18. The combination according to claim 1, characterized in that two or more immune checkpoint inhibitors are administered.

19. The combination according to claim 15, wherein the at least one additional anticancer treatment is a histone deacetylase (HDAC) inhibitor.

20. A pharmaceutical composition comprising (a) a nucleic acid encoding p53 and / or a nucleic acid encoding MDA-7, (b) at least one CD122 / CD132 agonist, and (c) at least one immune checkpoint inhibitor, wherein the at least one CD122 / CD132 agonist comprises an IL-2 / anti-IL-2 immune complex or an IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, and the at least one immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.

21. A composition for use in a method of treating cancer in a subject, wherein the composition comprises a nucleic acid encoding p53 and / or a nucleic acid encoding MDA-7, the method comprising administering the composition to the subject in combination with at least one CD122 / CD132 agonist and at least one immune checkpoint inhibitor, wherein the at least one CD122 / CD132 agonist selectively binds to CD122 / CD132, the at least one CD122 / CD132 agonist comprises an IL-2 / anti-IL-2 immune complex or an IL-15 / IL-15 receptor α-IgG1-Fc (IL-15 / IL-15Rα-IgG1-Fc) immune complex, and the at least one immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1.

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