HK40042181B - Methods and compositions comprising tumor suppressor gene therapy and cd122/cd132 agonists for the treatment of cancer - Google Patents

Description

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

This application claims the benefit of U.S. Provisional Patent Application No. 62/645,022, filed March 19, 2018, and U.S. Provisional Patent Application No. 62/803,887, filed February 11, 2019, the entire contents of which are incorporated herein by reference.

Technical Field

This invention generally relates to the fields of biology and medicine. More specifically, this invention relates to methods and compositions for combining the restoration or amplification of tumor suppressor function with preferred CD122/CD132 agonists.

Background Technology

Malignant cells often develop resistance to DNA-damaging agents, such as chemotherapy and radiation-induced programmed cell death or apoptosis. This resistance is usually a result of aberrant expression of certain oncogenes or loss of expression of tumor suppressor genes in the control of apoptosis. Strategies designed to replace defective tumor suppressor genes and force the expression of apoptosis-inducing genes offer hope for restoring this cell death pattern in tumor cells.

One of the most studied tumor suppressor genes is p53, which plays a crucial role in multiple processes, including cell cycle regulation and apoptosis control (Hartwell et al., 1994). p53 mutations are common in tumor cells and are associated with cancer progression and the development of resistance to chemotherapy and radiotherapy (Spitz et al., 1996). Both in vitro and in vivo preclinical studies have shown that restoring wild-type (wt) p53 function can induce apoptosis in cancer cells. Intratumoral injection in animal models of retroviral or adenoviral wt-p53 constructs has resulted in tumor regression in a variety of tumor histologies, including non-small cell lung cancer (NSCLC), leukemia, glioblastoma, as well as breast cancer, liver cancer, ovarian cancer, colon cancer, and kidney cancer (Fujiwara et al., 1994). Promising preclinical and clinical data have led to the initiation of international randomized phase II/III trials of p53 gene therapy for first-line treatment of ovarian cancer patients (Buller et al., 2002). However, the study ended after the first interim analysis because it did not show sufficient therapeutic benefit (Zeimet and Marth, 2003).

Therefore, despite significant progress in tumor suppressor gene therapy, several obstacles remain limiting clinical success, including nonspecific expression, inefficient delivery, and biosafety. Furthermore, multiple genetic variations in cancer and epigenetic regulatory abnormalities lead to aberrant gene silencing; thus, monogenic gene therapy may not be a suitable strategy for treating cancer. Therefore, there is a need for targeted approaches that combine multiple tumor suppressor factors with other anticancer agents to achieve enhanced antitumor activity and efficient delivery of gene therapy.

Summary of the Invention

In one embodiment, this disclosure provides a method and composition for treating a subject with cancer, the method and composition 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 preferred CD122/CD132 agonist).

In some respects, nucleic acids encoding p53 are administered to the subjects. In some respects, nucleic acids encoding MDA7 are administered to the subjects. In some respects, nucleic acids encoding both p53 and MDA7 are administered to the subjects.

Specifically, the delivery of nucleic acids encoding p53 and/or MDA-7 and/or CD122/CD132 agonists in amounts that effectively restore or amplify tumor suppressor function. Specifically, the delivery of nucleic acids encoding p53 and/or MDA-7 to one or more tumor sites. In some respects, more than one CD122/CD132 agonist is administered. Specifically, the subjects are humans.

In some respects, CD122/CD132 agonists preferentially bind to CD122/CD132 receptor complexes and have a lower affinity for CD25 or IL15α receptors compared to affinity binding to CD122/CD132 receptor complexes. In specific respects, 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-15Ra-IgG1-Fc) immune complexes, PEGylated IL-2, PEGylated IL-15, IL-2 mutants, and/or IL-15 mutants. CD122/CD132 agonists may be IL-15 mutants (e.g., IL-15N72D) that bind to IL-15 receptor α/IgG1 Fc fusion proteins (such as ALT-803). In some respects, IL-15 pre-conjugates with IL-15Rα to preferentially bind to CD122/CD132. Specifically, the IL-2 receptor agonist is not F42K.

In some respects, nucleic acids encoding p53 and/or MDA-7 are delivered via viral and/or non-viral methods. In some respects, the nucleic acids encoding p53 and/or MDA-7 are delivered in expression cassettes (such as in viral vectors). In some respects, p53 and MDA-7 are under the control of a single promoter (such as cytomegalovirus (CMV), SV40, or PGK). In some respects, the viral vectors are adenovirus vectors (e.g., adenovirus vectors overexpressing ADP), retroviral vectors, vaccinia virus vectors (e.g., vaccinia virus vectors lacking NIL), adeno-associated virus vectors, herpesvirus vectors, vesicular stomatitis virus vectors, and polyomavirus vectors. In some respects, the nucleic acids encoding p53 and/or MDA-7 are delivered via gene editing methods (such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly spaced short palindromic repeats (CRISPR)) to restore or amplify the expression of tumor suppressor genes. This disclosure considers combinations of viral and nonviral gene delivery and expression and/or gene editing methods. In some aspects, the adenovirus p53 (Ad-p53) injection dose (mL) is such that the Ad-p53 dose received per tumor lesion is at least 1 × ^10¹¹ viral particles (vp)/ ^cm³ tumor volume. In some aspects, nucleic acids encoding p53 and/or encoding MDA-7 are administered to the subject in the form of a lipoplex. In some aspects, the lipoplex comprises DOTAP and at least one cholesterol, cholesterol derivative, or mixture of cholesterol.

In some aspects, the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 are administered to the subject intravenously, intraarterially, intravascularly, intrapleurally, intraperitoneally, intratracheally, intratumorally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, stereotactically, or by direct injection or infusion. In specific aspects, the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 are administered to the subject intratumorally. In some aspects, administration includes local or regional injection. In some aspects, administration is performed by continuous infusion, intratumoral injection, or intravenous injection.

In some respects, the subject is administered nucleic acid encoding p53 and/or nucleic acid encoding MDA-7 more than once. In some respects, the subject is administered at least one CD122/CD132 agonist more than once. In some respects, the subject is administered nucleic acid encoding p53 and/or nucleic acid encoding MDA-7 before, simultaneously with, or after at least one CD122 agonist and CD132 agonist.

In some respects, the cancers mentioned are melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, 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, respiratory cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, malignant epithelial tumors, sarcomas, central nervous system cancers, peripheral nervous system cancers, lymphoma, brain cancer, colon cancer, or bladder cancer. In specific respects, the cancers mentioned are metastatic.

In some aspects, the method further includes administering at least one additional anticancer treatment. In some aspects, the at least one additional anticancer treatment is surgical therapy, chemotherapy, radiotherapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, cryotherapy, radiation ablation, or biotherapy. In some aspects, biotherapy is monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes, gene editing, cell therapy, or gene therapy.

In some aspects, at least one adjunctive anticancer therapy is an immune checkpoint inhibitor. In some aspects, the immune checkpoint inhibitor is CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some aspects, at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some aspects, the anti-CTLA-4 antibody is trimelimumab or ipilimumab. In some aspects, at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. In some embodiments, the anti-KIR antibody is lirilumab. In some aspects, the PD-L1 inhibitor is durvalumab, atezolizumab, or avelumab. In some aspects, the PD-L2 inhibitor is rHIgM12B7. In some aspects, the LAG3 inhibitor is IMP321 or BMS-986016. In some aspects, the A2aR inhibitor is PBF-509. In some aspects, at least one immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) axis binding antagonist. In some aspects, 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 aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some aspects, the PD-1 binding antagonist inhibits the binding of PD-1 to PDL1 and/or PDL2. Specifically, 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.

In some aspects, at least one additional therapy is a histone deacetylase (HDAC) inhibitor. In some aspects, the HDAC inhibitor is tractinostat (CHR-3996 or VRx-3996). In some aspects, the method further includes providing extracellular matrix degrading proteins, such as relaxin, hyaluronidase, or core proteoglycans.

In some aspects, at least one adjunctive anticancer therapy is an oncolytic virus. In some aspects, the oncolytic virus is engineered to express p53, MDA-7, IL-12, TGF-β inhibitors and/or IL-10 inhibitors. In some aspects, the oncolytic 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’s diseasevirus, Epstein-Barr virus, influenza virus, reovirus, myxoma virus, Malabar virus, rhabdovirus, enadenotucirev, or Coxsackie virus. In some aspects, the oncolytic virus is engineered to express cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-12. In some aspects, the oncolytic virus is further defined as talimogene heherparepvec (T-VEC). In some respects, oncolytic adenovirus vectors are derived from adenoviruses lacking E1b, as well as adenoviruses in which the Ad E1a gene is driven by an alpha-fetoprotein (AFP) promoter, modified TERT promoter oncolytic adenoviruses, HRE-E2F-TERT heterozygous promoter oncolytic adenoviruses, and/or adenoviruses with modified E1a regulatory sequences, wherein at least one Pea3 binding site or its functional portion is deleted using an Elb-19K cloning insertion site. All of these adenoviruses can be modified to express therapeutic genes.

In some aspects, the at least one additional anticancer therapy is an inhibitor of a protein kinase or growth factor signaling pathway. In some aspects, the inhibitor of the protein kinase or growth factor signaling pathway is afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, or ruxo. Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, CAL101, PX-866, LY294002, Rapamycin, Temsirolimus, Everolimus, Ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, or GW572016. In some respects, protein kinase inhibitors are PI3K inhibitors, such as PI3Kδ inhibitors.

In some respects, immunotherapy includes cytokines such as GM-CSF, interleukins (e.g., IL-2), and/or interferons (e.g., IFNα) or heat shock proteins. In some respects, immunotherapy includes co-stimulatory receptor agonists, innate immune cell stimulants, or innate immune activators. In some respects, co-stimulatory receptor agonists are anti-OX40 antibodies, anti-GITR antibodies, anti-CD137 antibodies, anti-CD40 antibodies, or anti-CD27 antibodies. In some respects, immunocellular stimulants are inhibitors of cytotoxic inhibitory receptors or agonists of immunostimulatory Toll-like receptors (TLRs). In some respects, cytotoxic inhibitory receptors are inhibitors of NKG2A/CD94 or CD96 tactile receptors. In some respects, TLR agonists are TLR7 agonists, TLR8 agonists, or TLR9 agonists. In some respects, immunotherapy includes combinations of PD-L1 inhibitors, 4-1BB agonists, and OX40 agonists. In some respects, immunotherapy includes interferon gene-stimulating factor (STING) agonists. In some respects, innate immune activators are IDO inhibitors, TGFβ inhibitors, or IL-10 inhibitors. In some respects, when these immunotherapies are proteins, they can be delivered as peptides or their corresponding nucleic acids administered via replicating viruses and/or non-replicating viruses and/or non-viral gene therapies. In some respects, chemotherapy includes DNA damaging agents such as gamma radiation, X-rays, UV radiation, microwaves, electron emission, doxorubicin, 5-fluorouracil (5FU), capecitabine, etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), or hydrogen peroxide.

In another embodiment, a method of treating a subject's cancer is provided, the method comprising administering to the subject an effective amount of at least one oncolytic virus and at least one CD122/CD132 agonist and at least one immune checkpoint inhibitor. In some aspects, the at least one oncolytic virus is an adenovirus engineered to overexpress adenoviral death protein (ADP), such as VirRx007. In some aspects, the at least one oncolytic virus is genetically modified to express p53, MDA-7, cytokines, and/or immunostimulatory genes. In specific aspects, the cytokine is GM-CSF or IL-12. In some aspects, the immunostimulatory gene is an inhibitor of TGFβ or IL-10.

In some aspects, the at least one oncolytic 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, Ebola viruses, influenza viruses, reoviruses, myxoma viruses, Malaba viruses, rhabdoviruses, enadenotucirev, and Coxsackieviruses.

In some aspects, the viruses used in the above embodiments include viruses with replication capability and/or replication-deficient viruses. In some aspects, the viruses with and without replication capability are 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, myxoma viruses, Ebola viruses, influenza viruses, reoviruses, Malaba viruses, rhabdoviruses, enadenotucirev, or Coxsackieviruses. In some aspects, one or more viruses are used. In some aspects, the viral composition comprises a combination of viruses with replication capability and viruses without replication capability.

In other respects, the replicative viruses in the above embodiments can be one or more oncolytic viruses. These oncolytic viruses can be engineered to express p53 and/or IL24 and/or other genes besides p53 and/or IL24, such as cytokines (e.g., IL12) and/or another immunostimulatory gene (e.g., a TGF-β inhibitor or an IL10 inhibitor or heat shock protein). In some respects, oncolytic viruses can be used in place of or as a complement to p53 and/or IL24 tumor suppressor therapy. Examples of oncolytic viruses include single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia virus, vesicular stomatitis virus, polioviruses, Newcastle disease virus, Ebola virus, influenza virus and reovirus, myxoma virus, Malaba virus, rhabdovirus, enadenotucirev, or Coxsackie virus. Exemplary oncolytic 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, Toca 511, Ankerui (H101), Onyx-015, H102, H103, RIGVIR, adenoviruses overexpressing adenovirus death protein (ADP) (such as VirRx007), vaccinia virus lacking N1L, or vaccinia virus lacking N1L expressing IL12.

In some respects, viral and nonviral nucleic acid and gene-editing compositions induce local and/or systemic effects.

In some respects, the subjects treated are mammals or humans. In some respects, treatment is provided to prevent or treat pre-malignant or malignant hyperproliferative disorders. In some respects of prevention, the subjects are healthy subjects. In other respects of prevention, subjects include precancerous lesions, such as leukoplakia or developmental abnormalities. In other respects of prevention, subjects are at risk of developing cancer, for example, due to smoking or a family history of cancer. In some respects, treatment is for initial or recurrent hyperproliferative disorders. In some respects, treatment is administered to enhance or reverse resistance to another therapy. In some respects, resistance to treatment is historically known to be specific to a particular group of patients with hyperproliferative disorders. In some respects, resistance to treatment has been observed in individual patients with hyperproliferative disorders.

In some aspects of the above embodiments, the method further includes providing an extracellular matrix degrading protein. In some aspects, this includes administering an expression cassette encoding an extracellular matrix degrading protein. In some embodiments, the extracellular matrix degrading protein is relaxin, hyaluronidase, or core proteoglycan. In a specific aspect, the extracellular matrix degrading protein is relaxin. In some aspects, the expression cassette is in a viral vector. In some aspects, the viral vector is an adenovirus vector, a retroviral 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-viral gene therapy vector.

In some respects, expression cassettes encoding extracellular matrix degradation proteins are administered intratumorally, intraarterially, intravenously, intravascularly, intrapleurally, intraperitoneally, intratracheally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, stereotactically, or by direct injection or infusion. In some respects, nucleic acids encoding p53 and/or encoding MDA-7 are administered to the subject following at least one CD122/CD132 agonist. In some respects, nucleic acids encoding p53 and/or encoding MDA-7 are administered to the subject prior to at least one CD122/CD132 agonist. In some respects, nucleic acids encoding p53 and/or encoding MDA-7 are administered to the subject concurrently with at least one CD122/CD132 agonist. In a specific respect, an adenovirus vector is administered intratumorally to the subject. In some respects, nucleic acids encoding p53 and/or encoding MDA-7, as well as at least one CD122/CD132 agonist, induce distant (systemic) effects on distal tumors that have not been injected with nucleic acids encoding p53 and/or encoding MDA-7.

In some respects, the cancers referred to are melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, 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, respiratory cancer, hematopoietic cancer, musculoskeletal cancer, neuroendocrine cancer, malignant epithelial tumors, sarcomas, central nervous system cancers, peripheral nervous system cancers, lymphoma, brain cancer, colon cancer, or bladder cancer. In some respects, the cancers are metastatic.

In some aspects, the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 are in the expression cassette. In other aspects, the expression cassette is in 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 a particular aspect, the viral vector is an adenovirus vector.

In some respects, the viral vector is administered as viral particles ranging from approximately ^10³ to approximately ^10¹³ . In some respects, the adenovirus vector is administered to the subject intravenously, intraarterially, intravascularly, intrapleurally, intraperitoneally, intratracheally, intratumorally, intrathecally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, stereotactically, or by direct injection or infusion. In some respects, the adenovirus vector is administered to the subject more than once.

In some aspects, a nucleic acid encoding p53 is administered to the subject. In other aspects, a nucleic acid encoding MDA-7 is administered to the subject. In some aspects, both nucleic acids encoding p53 and MDA-7 are administered to the subject. In some aspects, p53 and MDA-7 are under the control of a single promoter. In some embodiments, the promoter is cytomegalovirus (CMV), SV40, or PGK.

In some respects, nucleic acids are administered to subjects in the form of liposome complexes. In some respects, the liposome complex comprises DOTAP and at least one cholesterol, cholesterol derivative, or mixture of cholesterol. In some respects, nucleic acids are administered in the form of nanoparticles.

In some cases, administration involves local or regional injection. In others, administration is performed via continuous infusion, intratumoral injection, or intravenous injection.

In some aspects, the method further includes administering at least one additional anticancer treatment. In some aspects, at least one additional anticancer treatment is surgical therapy, chemotherapy (e.g., administration of a protein kinase inhibitor or EGFR-targeted therapy), embolization therapy, chemoembolization therapy, radiotherapy, cryotherapy, thermotherapy, phototherapy, radiation ablation therapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, or biological therapy (such as monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes, or gene therapy).

In some respects, immunotherapy includes cytokines. Specifically, cytokines include granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukins (such as IL-2), and/or interferons (such as IFN-α). Additional methods to enhance the immune response targeting tumors include additional immune checkpoint inhibition. In some respects, immune checkpoint inhibition includes 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 respects, immunotherapy includes co-stimulatory receptor agonists, such as anti-OX40 antibodies, anti-GITR antibodies, anti-CD137 antibodies, anti-CD40 antibodies, and anti-CD27 antibodies. In some respects, immunotherapy includes the suppression of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs). In other respects, immunotherapy includes the stimulation of innate immune cells, such as natural killer (NK) cells, macrophages, and dendritic cells. Other immunostimulatory therapies may 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-cell vaccines, peptides, and recombinant tumor-associated antigen vaccines), and adoptive cell therapy (ACT) (e.g., T cells, natural killer cells, TILs, and LAK cells), as well as ACT using genetically engineered receptors (e.g., chimeric antigen receptors (CARs) and T-cell receptors (TCRs)). In some respects, combinations of these agents may be used, such as combinations of immune checkpoint inhibitors, checkpoint inhibition plus T-cell co-stimulatory receptor agonism, and checkpoint inhibition plus TIL ACT. In some respects, adjunctive anticancer therapies include combinations of immune checkpoint inhibitors (e.g., avermab), 4-1BB (CD-137) agonists (e.g., utomilumab), and OX40 (TNFRS4) agonists.

In some aspects, chemotherapy includes DNA damaging agents. In some embodiments, DNA damaging agents are gamma radiation, X-rays, UV radiation, microwaves, electron emission, doxorubicin, 5-fluorouracil (5FU), capecitabine, etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), or hydrogen peroxide. In specific aspects, the DNA damaging agent is 5FU or capecitabine. In some respects, chemotherapy includes cisplatin (CDDP), carboplatin, procarbazine, dichloromethyldiethylamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, actinomycin D, daunorubicin, doxorubicin, bleomycin, procainoxam, mitomycin, etoposide (VP16), tamoxifen, docetaxel, paclitaxel, antiplatinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, HDAC inhibitors, or any analogues or derivatives thereof.

In some aspects, at least one additional cancer treatment is a protein kinase inhibitor or a monoclonal antibody that inhibits receptors involved in protein kinase or growth factor signaling pathways. For example, the protein kinase or receptor inhibitor may be an inhibitor of 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. In a specific aspect, 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 can be afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fotatinib, gefitinib, imatinib, lapatinib, lenvatinib, lilotinib, nilotinib, panitumab, pazopanib, pilgatanib, ranitumab, ruxotinib, cicatinib, sorafenib, sunitinib, trastuzumab, vandestatinib, etc. Nifedipine, AP23451, vemurafenib, CAL101, PX-866, LY294002, rapamycin, tesiromoxim, everolimus, desfolimox, avozidil, genistein, selmetinib, AZD-6244, vastarani, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof. In some respects, protein kinase inhibitors are AKT inhibitors (e.g., MK-2206, GSK690693, A-443654, VQD-002, miltefosine, or perifosine). In some respects, the EGFR-targeting therapies used 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 and include, but are not limited to, tyrosine kinase inhibitors active against one or more receptors and EGFR-binding antibodies or aptamers. For example, EGFR inhibitors may be gefitinib, erlotinib, cetuximab, matuzumab, panitumumab, AEE788, CI-1033, HKI-272, HKI-357, or EKB-569. Protein kinase inhibitors may be BRAF inhibitors, such as dabrafenib, or MEK inhibitors, such as trametinib.

Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while preferred embodiments of the invention are indicated, the detailed description and specific embodiments are given by way of illustration only, 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.

Attached Figure Description

The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these drawings, in conjunction with the detailed description of the specific embodiments presented herein.

Figure 1: Effective Ad-p53 dosing and tumor response. Waterfall plot of tumor response in a subgroup of patients treated with Ad-p53 > 7 × ^10¹⁰ viral particles/ ^cm³ compared to those treated with Ad-p53 doses < 7 × ^10¹⁰ viral particles/ ^cm³ (left inset). Detailed examination of Ad-p53 responders revealed that the majority of responders (7/9 patients) had received Ad-p53 doses close to or exceeding 1 × ^10¹¹ vp/ ^cm³ (range 7.81 to 333.2 × ^10¹⁰ vp/ ^cm³ ). Therefore, the dose used for clinical application of Ad-p53 may exceed 1 × ^10¹¹ vp/ ^cm³ injected into the tumor volume.

Figure 2: Superior overall survival with Ad-p53 biomarker/dose-optimized therapy. Patients with a favorable tumor p53 biomarker profile treated with Ad-p53 at doses >7 × ^10¹⁰ viral particles/ ^cm³ had superior one-year and overall survival compared to patients treated with methotrexate. The results indicate that favorable tumor p53 biomarkers and Ad-p53 dose-optimized therapy significantly increased overall survival compared to methotrexate (median survival of Ad-p53 was 11.5 months vs. 4.3 months with methotrexate; p < 0.016, HR 1.9767).

Figure 3: QUADRA-FUSE ^™ Infusion Device. The QUADRA-FUSE ^™ (from the acquired image) is a multiple-tine infusion device with three heads extending from the cannula needle axis (upper right inset). These three heads are deployed around the central needle axis with an adjustable diameter of 1–5 cm, depending on the shorter width (W) diameter of the tumor lesion. This lateral extension allows for broad drug dispersion throughout the lesion. Each head has two delivery orifices (four fluid outlets); thus, each infusion produces 12 delivery points.

Figure 4: Efficacy of Ad-p53+CD122/132 agonist + anti-PD-1: Tumor volume. A graph illustrating primary tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, Ad-p53, or combinations of CD122/132+anti-PD-1, Ad-p53+CD122/132, Ad-p53+anti-PD-1, or Ad-p53+CD122/132+anti-PD-1. Severe tumor progression was observed during CD122/132, anti-PD-1, and CD122/132+anti-PD-1 therapy, which was reversed by combination with Ad-p53 therapy. The results also showed that the efficacy of Ad-p53+CD122/132, Ad-p53+anti-PD-1, and Ad-p53+CD122/132+anti-PD-1 treatments was improved compared to any of the aforementioned therapies. By day 21, the mean tumor volume in the groups treated with (PBS), CD122/132, anti-PD-1, CD122/132+anti-PD-1, and Ad-p53 all exceeded 2,000 ^mm³ . In contrast, each of the following combinations of treatments—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 non-Ad-p53 therapy or Ad-p53 therapy alone. ANOVA comparison of tumor volume on day 21 confirmed a synergistic antitumor effect between 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, ANOVA comparison of tumor volume on day 30 confirmed that the synergistic antitumor effect was maintained only in the Ad-p53+CD122/132+anti-PD-1 treatment combination (overall p < 0.0001, and individual p < 0.0001 compared to each of the other treatment groups).

Figure 5: Complete tumor response rate. Complete tumor response to therapy is generally considered to be associated with significant treatment benefit and is essential for a curative outcome. As shown in Figure 5, for the p53 treatment group and its control, only Ad-p53+CD122/132+anti-PD-1 therapy resulted in complete tumor regression of both the primary and contralateral tumors. Complete tumor responses to both primary and contralateral tumors were observed in 60% (6 out of 10 animals) of the Ad-p53+CD122/132+ anti-PD-1 treatment group, while no complete tumor responses were observed in any of the 70 animals in the other treatment groups (p < 0.0001 by two-sided Fisher’s Exact Test compared to animals in the Ad-p53+CD122/132+ anti-PD-1 treatment group and all other treatment groups; p ≤ 0.011 by two-sided Fisher’s Exact Test compared to animals in the Ad-p53+CD122/132+ anti-PD-1 treatment group and any other treatment group). Unexpectedly, the complete tumor response was durable and maintained in 50% of the Ad-p53+CD122/132+ anti-PD-1 treatment group after 40 days, presumably curing these tumors in these animals.

Figures 6A and 6B: Systemic/distant therapeutic effects on contralateral tumor growth. The systemic/distant effects of primary tumor treatment on contralateral implanted tumors were assessed in rodents whose primary tumors had been treated with one of the Ad-p53 intratumoral therapies. Consistent with the unexpectedly and significantly enhanced synergistic effect of Ad-p53+CD122/132+anti-PD-1 treatment on primary tumor growth and complete regression, we also observed a surprisingly strong and statistically significant distant 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 was eliminated in 90% (9 out of 10) of animals receiving Ad-p53+CD122/132+anti-PD-1 primary tumor treatment. In contrast, contralateral tumor growth was observed in 62.5% to 100% of animals in other Ad-p53 treatment groups. The difference in contralateral tumor growth was statistically significant (p = 0.0004 by chi-square analysis of all treatment groups; p ≤ 0.0430 by two-sided Fisher exact test comparing the Ad-p53+CD122/132+anti-PD-1 treatment group with any other treatment group). Figure 6B depicts a graph showing the contralateral tumor volume over time in rodents receiving the three most effective primary tumor treatments: Ad-p53+CD122/132, Ad-p53+anti-PD-1, or Ad-p53+CD122/132+anti-PD-1. ANOVA comparison of these contralateral tumor volumes on day 22 determined the synergistic antitumor effect of Ad-p53+CD122/132+anti-PD-1 treatment (overall p = 0.0435). Compared to the Ad-p53+ anti-PD-1 group, only the Ad-p53+CD122/132+ anti-PD-1 group showed a statistically significant reduction in contralateral tumor growth (p = 0.0360). In summary, these findings suggest that among all Ad-p53 therapies, only the triple combination Ad-p53+CD122/132+ anti-PD-1 therapy produced curative efficacy by inducing robust local and systemic anti-tumor immunity that mediates substantial distant effects.

Figure 7: Efficacy of Ad-p53+CD122/132+anti-PD-1: Prolonged survival. Kaplan-Meier survival curves of mice treated with PBS, CD122/132+anti-PD-1, Ad-p53, or the combination of Ad-p53+CD122/132, Ad-p53+anti-PD-1, and Ad-p53+CD122/132+anti-PD-1. These survival curves were statistically significant by log rank test (overall p < 0.0001; p ≤ 0.0003 for Ad-p53+CD122/132+anti-PD-1 treatment group compared with any other treatment group). These results also demonstrate the unexpected substantial synergy of Ad-p53+CD122/132+anti-PD-1 therapy. The median survival of the Ad-p53+CD122/132+anti-PD-1 therapy group had not been reached after 40 days, and 80% of the animals in this group remained alive without any residual tumor. In stark contrast, 98% (49/50) of the animals in the other treatment groups died by day 30, with median survival ranging from 10 to 28 days.

Figure 8: VirRx007 + CD122/132 agonist + anti-PD-1 efficacy: tumor volume. A graph showing the primary tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, CD122/132 + anti-PD-1, VirRx007, or combinations of VirRx007 + CD122/132, VirRx007 + anti-PD-1, and VirRx007 + CD122/132 + anti-PD-1. Severe tumor progression was observed in the groups treated with CD122/132, anti-PD-1, and CD122/132 + anti-PD-1 therapy, which was reversed by combination with VirRx007 therapy. The results also showed that the efficacy of VirRx007 + anti-PD-1 and VirRx007 + CD122/132 + anti-PD-1 treatments was enhanced compared to any of the individual therapies described. Contrary to the findings with Ad-p53, VirRx007 did not demonstrate a synergistic effect with CD122/CD132 treatment. 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, each of the combinations 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 alone or any of the VirRx007 treatments alone. ANOVA analysis of tumor volume on day 30 confirmed the synergistic antitumor effects of VirRx007 + antiPD-1 and VirRx007 + CD122/132 + antiPD-1 therapy (p < 0.0001 for the overall or individual treatments compared to VirRx007). VirRx007 + CD122/132 + antiPD-1 therapy was superior to VirRx007 + antiPD-1 (p = 0.0002). Surprisingly, although the combination therapy of VirRx007 + CD122/132 did not show a significant benefit compared to VirRx007 monotherapy, it demonstrated the synergistic effect of triple therapy with VirRx007 + CD122/132 + antiPD-1.

Figure 9: Complete tumor response rate. Complete tumor response to therapy is generally considered to be associated with significant treatment benefit and is essential for a curative outcome. As shown in Figure 9, for both the VirRx007 treatment group and its controls, only VirRx007+CD122/132+anti-PD-1 treatment resulted in complete tumor regression of both the primary and contralateral tumors. Complete tumor response of both the primary and contralateral tumors was observed in 60% of animals in the VirRx007+CD122/132+anti-PD-1 treatment group, and no complete tumor response was observed in any of the 70 animals in the other treatment groups (p < 0.0001 by two-sided Fisher exact test comparing the VirRx007+CD122/132+anti-PD-1 treatment group with all other treatment groups; p < 0.011 by two-sided Fisher exact test comparing the VirRx007+CD122/132+anti-PD-1 treatment group with any other treatment group). Surprisingly, the complete tumor response was durable and maintained in 50% of animals treated with VirRx007+CD122/132+anti-PD-1 after 40 days, thus presumably curing these tumors in these animals.

Figures 10A and 10B: Systemic/distant therapeutic effects on contralateral tumor growth. The systemic/distant effects of primary tumor treatment on contralateral implanted tumors were assessed in rodents whose primary tumors had been treated with one of the VirRx007 intratumoral therapies. Consistent with the unexpectedly and significantly enhanced synergistic effect of VirRx007+CD122/132+anti-PD-1 treatment on primary tumor growth and complete regression, we also observed a surprisingly strong and statistically highly significant distant effect of VirRx007+CD122/132+anti-PD-1 treatment compared to other VirRx007 treatment groups. As shown in Figure 10A, contralateral tumor growth was eliminated in 80% of animals whose primary tumors received VirRx007+CD122/132+anti-PD-1 treatment. In contrast, contralateral tumor growth was observed in 80% to 100% of animals in other VirRx007 treatment groups. The difference in contralateral tumor growth was statistically significant (p = 0.0002 by chi-square analysis comparing all treatment groups; p ≤ 0.0230 by two-sided Fisher exact test comparing the VirRx007+CD122/132+anti-PD-1 treatment group with any other treatment group). These findings suggest that the combination of VirRx007+CD122/132+anti-PD-1 induces robust systemic anti-tumor immunity and mediates substantial distant effects with potential curative efficacy. Figure 10B depicts a plot showing the contralateral tumor volume over time in rodents treated with the three most effective combinations of primary tumors: VirRx007+CD122/132, VirRx007+anti-PD-1, or VirRx007+CD122/132+anti-PD-1. ANOVA comparison of contralateral tumor volumes on day 22 confirmed the synergistic antitumor effect of VirRx007+CD122/132+antiPD-1 therapy (overall p = 0.0171). Only the VirRx007+CD122/132+antiPD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the VirRx007+antiPD-1 group (p = 0.0115). In summary, these findings suggest that among all VirRx007 therapies, only the triple combination VirRx007+CD122/132+antiPD-1 therapy produced curative efficacy by inducing robust local and systemic antitumor immunity mediating substantial distant effects.

Figure 11: Efficacy of VirRx007+CD122/132+Anti-PD-1: Prolonged survival. Kaplan-Mel survival curves of mice treated with PBS, CD122/132+Anti-PD-1, VirRx007, or the combination of VirRx007+CD122/132, VirRx007+Anti-PD-1, and VirRx007+CD122/132+Anti-PD-1. These survival curves were statistically significant by log-rank test (overall p < 0.0001; p ≤ 0.0005 for VirRx007+CD122/132+Anti-PD-1 treatment group compared with any other treatment group). The results also demonstrated an unexpectedly substantial synergistic effect of VirRx007+CD122/132+Anti-PD-1 therapy. The median survival of the VirRx007+CD122/132+anti-PD-1 therapy group had not been reached after 40 days, and 90% of animals in this group remained alive. In stark contrast, 98% (49/50) of the animals in the other treatment groups died by day 40, with median survival ranging from 10 to 33 days. Remarkably, although the combination therapy of VirRx007+CD122/132 did not demonstrate a significant survival benefit compared to VirRx007 monotherapy, it did demonstrate the synergistic effect of the triple therapy of VirRx007+CD122/132+anti-PD-1.

Figure 12: Efficacy of Ad-IL24+CD122/132 agonist + anti-PD-1: Tumor volume. A schematic diagram showing the primary tumor volume over time in rodents receiving 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 and Ad-IL24+CD122/132+anti-PD-1. Severe tumor progression occurred during CD122/132, anti-PD-1, and CD122/132+anti-PD-1 therapy, which was reversed by combination with Ad-IL24 therapy. The efficacy of Ad-IL24+CD122/132+anti-PD-1 therapy was enhanced compared to any of the individual therapies described. By day 16, the mean tumor volume in the groups treated with (PBS), CD122/132, anti-PD-1, CD122/132+anti-PD-1, and Ad-IL24 all exceeded 2,000 ^mm³ . In contrast, the combination therapy of Ad-IL24+CD122/132+anti-PD-1 induced a significant reduction in tumor volume compared to either non-Ad-IL24 therapy alone or Ad-IL24 therapy alone. ANOVA comparisons of tumor volumes at day 16 confirmed the synergistic antitumor effect of Ad-IL24+CD122/132+anti-PD-1 therapy (p < 0.0001). Compared with 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.

Figure 13: Efficacy of Ad-IL24+CD122/132+Anti-PD-1: Prolonged Survival. Kaplan-Mel survival curves of mice treated with PBS, CD122/132+Anti-PD-1, Ad-IL24, or the combination of Ad-IL24+CD122/132+Anti-PD-1. These survival curves showed statistically significant differences by log-rank test (p < 0.0001). The results demonstrate the unexpected substantial synergy of Ad-IL24+CD122/132+Anti-PD-1 therapy. Median survival was synergistically improved in the Ad-IL24+CD122/132+Anti-PD-1 therapy group. All animals in the PBS, CD122/132+Anti-PD-1, and IL24 treatment groups died on day 16, while 50% of the animals in the Ad-IL24+CD122/132+Anti-PD-1 therapy group survived on day 19. Compared with Ad-IL24 alone (p = 0.0003) or CD122/132+ anti-PD-1 therapy alone (p < 0.0001), the Ad-IL24+CD122/132+ anti-PD-1 therapy group showed a statistically significant prolonged survival. Interestingly, the Ad-IL24+CD122/132 dual therapy showed a surprising superior efficacy compared with the CD122/132+ anti-PD-1 dual therapy (p = 0.0002 by log-rank test, data not shown).

Figure 14: Efficacy of Ad-Luciferase (Ad-Luc) Negative Control + CD122/132 Agonist + Anti-PD-1: Tumor Volume. This figure shows the primary tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, CD122/132 + anti-PD-1, Ad-Luc control, or combinations of Ad-Luc control + CD122/132, Ad-Luc control + anti-PD-1, and Ad-Luc control + CD122/132 + anti-PD-1. There was no significant increase in efficacy when Ad-Luc was combined with anti-PD-1, CD122/132, or CD122/132 + anti-PD-1 treatment compared to treatment with Ad-p53, VirRx007, and Ad-IL24. By day 16, the mean tumor volume in all groups exceeded 2,000 ^mm³ . The comparison of tumor volume by ANOVA on day 16 was not statistically significant (p = 0.1212; mean tumor volume was not statistically significant between any treatment groups).

Figure 15: Triple therapy, combining Ad-p53, VirRx007, and Ad-IL24 with CD122/132+ anti-PD-1, prolonged survival compared to the Ad-Luc control plus CD122/132+ anti-PD-1. Kaplan-Mel survival curves for mice treated with CD122/132+ anti-PD-1 in combination with Ad-p53, VirRx007, Ad-IL24, or the Ad-Luc control. These survival curves showed statistically significant differences using a log-rank test (p < 0.0001). Compared with the triple therapy of Ad-Luc + CD122/132 + anti-PD-1 (p < 0.0001 for both Ad-p53 and VirRx007 with CD122/132 + anti-PD-1, and p < 0.015 for Ad-IL24 with CD122/132 + anti-PD-1), the triple therapy of each of Ad-p53, VirRx007 and Ad-IL24 with CD122/132 + anti-PD-1 showed a statistically significant increase in survival.

Figure 16: Efficacy of Ad-p53 + CD122/132 (IL15) agonist + anti-PD-1: tumor volume. The preferred CD122/CD132 agonist for concomitant use with tumor suppressor therapy is an immune complex consisting of recombinant IL15 and IL-15-Rα-Fc. A graph showing the primary tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132 + anti-PD-1, Ad-p53 alone, or the combination of Ad-p53 + CD122/132 (IL15) + anti-PD-1. Severe tumor progression occurred during PBS, CD122/132 + anti-PD-1, and Ad-p53 therapy. Consistent with the earlier Ad-p53 combination therapy results described above, the efficacy of Ad-p53 + CD122/132 (IL15) + anti-PD-1 therapy was significantly improved compared to any of the aforementioned therapies. 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 of Ad-p53+CD122/132(IL15)+anti-PD-1 induced a significant reduction in tumor volume. Statistical analysis of variance (ANOVA) of tumor volumes confirmed the synergistic antitumor effect of Ad-p53+CD122/132(IL15)+anti-PD-1 therapy (overall p < 0.0001, and individual p < 0.0001 compared to each of the other treatment groups).

Figure 17: Systemic/distant therapeutic effects of Ad-p53+CD122/132(IL15)+anti-PD-1 on contralateral tumor growth. The preferred CD122/CD132 agonist for concomitant tumor growth therapy is an immune complex consisting of recombinant IL15 and IL-15-Rα-Fc. The systemic/distant effects of primary tumor treatment on contralateral implanted tumors were assessed in rodents whose primary tumors had been treated with Ad-p53+CD122/132(IL15)+anti-PD-1. Consistent with the unexpectedly and significantly enhanced synergistic effect of Ad-p53+CD122/132(IL15)+anti-PD-1 treatment on primary tumor growth shown in Figure 16, we also observed a surprisingly potent and statistically significant distant effect of Ad-p53+CD122/132(IL15)+anti-PD-1 treatment compared to other Ad-p53 treatment groups. Figure 17 depicts a graph showing the contralateral tumor volume over time in rodents receiving primary tumor treatment with the combination of Ad-p53+CD122/132, Ad-p53+anti-PD-1, or Ad-p53+CD122/132(IL15)+anti-PD-1. ANOVA comparison of these contralateral tumor volumes at day 22 determined the synergistic antitumor effect of Ad-p53+CD122/132(IL15)+anti-PD-1 treatment (overall p = 0.0433). 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 = 0.0359). In summary, these findings indicate that among all Ad-p53 therapies, only the triple combination of Ad-p53 + CD122/132 + anti-PD-1 therapy produced a curative effect by inducing a powerful local and systemic anti-tumor immune response that mediates substantial distant effects.

Figure 18: Efficacy of Ad-p53+CD122/132(IL15)+anti-PD-1: Prolonged survival. The preferred CD122/CD132 agonist for concomitant use with tumor suppressor therapy is an immune complex consisting of recombinant IL15 and IL-15-Rα-Fc. Kaplan-Mel survival curves for mice treated with PBS, CD122/132+anti-PD-1, Ad-Luc+CD122/132+anti-PD-1 control, Ad-p53, or the combination of Ad-p53+CD122/132(IL15)+anti-PD-1 are shown in Figure 18. These survival curves were statistically significant by log-rank test (overall p < 0.0001; p < 0.0001 for Ad-p53+CD122/132(IL15)+anti-PD-1 treatment group compared with any other treatment group). The results also demonstrated an unexpectedly substantial synergistic effect of Ad-p53+CD122/132(IL15)+anti-PD-1 therapy. In the Ad-p53+CD122/132(IL15)+anti-PD-1 therapy group, 50% of the animals were alive by day 36. In stark contrast, all animals in the other treatment groups died by day 22, with a median survival ranging from 10 to 18 days.

Detailed Implementation

It is well known that tumors evolve during their initiation and progression to evade the destruction of the immune system. Although recent use of immune checkpoint inhibitors to reverse this resistance has shown some success, most patients do not respond to these treatments. Therefore, in some embodiments, the methods and compositions of this disclosure are used to alter the tumor microenvironment to overcome drug resistance and enhance the anti-tumor immune response. In one embodiment, a method for treating cancer is provided 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, 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 mutants, and/or IL-15 mutants. CD122/CD132 agonists can also be IL-15 mutants (e.g., IL-15N72D) that bind to IL-15 receptor α/IgG1 Fc fusion proteins (such as ALT-803) (Rhode et al., 2016).

In addition, the inventors have determined that administering adjunctive therapies (such as immune checkpoint inhibitors, like anti-PD1 antibodies) before, during, or after administration of p53 and/or MDA-7 gene therapy in combination with a preferred CD122/CD132 agonist enhances anti-tumor immunity.

Furthermore, the inventors have determined that administering an additional therapy to degrade the extracellular matrix of tumor cells enhances the tumor penetration of the combination therapy. Specifically, an extracellular matrix degradation therapy is administered prior to the combination therapy. In one method, the extracellular matrix degradation therapy is a relaxin gene therapy, such as adenoviral relaxin. Specifically, adenoviral relaxin is administered intratumorally or intraarterially.

In addition, treatment methods may include adjunctive anticancer therapies, such as cytokines or chemotherapy, to enhance the antitumor effects of the combination therapies presented herein. For example, cytokines may be granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemotherapy may be 5-fluorouracil (5FU), capecitabine, cyclophosphamide, or a PI3K inhibitor.

I. Definition

As used herein, "substantially free of" with respect to a particular component means that the particular component was not intentionally formulated into the composition and/or that the particular component exists only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is in which the amount of the particular component cannot be detected using standard analytical methods.

As used herein, "a" or "an" may mean one or more species. As used herein in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more species.

Unless explicitly stated otherwise, referring only to alternatives or that alternatives are mutually exclusive, the use of the term "or" in the claims means "and/or," although this disclosure supports the definition of "and/or" referring only to alternatives. As used herein, "another" may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that a value includes inherent error variations in the equipment or method used to determine that value, or variations that exist between the subjects under study.

As used herein, “wildtype” refers to the naturally occurring sequence of a nucleic acid at a genetic locus in an organism’s genome, as well as the sequence transcribed or translated from such nucleic acid. Therefore, the term “wildtype” can also refer to the amino acid sequence encoded by said nucleic acid. Since a genetic locus may have more than one sequence or allele in an individual population, the term “wildtype” encompasses all such naturally occurring alleles. As used herein, the term “polymorphism” refers to the presence of variation (i.e., the presence of two or more alleles) at a genetic locus in an individual within a population. As used herein, “mutant” refers to a change in the sequence of a nucleic acid or the protein, polypeptide, or peptide it encodes due to recombinant DNA technology.

When used in relation to proteins, genes, nucleic acids, or polynucleotides in cells or organisms, the term "exogenous" means a protein, gene, nucleic acid, or polynucleotide that has been introduced into a cell or organism by artificial or natural means; or when used in relation to cells, the term means a cell that has been isolated and subsequently introduced into other cells or organisms by artificial or natural means. Exogenous nucleic acids can originate from different organisms or cells, or they can be one or more additional copies of nucleic acids naturally present in an organism or cell. Exogenous cells can originate from different organisms, or they can originate from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid located at a chromosomal position different from its position on the chromosome in a natural cell, or a nucleic acid with a side sequence different from that found in nature.

An "expression construct" or "expression cassette" is a nucleic acid molecule that directs transcription. An expression construct includes at least one or more transcriptional control elements (such as promoters, enhancers, or their functional equivalents) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements, such as transcription termination signals, may also be included.

A “carrier” or “construction” (sometimes referred to as a gene delivery system or gene transfer “medium”) is a macromolecule or molecular complex containing polynucleotides to be delivered to host cells in vitro or in vivo.

A common type of vector, the "plasmid," is an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of chromosomal DNA. In some cases, it is round and double-stranded.

The “ori” or “ori origin” is, for example, a DNA sequence in lymphotropic herpesviruses that, when present in a plasmid within a cell, is capable of maintaining the linked sequence at or near the site of DNA synthesis in the plasmid and/or the origin of DNA synthesis. For example, the ori of EBV includes the FR sequence (20 imperfect copies of a 30 bp repeat sequence), preferably the DS sequence; however, other sites in EBV bind EBNA-1, for example, the Rep* sequence can serve as the origin of replication instead of DS (Kirshmaier and Sugden, 1998). Therefore, the origin of replication of EBV includes the FR, DS, or Rep* sequences, or any functionally equivalent sequences obtained through nucleic acid modification, or synthetic combinations derived from them. For example, the present invention can also use genetically engineered (e.g., through the insertion or mutation of a single element) origin of replication of EBV, as specifically described in Lindner et al., 2008.

A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgenic” that “encodes” a specific protein is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed in vitro or in vivo and optionally also translated into a gene product (e.g., a polypeptide). The coding region can exist as cDNA, genomic DNA, or RNA. When existing as DNA, the nucleic acid molecule can be single-stranded (i.e., sense strand) or double-stranded. The boundaries of the coding region are defined by a start codon at the 5’ (amino) end and a translation stop codon at the 3′ (carboxyl) end. Genes can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence will typically be located at the 3’ end of the gene sequence.

The term "control element" collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, splice sites, etc., that collectively provide for the replication, transcription, post-transcriptional processing, and translation of coding sequences in recipient cells. The presence of all these control elements is not necessary as long as the selected coding sequence can replicate, transcribe, and translate in a suitable host cell.

The term "promoter" is used in its general sense herein to refer to a nucleotide region containing a DNA regulatory sequence derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. It may contain genetic elements that regulate the binding of proteins and molecules, such as RNA polymerase and other transcription factors, to initiate specific transcription of the nucleic acid sequence. The phrases "operably positioned," "operably linked," "under the control of," and "under transcriptional control" refer to a promoter being in the correct functional position and/or orientation associated with the nucleic acid sequence to control transcription initiation and/or expression of that sequence.

An "enhancer" is a nucleic acid sequence that, when located near a promoter, imparts increased transcriptional activity relative to the transcriptional activity produced by the promoter in the absence of an enhancer domain.

The term "operably linked" or "co-expressed" of nucleic acid molecules refers to the linking of two or more nucleic acid molecules (e.g., nucleic acid molecules to be transcribed, promoters, and enhancer elements) in a manner that allows the nucleic acid molecules to be transcribed. The term "operably linked" or "co-expressed" of peptides and/or polypeptide molecules refers to the linking of two or more peptides and/or polypeptide molecules in a manner that produces a single polypeptide chain (i.e., a fusion polypeptide) having at least one property of each fused peptide and/or polypeptide component. The fusion polypeptide is preferably chimeric, i.e., composed of heterologous molecules.

"Homology" refers to the percentage of identity between two polynucleotides or two polypeptides. The correspondence between one sequence and another can be determined using techniques known in the art. For example, homology can be determined by directly comparing the sequence information of two polypeptide molecules using readily available computer programs. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that promote the formation of stable duplexes between homologous regions, followed by digestion with one or more single-strand-specific nucleases, and determining the size of the digested fragment. Two DNA sequences or two polypeptide sequences are "substantially homologous" to each other when, as determined by the methods described above, at least about 80%, preferably at least about 90%, and most preferably at least about 95%, of the nucleotides or amino acids match on molecules of a defined length.

The term "nucleic acid" generally refers to at least one molecule or strand of DNA, RNA, or a derivative or analog thereof, containing at least one nucleobase, such as naturally occurring purine or pyrimidine bases found in DNA (e.g., adenine "A", guanine "G", thymine "T", and cytosine "C") or naturally occurring purine or pyrimidine bases found in RNA (e.g., A, G, uracil "U", and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide". The term "oligonucleotide" refers to at least one molecule with a length between about 3 and about 100 nucleobases. The term "polynucleotide" refers to at least one molecule with a length greater than about 100 nucleobases. These definitions generally refer to at least one single-stranded molecule, but in certain embodiments, they will also encompass at least one additional strand that is partially, substantially, or completely complementary to the at least one single-stranded molecule. Therefore, nucleic acids may encompass at least one double-stranded molecule or at least one triple-stranded molecule, which contains one or more complementary strands or "complements" of a specific sequence of the strands constituting the molecule.

The term "therapeutic benefit" as used throughout this application refers to anything that promotes or enhances a patient's health in relation to cancer medical treatment. A non-exhaustive list of examples of therapeutic benefits includes: prolonging a patient's lifespan over any period of time; reducing or delaying the development of the disease; reducing excessive proliferation; reducing tumor growth; delaying metastasis; reducing the proliferation rate of cancer cells or tumor cells; inducing apoptosis in any treated cells or any cells affected by treated cells; and alleviating pain attributable to the patient's condition.

An "effective dose" is at least the minimum amount required to achieve a measurable improvement or prevention of a particular disease. The effective dose described herein can vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit the desired response in an individual. An effective dose is also the amount by which the beneficial effect of treatment outweighs any toxic or adverse effects of treatment. For prophylactic use, beneficial or desired outcomes include results such as: elimination or reduction of risk, reduction of severity, or delay of disease onset, which includes biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during the development of the disease. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as: reduction of one or more symptoms caused by the disease, improvement of the quality of life of those suffering from the disease, reduction of the dosage of other medications required to treat the disease, enhancement of the effect of another medication (e.g., by targeting), delay of disease progression, and/or prolongation of survival. In the case of cancer or tumor, an effective amount of a drug may have the following effects: reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., to some extent slowing down or ideally stopping) the infiltration of cancer cells into surrounding organs; inhibiting (i.e., to some extent slowing down and ideally stopping) tumor metastasis; inhibiting tumor growth to some extent; and/or alleviating one or more symptoms associated with the disease to some extent. An effective amount may be administered in one or more doses. For the purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be combined with another drug, compound, or pharmaceutical composition. Therefore, "effective amount" can be considered in the context of administering one or more therapeutic agents, and if a single agent, when combined with one or more other agents, yields or achieves the desired result, it can be considered that the single agent is administered in an effective amount.

As used herein, "carrier" includes any and all solvents, dispersion media, mediators, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption-retarding agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is considered. Additional active ingredients may also be incorporated into the composition.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the bioactivity of the active ingredient to be effective and that does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (mediators, additives) are those excipients that can be reasonably administered to test mammals to provide an effective dose of the active ingredient used.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural processes of the treated individual or cells during a clinicopathological process. Desired therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and reversing or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including but not limited to reducing the proliferation of cancer cells (or destroying cancer cells), reducing symptoms caused by the disease, improving the quality of life of individuals with the disease, reducing the dosage of other medications required to treat the disease, and/or prolonging the individual's survival.

"Anti-cancer" agents can negatively affect cancer cells/tumors in a subject's body, for example, by: promoting the killing of cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting immune responses against cancer cells or tumors, preventing or inhibiting cancer progression, or prolonging the lifespan of a subject with cancer.

The term “antibody” is used in the broadest sense in this article, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, the individual antibodies constituting that population are identical except for the possible presence of small amounts of mutations (e.g., naturally occurring mutations). Therefore, the modifier "monoclonal" indicates that the antibody's properties are not those of a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies typically comprise antibodies containing a polypeptide sequence that binds to a target, wherein the polypeptide sequence that binds to the target is obtained by a method comprising selecting a single polypeptide sequence that binds to the target from a pool of polypeptide sequences. For example, the selection method may be to select a unique clone from a pool of clones (e.g., hybridoma clones, phage clones, or recombinant DNA clones). It should be understood that the selected target-binding sequence may be further modified, for example, to increase affinity for the target, to humanize the target-binding sequence, to improve its production in cell cultures, to reduce its immunogenicity in vivo, to create multispecific antibodies, etc., and antibodies containing said modified target-binding sequences are also monoclonal antibodies of the present invention. Unlike polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), monoclonal antibody preparations target a single determinant on the antigen for each monoclonal antibody. In addition to their specificity, monoclonal antibody preparations are advantageous because they are generally unaffected by contamination from other immunoglobulins.

The term “CD122/CD132 agonist” or “preferred CD122/CD132 agonist” refers to an agent that preferentially binds to the CD122/CD132 receptor complex and has a low affinity for the IL-2α receptor (CD25) or IL-15α receptor. Known preferred CD122/CD132 agonists include IL2/anti-IL2 monoclonal antibody immune complexes (see, for example, U.S. Patent Publication No. US20170183403A1; which is incorporated herein by reference in its entirety); genetically engineered IL-2 mutant proteins having a modified amino acid sequence compared to wild-type IL-2 (see, for example, U.S. Patent Publication No. US 2017/0044229 A1; which is incorporated herein by reference in its entirety); genetically engineered IL-2 mutant proteins having a modified amino acid sequence compared to a combination of wild-type IL-2 and anti-IL2 monoclonal antibody immune complexes (see, for example, International Patent Publication No. WO2014100014A1; which is incorporated herein by reference in its entirety); and pegylated forms of IL-2, such as NKTR-214 (see, for example, Chary). ch et al., 2016; the entire contents of which are incorporated herein by reference); IL-15/anti-IL-15 monoclonal antibody immune complex; IL15/IL15 receptor α-IgG1-Fc (IL15/IL15Rα-IgG1-Fc) immune complex (see, for example, U.S. Patent Publication Nos. US20060257361A1, EP2724728A1 and Dubois et al., 2008; all of which are incorporated herein by reference); genetically engineered IL-15 mutant proteins having modified amino acid sequences compared to the combination of wild-type IL-15 and IL15Rα-IgG1-Fc immune complexes (see, for example, U.S. Patent Publication No. US20070160578; the entire contents of which are incorporated herein by reference); or a PEGylated form of IL-15 having preferred binding to CD122/CD132.

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

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 removing T cell dysfunction caused by signal transduction along the PD-1 signaling axis—resulting in the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists may include PD-1 binding antagonists, PD-L1 binding antagonists, or PD-L2 binding antagonists.

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, 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 with one or more binding partners. In one specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, 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 co-stimulatory signals mediated by or via cell surface proteins expressed during PD-1-mediated signal transduction in T lymphocytes, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In one particular aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another particular aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In yet another particular aspect, the PD-1 binding antagonist is CT-011 (pilizumab). In yet another particular aspect, the PD-1 binding antagonist is AMP-224.

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more binding partners (such as PD-1 or B7-1). In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 with its binding partner. In one particular aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, a PD-L1 binding antagonist includes an anti-PD-L1 antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more binding partners (such as PD-1 or B7-1). In one embodiment, a PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed during PD-L1-mediated signaling in T lymphocytes, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In one particular aspect, the anti-PD-L1 antibody is YW243.55.S70. In another particular aspect, the anti-PD-L1 antibody is MDX-1105. In another particular aspect, the anti-PD-L1 antibody is MPDL3280A. In yet another particular aspect, the anti-PD-L1 antibody is MEDI4736.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L2 with one or more binding partners (such as PD-1). In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 with one or more binding partners. In one particular aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, a PD-L2 antagonist includes an anti-PD-L2 antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L2 with one or more binding partners (such as PD-1). In one embodiment, a PD-L2 binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed during PD-L2-mediated signal transduction in T lymphocytes, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesive.

"Immune checkpoint inhibitors" are any compounds that inhibit the function of immune checkpoint proteins. Inhibition includes both reduction and complete blockage. Specifically, immune checkpoint proteins are human immune checkpoint proteins. Therefore, immune checkpoint protein inhibitors are specifically inhibitors of human immune checkpoint proteins.

"Extracellular matrix degrading protein" or "extracellular matrix degrading protein" refers to any protein that acts on the integrity of the cell matrix, particularly on at least one of the components of the matrix or on the bonds binding these various components, either wholly or partially degrading or destabilizing.

In this paper, "distant effect" refers to tumor shrinkage beyond the local treatment area. For example, a combination of local treatment using p53 and/or IL-24 with systemic treatment using immune checkpoint therapy can cause a distant effect at a distant, untreated tumor site.

II. Tumor suppressor factors

In some embodiments, a tumor suppressor therapy, such as p53 and/or MDA-7 therapy, is administered to the subject. Nucleic acids encoding p53 and/or MDA-7 can be provided by various methods known in the art.

In some respects, p53 and MDA-7 tumor suppressor therapy incorporates nucleic acid variants to enhance their activity. In some respects, the variant tumor suppressor nucleic acid is a p53 variant that resists negative regulation (Yun et al., 2012; this article is incorporated herein by reference in its entirety).

A.p53

In some embodiments, this disclosure provides combination therapies for treating cancer. Some of the combination therapies provided herein include p53 gene therapy, which involves administering a wild-type p53 gene to a subject. Wild-type p53 is considered an important growth regulator in many cell types. The p53 gene encodes a 375-amino acid phosphoprotein that can form complexes with host proteins such as large T antigen and E1B. This protein is present in normal tissues and cells, but at very low concentrations compared to transformed cells or tumor tissue.

Missense mutations are common in the p53 gene and are crucial for its oncogene transformation ability. A single genetic change caused by a point mutation can produce an oncogenic p53. However, unlike other oncogenes, p53 point mutations are known to occur in at least 30 different codons, generally producing dominant alleles that produce phenotypic shifts in cells without reducing homozygosity. Furthermore, many of these dominant-negative alleles appear to be tolerated in the organism and transmitted germlineally. The variety of mutant alleles appears to range from minimal functional impairment to strongly penetrating dominant-negative alleles (Weinberg, 1991). High levels of mutant p53 have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses.

In some respects, p53 biomarkers are used to select patients for p53 treatment. Specifically, a favorable tumor p53 biomarker profile is defined by immunohistochemistry, based on wild-type p53 gene configuration or <20% p53-positive cells (US Patent No. 9,746,471 and Nemunaitis et al., 2009; both are incorporated herein by reference in their entirety).

R.MDA-7

The combination therapy described herein may also include MDA-7 gene therapy, which involves administration of the full-length or truncated MDA-7 gene. The protein product of the mda-7 gene, interleukin (IL)-24, is a cytokine belonging to the IL-10 cytokine family and is also a tumor suppressor. Jiang et al., 1995 (WO1995011986), described the cDNA encoding the MDA-7 protein. The MDA-7 cDNA encodes an evolutionarily conserved protein with a predicted size of 23.8 kDa and 206 amino acids.

The nucleic acid encoding MDA-7 provided herein may encode a full-length or truncated human IL-24 protein or polypeptide. A truncated version of MDA-7 will contain one or more portions of a continuous amino acid region of the full-length sequence, but will not contain the entire sequence. The truncated version may be shortened by any number of continuous amino acids at any site on the polypeptide. For example, a truncated version of MDA-7 may encode amino acids from about 49 to about 206; about 75 to about 206; about 100 to about 206; about 125 to about 206; about 150 to about 206; about 175 to about 206; or about 182 to about 206. It is also contemplated that polypeptides containing at least about 85%, 90%, and 95% of human wild-type MDA-7 are within the scope of this invention.

C. Other tumor suppressor factors

Additional tumor suppressor factors may be utilized in this disclosure. Gene therapy vectors for use in this disclosure that incorporate other tumor suppressor genes include, but are not limited to, those listed in Table 1.

Table 1: Tumor Suppressor Genes

III. Extracellular matrix degradation

This article also provides methods for enhancing the antitumor effects of tumor suppressor gene therapies and/or immune checkpoint inhibitors. In one aspect, gene therapy delivery (e.g., viral distribution) and tumor penetration are enhanced by degrading proteins or agents in the extracellular matrix (ECM) or its components of tumor cells.

The extracellular matrix (ECM) is a collection of extracellular molecules secreted by cells that provide structural and biochemical support to surrounding cells. Because multicellularity has evolved independently across different multicellular lineages, the composition of the ECM varies among multicellular structures; however, cell adhesion, cell-cell communication, and differentiation are common functions of the ECM. ECM components that may be targeted by ECM degradative proteins include collagen, elastin, hyaluronic acid, fibronectin, and laminin.

A. Relaxin

One extracellular matrix degrading protein that can be used in the methods described herein is relaxin. Relaxin is a 6 kDa peptide hormone structurally associated with insulin and insulin-like growth factor. It is primarily produced in the corpus luteum and endometrium, and its serum levels increase significantly during pregnancy (Sherwood et al., 1984). Relaxin is an effective inhibitor of collagen expression when collagen is overexpressed, but it does not significantly alter the basal level of collagen expression compared to other collagens. It promotes the expression of various MMPs (such as MMP2, MMP3, and MMP9) to degrade collagen, leading to the degradation of connective tissue and basement membrane, resulting in the destruction of the extracellular matrix of the birth canal. In addition, relaxin has been observed to promote the expression of MMP1 and MMP3 in the lungs, heart, skin, intestine, mammary glands, blood vessels, and seminal ducts, where relaxin acts as an inhibitor of collagen overexpression (Qin, X. et al., 1997a; Qin, X. et al., 1997b).

Application of relaxin protein or nucleic acids encoding said 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 causing extracellular matrix degradation. Specifically, when applied to tumor tissue tightly enclosed by connective tissue, the combined administration of tumor suppressor gene therapy with relaxin has shown improved antitumor efficacy.

The relaxin protein can be the full-length relaxin or a biologically active portion of the relaxin molecule, as described in U.S. Patent No. 5,023,321. Specifically, relaxin is recombinant human relaxin (H2) or other active agents having relaxin-like activity, such as agents that competitively displace relaxin bound to a receptor. Relaxin can be prepared by any method known to those skilled in the art, preferably as described in U.S. Patent No. 4,835,251. Relaxin analogues or derivatives thereof are described in US5,811,395, and peptide synthesis is described in U.S. Patent Publication No. US2,011,003,9778.

Kim et al. (2006) described an exemplary adenovirus relaxin that can be used in the methods presented herein. In short, a replicative (Ad-ΔE1B-RLX) adenovirus expressing relaxin is generated by inserting the relaxin gene into the E3 adenovirus region.

B. Hyaluronidase

In some embodiments, any substance capable of hydrolyzing polysaccharides (such as hyaluronic acid) commonly found in the extracellular matrix can be applied. Specifically, the extracellular matrix degrading protein used in this invention can be hyaluronidase. Hyaluronic acid is a ubiquitous component of the vertebrate extracellular matrix. This glucuronic acid and glucosamine-based linear polysaccharide [D-glucuronic acid 1-β-3)N-acetyl-D-glucosamine (1-β-4)] can influence the physicochemical properties of the matrix by virtue of its ability to form a very viscous solution. Hyaluronic acid also interacts with various receptors and binding proteins located on the cell surface. These interactions are involved in a wide range of biological processes, such as fertilization, embryonic development, cell migration and differentiation, wound healing, inflammation, tumor growth, and the formation of metastases.

Hyaluronic acid is hydrolyzed by hyaluronidase, and this hydrolysis causes the breakdown of the extracellular matrix. Therefore, any substance possessing hyaluronidase activity is contemplated to be suitable for use in the methods of this invention, such as the hyaluronidase described in Kreil (Protein Sci., 1995, 4: 1666-1669). The hyaluronidase can be a hyaluronidase derived from mucins of mammals, reptiles, or hymenopterans; a hyaluronidase derived from mucins from the salivary glands of leeches; or a hyaluronidase derived from hyaluronic acid lysins of bacteria, particularly streptococci, pneumococci, and clostridium. The enzymatic activity of hyaluronidase can be assessed using conventional techniques, such as those described in Hynes and Ferretti (Methods Enzymol., 1994, 235: 606-616) or Bailey and Levine (J. Pharm. Biomed. Anal., 1993, 11: 285-292).

C. Core proteoglycans

Decorin, a leucine-rich small proteoglycan, is a ubiquitous component of the extracellular matrix and is preferentially found to associate with collagen fibrils. Decorin binds to collagen fibrils and delays the lateral assembly of individual triple-helical collagen molecules, thereby reducing the fibril diameter. Furthermore, decorin regulates the interactions between extracellular matrix components such as fibronectin and platelet-reactive proteins and cells. In addition, decorin can influence extracellular matrix remodeling by inducing matrix metalloproteinase collagenase. These observations suggest that decorin regulates extracellular matrix production and assembly at several levels and therefore plays an important role in the remodeling of connective tissue, as described by Choi et al. (Gene Therapy, 17:190-201, 2010) and by Xu et al. (Gene Therapy, 22(3):31-40, 2015).

An exemplary adenoviral core proteoglycan that can be used in the methods provided herein is described by Choi et al. (GeneTherapy, 17: 190-201, 2010). Briefly, a core proteoglycan-expressing, replicative (Ad-ΔE1B-DCNG) adenovirus is generated by inserting the core proteoglycan gene into the E3 adenovirus region. Another exemplary adenoviral core proteoglycan that can be used in the methods provided herein is described by Xu et al. (GeneTherapy, 22(3): 31-40, 2015). Similarly, a core proteoglycan-expressing, replicative (Ad.dcn) adenovirus is generated by inserting the core proteoglycan gene into the E3 adenovirus region.

IV. Nucleic Acids

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

The one or more nucleic acids 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, regardless of their sequence length, to create one or more nucleic acid constructs. There can be significant differences in the total length between nucleic acid constructs. Therefore, nucleic acid segments of almost any length can be used, wherein the total length is preferably limited by ease of preparation or use in the intended recombinant nucleic acid protocol.

A. Nucleic acid delivery via expression vectors

The vectors provided herein are primarily designed for the expression of 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 promoters (i.e., constitutive, inducible, repressible, and tissue-specific promoters). In some respects, p53 and MDA-7 may be co-expressed in the vector. In other respects, p53 and/or MDA-7 may be co-expressed with extracellular matrix degrading genes. Similarly, the vectors may contain selective markers to facilitate their in vitro manipulation unless otherwise specified.

Those skilled in the art should be well equipped to construct 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). Vectors include, but are not limited to, plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., those derived from Moloney's murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (Ad) vectors (including their replicative, replication-deficient, and non-viral gene forms), adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Ebola virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey's murine sarcoma virus vectors, murine mammary tumor virus vectors, and Rous sarcoma virus vectors.

1. Viral vector

In certain aspects of the invention, viral vectors encoding tumor suppressor factors and/or extracellular matrix degradation genes may be provided. In the generation of recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences of heterologous (or non-natural) proteins. A viral vector is an expression construct that utilizes a viral sequence to introduce nucleic acids and, possibly, proteins into a cell. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes makes them attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids of certain aspects of the invention are described below.

Lentivirals are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. 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. Patents 6,013,516 and 5,994,136).

Recombinant lentiviral vectors can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and nucleic acid sequence expression. For example, U.S. Patent 5,994,136 describes a recombinant lentivirus capable of infecting non-dividing cells, wherein suitable host cells are transfected with two or more vectors carrying packaging functions (i.e., gag, pol, and env, and rev and tat), which is incorporated herein by reference.

a. Adenovirus vector

A method for delivering tumor suppressor factors and/or extracellular matrix degrading genes involves the use of an adenoviral expression vector. Although adenoviral vectors are known to have a low capacity for integration into genomic DNA, the high efficiency of gene transfer provided by these vectors offsets this characteristic. The adenoviral expression vector comprises a construct containing an adenoviral sequence sufficient to (a) support the packaging of the construct and (b) ultimately express a recombinant gene construct cloned therein.

Adenoviruses are known to those skilled in the art for their growth and manipulation, and exhibit a wide host range both in vitro and in vivo. This group of viruses can be obtained at high titers, such as 10⁹–10¹¹ plaque-forming units per ml, and they possess high infectivity. The life cycle of adenoviruses does not require integration into the host cell genome. The exogenous genes delivered by adenoviral vectors are free-form, thus exhibiting low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenoviruses (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Understanding the genetic organization of adenoviruses—a 36kb linear double-stranded DNA virus—allows for the replacement of large segments of adenoviral DNA with foreign sequences up to 7kb (Grunhaus and Horwitz, 1992). Unlike retroviruses, adenoviral infection of host cells does not lead to chromosomal integration because adenoviral DNA can replicate in a free manner without potential genotoxicity. Furthermore, adenoviruses are structurally stable, and no genomic rearrangements have been detected after extensive amplification.

Adenoviruses are particularly well-suited as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titers, wide target cell range, and high infectivity. The viral genome contains inverted repeat sequences (ITRs) of 100-200 base pairs at both ends; these ITRs are cis-elements essential for viral DNA replication and packaging. Early (E) and late (L) regions of the genome contain distinct transcriptional units separated by the initiation of viral DNA replication. The E1 regions (E1A and E1B) encode proteins responsible for regulating the transcription of the viral genome and some cellular genes. Expression of the E2 regions (E2A and E2B) leads to the synthesis of proteins used for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutdown (Renan, 1990). The products of late genes, including most viral capsid proteins, are expressed only after extensive processing of a single primary transcript emitted by the major late promoter (MLP). The MLP (located at 16.8 m.u.) is particularly effective during the late stage of infection, and all mRNAs emitted from this promoter have a 5′-triple leader (TPL) sequence that makes the mRNA a specific mRNA for translation.

The recombinant adenovirus described herein can be generated through homologous recombination between a shuttle vector and a proviral vector. Due to the potential recombination between the two proviral vectors, wild-type adenovirus can be generated from this process. Therefore, a single clone of the virus is isolated from a single plaque, and the genomic structure of said clone is examined.

The adenovirus vector can be replicative, replication-deficient, or conditionally deficient; the nature of the adenovirus vector is not considered essential to the successful practice of this invention. The adenovirus can be any of 42 different known serotypes or subgroups A-F. Subgroup C type 5 adenovirus is a specific starting material to obtain a conditionally replication-deficient adenovirus vector for use in this invention. This is because type 5 adenovirus is a human adenovirus about which a great deal of biochemical and genetic information is known, and which has historically been used in most constructions employing adenovirus as a vector.

Nucleic acids can be introduced into adenoviral vectors at locations where coding sequences have been removed. For example, the E1 coding sequence can be removed from replication-defective adenoviral vectors. Polynucleotides encoding the gene of interest can also be inserted into E3-replacement vectors to replace the missing E3 region, as described by Karlsson et al. (1986), or into the E4 region, in which helper cell lines or helper viruses supplement the E4 deficiency.

The generation and proliferation of replication-defective adenovirus vectors can be performed using helper cell lines. A unique helper cell line, named 293, is transformed from human embryonic kidney cells via the Ad5 DNA fragment and constitutively expresses the E1 protein (Graham et al., 1977). Since the E3 region is separable from the adenovirus genome (Jones and Shenk, 1978), adenovirus vectors can carry exogenous DNA in the E1 region, the E3 region, or both, with the aid of 293 cells (Graham and Prevec, 1991).

Helper cell lines can be derived from human cells, such as human embryonic 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 permit human adenovirus. Such cells include, for example, monkey kidney cells or other monkey embryonic mesenchymal or epithelial cells. As described above, a specific helper cell line is 293.

Methods for producing recombinant adenovirus are known in the art, such as U.S. Patent No. 6,740,320, which is incorporated herein by reference. Furthermore, Racher et al. (1995) disclosed an improved method for culturing 293 cells and propagating adenovirus. In one form, native cell aggregates are grown by inoculating single cells into 1-liter silicated roller flasks (Techne, Cambridge, UK) containing 100–200 ml of culture medium. Cell viability is estimated using trypan blue after stirring at 40 rpm. In another form, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/L) are used as follows. Cell inoculum resuspended in 5 ml of culture medium is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and allowed to stand for 1–4 hours with occasional agitation. The culture medium is then replaced with 50 ml of fresh culture medium and shaking is initiated. To produce the virus, allow the cells to grow to approximately 80% confluence, then replace the medium (to 25% final volume) and add adenovirus at 0.05 MOI. Let the culture stand overnight, then increase its volume to 100% and begin shaking again for 72 hours.

b. Retroviral vector

Additionally, tumor suppressor factors and/or extracellular matrix degradation genes can be encoded by retroviral vectors. Retroviruses are a group of single-stranded RNA viruses characterized by their ability to convert their RNA into double-stranded DNA in infected cells via reverse transcription (Coffin, 1990). The resulting DNA is then stably integrated into the cell chromosome as a provirus and directs the synthesis of viral proteins. This integration results in the retention of viral gene sequences in the recipient cell and its progeny. The retroviral genome contains three genes: gag, pol, and env, which encode capsid proteins, polymerases, and envelope components, respectively. A sequence found upstream of the gag gene contains signals for packaging the genome into the virion. Two long terminal repeats (LTRs) are present at the 5' and 3' ends of the viral genome. These sequences contain strong promoter and enhancer sequences and are essential for integration into the host cell genome (Coffin, 1990).

To construct retroviral vectors, nucleic acids encoding the genes of interest are inserted into the viral genome, replacing certain viral sequences to produce replication-defective viruses. To generate virions, packaging cell lines containing the gag, pol, and env genes but lacking the LTR and packaging components are constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA along with the retroviral LTR and packaging sequence is introduced into this cell line (by, for example, calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

A problem associated with the use of defective retroviral vectors is the potential for replication-capable wild-type viruses to appear in packaging cells. This could be due to recombination events in which the complete sequence from the recombinant virus is inserted upstream of the gag, pol, and env sequences integrated into the host cell genome. However, available packaging cell lines should significantly reduce the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

c. Adeno-associated virus vector

Adeno-associated virus (AAV) is an attractive vector system for use in this disclosure because of its high integration frequency and its ability to infect undivided cells, thereby making it suitable for gene delivery into mammalian cells (Muzyczka, 1992). AAV has a broad host range (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988), which means it can be used in this invention. Details regarding the production and use of the rAAV vector are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

AAV is a parvovirus-dependent infection because it requires co-infection with another virus (adenovirus or a member of the herpesvirus family) to achieve productive infection in cultured cells (Muzyczka, 1992). Without co-infection with a helper virus, the wild-type AAV genome integrates into human chromosome 19 via its ends, where it resides latently as a provirus (Kotin et al., 1990; Samulski et al., 1991). However, rAAV integration is not limited to chromosome 19 unless the AAV Rep protein is also expressed (Shelling and Smith, 1994). When cells carrying the AAV provirus are repeatedly infected with a helper virus, the AAV genome is "rescued" from the chromosome or recombinant plasmid, and normal productive infection is established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990; Muzyczka, 1992).

Typically, recombinant AAV (rAAV) viruses are prepared by co-transfection with a plasmid containing the gene of interest flanked by two AAV terminal repeat sequences (McLaughlin et al., 1988; Samulski et al., 1989; all of which are incorporated herein by reference) and an expression plasmid containing the wild-type AAV coding sequence but without the terminal repeat sequences, such as pIM45 (McCarty et al., 1991). Cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function. The rAAV virus stock prepared in this manner is contaminated with adenovirus, which must be physically separated from the rAAV particles (e.g., by density centrifugation with cesium chloride). 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 carrying rAAV DNA as a pre-integration virus can also be used (Flotte et al., 1995).

d. Other viral vectors

Other viral vectors can be used as constructs in this disclosure. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) and herpesvirus can be employed. They provide a variety of attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

Molecular clones of Venezuelan equine encephalitis (VEE) virus have been genetically modified into replicative vaccine vectors for expressing heterologous viral proteins (Davis et al., 1996). Studies have shown that VEE infection stimulates an effective CTL response, and VEE has been shown to be an extremely useful vector for immunization (Caley et al., 1997).

In other embodiments, the nucleic acid encoding chimeric CD154 is contained within an infectious virus engineered to express a specific binding ligand. Thus, the viral particle will specifically bind to the homologous receptor on the target cell and deliver its contents to the cell. Recently, a novel method designed to allow specific targeting of retroviral vectors has been developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification can allow specific infection of hepatocytes via salivary glycoprotein receptors.

For example, targeting of recombinant retroviruses has been designed using biotinylated antibodies against retroviral envelope proteins and specific cellular receptors. Antibodies are conjugated via biotinylated components using streptavidin (Roux et al., 1989). Antibodies targeting major histocompatibility complex class I and II antigens have demonstrated in vitro infection of various human cells carrying these surface antigens by ecotropic viruses (Roux et al., 1989).

2. Control element

Expression cassettes contained in vectors usable in this disclosure specifically contain (along the 5' to 3' direction) a eukaryotic transcription promoter operatively linked to a protein-coding sequence, a splicing signal containing an interpolated sequence, and a transcription termination/polyadenylation sequence. Promoters and enhancers controlling transcription of protein-coding genes in eukaryotic cells consist of a variety of genetic elements. Cellular mechanisms are able to collect and integrate the regulatory information conveyed by each element, thereby allowing different genes to evolve unique, often complex, patterns of transcriptional regulation. Promoters used in the context of this invention include constitutive, inducible, and tissue-specific promoters.

a. Promoters/enhancers

The expression constructs presented in this paper contain promoters to drive the expression of tumor suppressor factors and/or extracellular matrix degradation genes. Promoters typically contain sequences that locate the RNA synthesis initiation site. The most well-known example of such a sequence is the TATA box, but in some promoters lacking a TATA box, such as the promoters of mammalian terminal deoxynucleotidyl transferase genes and SV40 late genes, discrete elements covering the initiation site themselves help to fix the initiation location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these promoters are located in a region 30–110 bp upstream of the initiation site, but many promoters have been shown to also contain functional elements downstream of the initiation site. To bring the coding sequence “under promoter control,” the 5′ end of the transcription start site of the transcription reading frame is placed “downstream” (i.e., the 3′ end) of the selected promoter. The “upstream” promoter stimulates DNA transcription and promotes the expression of the encoded RNA.

The spacing between promoter elements is often flexible, so promoter function is preserved when elements are reversed or moved relative to each other. In the tk promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can act synergistically or independently to activate transcription. Promoters may or may not be used with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

Promoters can be promoters that naturally associate with a nucleic acid sequence, such as those obtained by isolating a 5′ non-coding sequence located upstream of a coding region and/or exon. Such promoters can be referred to as “endogenous.” Similarly, enhancers can be enhancers that are naturally associated with a nucleic acid sequence, located downstream or upstream of that sequence. Alternatively, certain advantages are gained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which is a promoter that does not normally associate with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that do not normally associate with a nucleic acid sequence in their natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cells, and promoters or enhancers that are not “naturally present” (i.e., contain different elements of different transcriptional regulatory regions and/or mutations that alter expression). For example, the promoters most commonly used for recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthetically generating nucleic acid sequences for promoters and enhancers, sequences can also be generated using recombinant cloning and/or nucleic acid amplification technologies (including PCR ^™ ) in conjunction with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, both of which are incorporated herein by reference). Furthermore, it is conceivable that control sequences guiding transcription and/or expression in non-nuclear organelles (such as mitochondria, chloroplasts, etc.) could also be employed.

Naturally, it will be important to employ promoters and/or enhancers that effectively direct the expression of DNA segments in the selected organelles, cell types, tissues, organs, or organisms for expression. Those skilled in the art of molecular biology are generally familiar with using combinations of promoters, enhancers, and cell types for protein expression (see, for example, Sambrook et al., 1989, which is incorporated herein by reference). The promoters employed can be constitutive, tissue-specific, inducible, and/or, under appropriate conditions, advantageous for directing high-level expression of the introduced DNA segment, such as in the large-scale production of recombinant proteins and/or peptides. Promoters can be heterologous or endogenous.

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

Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the β-actin promoter (Ng, 1989; Quitsche et al., 1989), the GADPH promoter (Alexander et al., 1988; Ercolani et al., 1988), and the metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and cascade response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter near the minimal TATA box (tre). Human growth hormone promoter sequences (e.g., the minimal human growth hormone promoter described with Genbank accession number X05244, nucleotides 283-341) or mouse mammary tumor promoters (available from ATCC, catalog number ATCC 45007) can also be used. In some embodiments, the promoters are CMV IE, dectin-1, dectin-2, human CD11c, F4/80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I, or MHC class II promoters; however, any other promoters that can drive the expression of the p53, MDA-7, and/or relaxin genes may be used in the practice of this invention.

In some respects, the methods of this disclosure also involve enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have the potential for cis-action even at relatively long distances (at most a few thousand bases from the target promoter), regardless of orientation. However, enhancer function is not necessarily limited to such long distances, as they can also function at very close proximity to a given promoter.

b. Initial signals and connection expressions

Specific start signals can also be used in the expression constructs provided in this disclosure to efficiently translate coding sequences. These signals include the ATG start codon or adjacent sequences. Exogenous translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will be able to readily determine this and provide the necessary signals. It is well known that the start codon must “frame-match” the reading frame of the desired coding sequence to ensure the translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be improved by including appropriate transcription enhancer elements.

In some embodiments, internal ribosome entry site (IRES) elements are used to create multi-gene or polycistronic information. IRES elements are able to bypass ribosome scanning models of 5′ methylation-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements from two members of the parvovirus family (poliovirus and encephalocardivirus) have been described (Pelletier and Sonenberg, 1988), as well as from mammalian information (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames (OPGs). Multiple OPGs can be transcribed together, each separated by an IRES, thereby creating polycistronic information. With the aid of IRES elements, ribosomes can access each OPG for efficient translation. A single information can be transcribed using a single promoter/enhancer to efficiently express multiple genes (see U.S. Patent Nos. 5,925,565 and 5,935,819, both of which are incorporated herein by reference).

Additionally, certain 2A sequence elements can be used to create linked or co-expressed genes in the constructs provided in this disclosure. For example, cleavage sequences can be used to co-express genes by linking open reading frames to form a single cistron. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or “2A-like” sequences (e.g., Thoseea asigna virus 2A; T2A) (Minskaia and Ryan, 2013).

c. The starting point of replication

In order for the vector to proliferate in host cells, the vector may contain one or more replication initiation sites (commonly referred to as "ori"), for example, nucleic acid sequences corresponding to the oriP of EBV as described above or genetically modified oriP with similar or enhanced functionality in programming, said nucleic acid sequences being specific nucleic acid sequences for initiating replication. Alternatively, replication origins or autonomous replication sequences (ARS) of other extrachromosomal replicating viruses as described above may be used.

3. Select and filter tags

In some embodiments, cells containing constructs of this disclosure can be identified in vitro or in vivo by including markers in the expression vector. Such markers confer identifiable alterations to the cells, thereby allowing easy identification of cells containing the expression vector. Typically, selection markers are markers that confer the property of allowing selection. Positive selection markers are those in which the presence of the marker allows selection, while negative selection markers are those in which the presence of the marker prevents selection. An example of a positive selection marker is a drug resistance marker.

Typically, drug-selective markers are helpful for cloning and identifying transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin (zeocin), and histamine are useful selection markers. Besides markers that confer resistance allowing for condition-based differentiation of transformant phenotypes, other types of markers are envisioned, including selection markers such as GFP, which are based on colorimetric analysis. Alternatively, selectable enzymes, such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), can be used as negative selection markers. Those skilled in the art will also know how to use immunological markers, possibly in conjunction with FACS analysis. The markers used are considered unimportant, provided that the marker can be co-expressed with the nucleic acid encoding the gene product. Other examples of selection and selection markers are well known to those skilled in the art.

B. Other nucleic acid delivery methods

In addition to viral delivery of nucleic acids encoding tumor suppressor factors and/or extracellular matrix degradation genes, the following are other methods for delivering recombinant genes to a given host cell, and are therefore considered in this disclosure.

As described herein or as will be known to those skilled in the art, the introduction of nucleic acids (such as DNA or RNA) can be performed using any suitable method for delivering nucleic acids to transform cells. Such methods include, but are not limited to, direct delivery of DNA via: in vitro transfection (Wilson et al., 1989; Nabel et al., 1989), via 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, all of which are incorporated herein by reference), said injection including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215 (which is incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, which is incorporated herein by reference; Tur-Kaspa et 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 DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct acoustic loading (Fechheimer et al., 1987); transfection via liposome-mediated methods (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated methods (Wu and Wu, 1987; Wu and Wu, 1988); transfection via particle bombardment (PCT applications WO 94/09699 and 95/06128; US patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,53 Patents 8,877 and 5,538,880 (all of which are incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, all of which are incorporated herein by reference); by transformation mediated by Agrobacterium (U.S. Patents 5,591,616 and 5,563,055, all of which are incorporated herein by reference); by DNA uptake mediated by drying/inhibition (Potrykus et al., 1985), and any combination of such methods. By applying techniques such as these, one or more organelles, one or more cells, one or more tissues, or one or more organisms can be transformed stably or transiently.

1. Electroporation

In certain specific embodiments of this disclosure, gene constructs are introduced into target overproliferating cells via electroporation. Electroporation involves exposing cells (or tissues) and DNA (or DNA complexes) to a high-voltage discharge.

Electroporation transfection of eukaryotic cells has been very successful. In this way, 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).

It is anticipated that electroporation conditions for overproliferating cells from different sources can be optimized. Optimization of parameters such as voltage, capacitance, time, and electroporation medium composition may be particularly desirable. The implementation of other conventional adjustments will be known to those skilled in the art. See, for example, Hoffman, 1999; Heller et al., 1996.

2. Lipid-mediated transformation

In another embodiment, tumor suppressor factors and/or extracellular matrix degrading genes can be embedded in liposomes or lipid formulations. Liposomes are vesicle structures characterized by a phospholipid bilayer and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. These multiple lipid layers spontaneously form when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, and water and dissolved solutes are entrained between the lipid bilayers (Ghosh and Bachhawat, 1991). Gene constructs complexed with Lipofectamine (Gibco BRL) are also envisioned.

Lipid-mediated delivery and expression of exogenous DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated the feasibility of lipid-mediated delivery and expression of exogenous DNA in cultured chicken embryo cells, HeLa cells, and hepatocellular carcinoma cells.

Lipid-based nonviral formulations offer an alternative to adenovirus gene therapy. Although numerous cell culture studies have documented lipid-based nonviral gene transfer, systemic gene delivery via lipid-based formulations remains limited. A major limitation of nonviral lipid-based gene delivery is the toxicity of the cationic lipids constituting the nonviral delivery medium. In vivo toxicity of liposomes partially explains the discrepancy between in vitro and in vivo gene transfer outcomes. Another factor contributing to this contradictory data is the difference in lipid medium stability in the presence and absence of serum proteins. The interaction between lipid mediums and serum proteins has a significant impact on the stability characteristics of lipid mediums (Yang and Huang, 1997). Cationic lipids attract and bind to negatively charged serum proteins. Lipid mediums associated with serum proteins are lysed or absorbed by macrophages, thereby facilitating the removal of said lipid mediums from circulation. Current in vivo lipid delivery methods utilize subcutaneous, intradermal, intratumoral, or intracranial injections to avoid the toxicity and stability problems associated with cationic lipids in circulation. The interaction between lipid mediators and plasma proteins is the reason for the difference in gene transfer efficiency between in vitro (Felgner et al., 1987) and in vivo (Zhu et 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).

Advances in lipid formulations have improved the efficiency of in vivo gene transfer (Templeton et al., 1997; WO 98/07408). Novel lipid formulations consisting of equimolar ratios of 1,2-bis(oleoyloxy)-3-(trimethylamine)propane (DOTAP) and cholesterol significantly enhanced systemic in vivo gene transfer by approximately 150-fold. The DOTAP:cholesterol lipid formulation forms a unique structure known as a “sandwich liposome.” This formulation reportedly sandwiches DNA between recessed bilayers or “vase” structures. Beneficial features of these lipid structures include positive p-coagulation, colloidal stabilization via cholesterol, two-dimensional DNA stacking, and increased serum stability. Patent applications 60/135,818 and 60/133,116 discuss formulations that can be used with the present invention.

The production of lipid formulations is typically accomplished through sonication or continuous extrusion of a liposome mixture following (I) reverse-phase evaporation; (II) dehydration-rehydration; (III) detergent dialysis; and (IV) membrane hydration. Once formed, lipid structures can be used to encapsulate compounds that are toxic (chemotherapeutic agents) or unstable (nucleic acids) when in circulation. Lipid encapsulation has contributed to lower toxicity and longer serum half-lives for such compounds (Gabizon et al., 1990). Lipid-based gene transfer strategies are being used to enhance conventional therapies or develop novel treatments for many diseases, particularly for the treatment of hyperproliferative disorders.

V. Preferred CD123/CD132 agonists

In some respects, administering at least one CD122/CD132 agonist to a subject, such as a CD122/CD132 agonist that preferentially binds to the CD122/CD132 receptor complex and has a low affinity for binding to the CD25 or IL15α receptor. CD122/CD132 may be selected from genetically engineered IL-22 mutant proteins that have a modified amino acid sequence compared to wild-type IL2 (US 2017/0044229; this patent is incorporated herein by reference in its entirety). In some respects, preferred CD122/CD132 agonists are IL-2/anti-IL-2 monoclonal antibody immune complexes (US20170183403A1; this patent is incorporated herein by reference in its entirety); or genetically engineered IL-2 mutant proteins with modified amino acids compared to a combination of wild-type IL-2 and anti-IL2 monoclonal antibody immune complexes (WO2014100014A1; this patent is incorporated herein by reference in its entirety); pegylated forms of IL-2, such as NKTR-214 (Charych et al., 2016); and IL15/anti-IL15 monoclonal antibody immune complexes. ; IL15/IL15 receptor α-IgG1-Fc (IL15/IL15Rα-IgG1-Fc) immune complex (US2006025736IA1, EP2724728A1, and Dubois et al., 2008); a genetically engineered IL-15 mutant protein having a modified amino acid sequence compared to the combination of wild-type IL-15 and IL15Rα-IgG1-Fc immune complex (US20070160578; this patent is incorporated herein by reference in its entirety); or a PEGylated form of IL15 that preferentially binds to CD122/CD132. In some embodiments, more than one CD122/CD132 agonist is used.

VI. Oncolytic viruses

In some aspects, this disclosure includes the application of at least one oncolytic virus. In some aspects, the oncolytic virus is engineered to express p53, MDA-7, IL-12, TGF-β inhibitors, ADP and/or IL-10 inhibitors. In some aspects, the oncolytic 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, Ebola virus, influenza virus, reovirus, myxoma virus, Malaba virus, rhabdovirus, enadenotucirev, or Coxsackie virus. In some aspects, the oncolytic virus is engineered to express cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-12. In some aspects, the oncolytic virus is further defined as talimogene heherparepvec (T-VEC). In some respects, oncolytic adenovirus vectors are derived from modified TERT promoter oncolytic adenoviruses (US Patent No. 8,067,567; which is incorporated herein by reference in its entirety) and/or HRE-E2F-TERT hybrid promoter oncolytic adenoviruses (PCT/KR2011/004693; which is incorporated herein by reference in its entirety) and/or adenoviruses having modified E1a regulatory sequences, wherein at least one Pea3 binding site or a functional portion thereof is deleted using an Elb-19K clone insertion site (EP2403951A2; which is incorporated herein by reference in its entirety), said oncolytic adenoviruses may be entirely modified to express therapeutic genes. In some respects, oncolytic adenovirus vectors are derived from E1b-deleted oncolytic adenoviruses (Yu and Fang, 2007; Li, 2009; both of which are incorporated herein by reference in their entirety).

Exemplary oncolytic 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, Toca511, Ancori, RIGVIR, adenoviruses overexpressing adenovirus death protein (ADP) as described in US 7589069 B1, which is incorporated herein by reference in its entirety, such as VirRx007, and vaccinia virus expressing IL12 without N1L as described in PCT/GB2015/051023, which is incorporated herein by reference in its entirety. Other exemplary oncolytic viruses are described, for example, in international patent publications WO2015/027163, WO2014/138314, WO2014/047350 and WO2016/009017, all of which are incorporated herein by reference.

In one particular aspect, the oncolytic virus agent is talimogene laherparepvec (T-VEC), which is a genetically engineered oncolytic herpes simplex virus expressing GM-CSF. Talimogene laherparepvec, HSV-1 [strain JS1] ICP34.5-/ICP47-/hGM-CSF (formerly known as OncoVEX ^{GM CSF)} , is an intratumorally delivered oncolytic immunotherapy containing immune-enhanced HSV-1 that selectively replicates in solid tumors. (Lui et al., 2003; U.S. Patent Nos. 7,223,593 and 7,537,924; these documents are incorporated herein by reference). In October 2015, the U.S. FDA approved T-VEC, marketed under the name IMLYGIC ^™ , for the treatment of melanoma in patients with unresectable tumors. The characteristics and administration methods of T-VEC are described in, for example, the IMLYGIC ^™ packaging insert (Amgen, 2015) and U.S. Patent Publication No. US2015/0202290, both of which are incorporated herein by reference. For example, talimogene laherparepvec is typically administered intratumorally to injectable skin, subcutaneous, and lymph node tumors at a dose of up to 4.0 ml at ^10⁶ plaque-forming units/mL (PFU/mL) on day 1 of week 1, followed by intratumoral injection at a dose of up to 4.0 ml at ^10⁸ PFU/mL on day 1 of week 4, and then every 2 weeks (±3 days) into injectable skin, subcutaneous, and lymph node tumors. The recommended volume of talimogene laherparepvec to be injected into one or more tumors depends on the size of those tumors and should be determined according to injection volume guidelines. Although T-VEC has shown clinical activity in patients with melanoma, many cancer patients do not respond to or cease responding to T-VEC treatment. In one embodiment, p53 and/or MDA-7 nucleic acids, along with at least one CD122/CD132 agonist, may be administered after, during, or before T-VEC therapy, such as to reverse treatment resistance.

In some embodiments, the E1b-deficient oncolytic adenovirus is combined with at least one preferred CD122/CD132 agonist and at least one immune checkpoint inhibitor. Exemplary E1b-deficient oncolytic adenoviruses are H101 (Ankerui); Onyx 015 or H103 expressing heat shock protein 70 (HSP70); or oncolytic adenovirus H102, wherein the expression of the Ad E1a gene is driven by the alpha-fetoprotein (AFP) promoter, resulting in preferential replication in hepatocellular carcinoma and other cancers that overexpress AFP compared to normal cells (Yu and Fang, 2007; Li, 2009; both are incorporated herein by reference in their entirety).

Other representative CD122/CD132 agonists used in this invention include, but are not limited to, the agents listed in Table 2 below:

Abbreviations: MAb = monoclonal antibody; Ab = antibody; r = recombinant; h = human; Rα = receptor α;

VII. Immune checkpoint inhibitors

In some embodiments, this disclosure provides a method of 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 that act as either on (e.g., co-stimulatory molecules) or off signals. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuating factor (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed cell death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and T cell activation V domain Ig inhibitor (VISTA). Specifically, immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.

Immune checkpoint inhibitors can be pharmaceuticals, such as small molecules, recombinant forms of ligands or receptors, or, in particular, antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogues can be used, specifically chimeric, humanized, or human forms of antibodies. As those skilled in the art will appreciate, alternative and/or equivalent names may be used for certain antibodies mentioned in this disclosure. In the context of this invention, such alternative and/or equivalent names are interchangeable. For example, lambolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

It is conceivable that any immune checkpoint inhibitor known in the art that stimulates an immune response can be used. Such immune checkpoint inhibitors include inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. These immune checkpoint inhibitors include, but are not limited to, agents targeting immune checkpoint proteins and pathways involving PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, LAG3 inhibitors known in the art include soluble LAG3 (IMP321 or LAG3-Ig disclosed in WO2009044273) and mouse or humanized antibodies that block human LAG3 (e.g., IMP701 disclosed in WO2008132601), or fully human antibodies that block human LAG3 (such as those disclosed in EP 2320940). Another example is provided by using a BTLA blocker, which includes, but is not limited to, antibodies that block the interaction of human BTLA with its ligands (such as 4C7 disclosed in WO2011014438). Another example is provided by using agents that neutralize B7H4, including but not limited to antibodies against human B7H4 (disclosed in WO 2013025779 and WO2013067492) or soluble recombinant forms of antibodies against B7H4 (such as those disclosed in US20120177645). Yet another example is provided by using agents that neutralize B7-H3, including but not limited to antibodies that neutralize human B7-H3 (e.g., MGA271 disclosed in US 20120294796 as BRCA84D and its derivatives). Another example is provided by agents that target TIM3, including but not limited to antibodies that target human TIM3 (e.g., the anti-human TIM3 blocking antibody F38-2E2 disclosed in WO 2013006490 A2, or by Jones et al., J Exp Med. 2008; 205(12): 2763-79).

Additionally, more than one immune checkpoint inhibitor (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody) can be combined with tumor suppressor gene therapy. For example, p53 gene therapy and an immune checkpoint inhibitor (e.g., anti-KIR antibody and/or anti-PD-1 antibody) can be administered to enhance innate anti-tumor immunity, followed by IL24 gene therapy and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody) to induce an adaptive anti-tumor immune response.

A. PD-1 axis antagonist

T cell dysfunction or non-responsiveness occurs concurrently with the induction and persistent expression of the inhibitory receptor programmed death 1 peptide (PD-1). Therefore, this article provides therapeutic targeting of PD-1 and other molecules that signal through interaction with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). 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, this article provides the inhibition of PD-L1/PD-1 interaction in combination with p53, ADP, and/or MDA-7 gene therapies, such as to enhance CD8 ⁺ T cell-mediated tumor killing.

This article provides a method for treating or delaying cancer progression in an individual, the method comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist in combination with p53, ADP (VirRx007), and/or MDA-7 gene therapy. This article also provides a method for enhancing the immune function of an individual in need, the method comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist in combination with p53, ADP (VirRx007), and/or MDA-7 gene therapy.

For example, PD-1 axis binding 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.

In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In one particular aspect, 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 one particular aspect, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In one particular aspect, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists used in the methods provided herein are known in the art, such as those described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

In some embodiments, the PD-1 binding 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 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin sequence) of PDL1 or PDL2). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and the anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck3475, lambolizumab, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342. Additional PD-1 binding antagonists include pilizumab (also known as CT-011), MEDI0680 (also known as AMP-514), and REGN2810.

In some respects, immune checkpoint inhibitors are PD-L1 antagonists, such as durvalumab, also known as MEDI4736; atezolizumab, also known as MPDL3280A; or avermab, also known as MSB00010118C. In some respects, immune checkpoint inhibitors are PD-L2 antagonists, such as rHIgM12B7. In some respects, immune checkpoint inhibitors are LAG-3 antagonists, such as, but not limited to, IMP321 and BMS-986016. Immune checkpoint inhibitors can also be adenosine A2a receptor (A2aR) antagonists, such as PBF-509.

In some respects, the antibodies described herein (such as anti-PD-1 antibodies, anti-PDL1 antibodies, or anti-PDL2 antibodies) also contain human or mouse constant regions. In another respect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another specific respect, the human constant region is IgG1. In yet another specific respect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In yet another specific respect, the antibody has reduced or minimal effector function. In yet another specific respect, minimal effector function is caused by production in prokaryotic cells. In yet another specific respect, minimal effector function is caused by "effector-less Fc mutation" or aglycosylation.

Therefore, the antibodies used in this paper can be deglycosylated. Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain, where X is any amino acid except proline. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid (most commonly serine or threonine), but 5-hydroxyproline or 5-hydroxylysine can also be used. The removal of the glycosylation site from the antibody (for N-linked glycosylation sites) is conveniently accomplished by altering the amino acid sequence to remove one of the aforementioned tripeptide sequences. This alteration can be achieved by replacing the asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conserved substitution).

Antibodies or antigen-binding fragments thereof can be prepared using methods known in the art, for example by a process comprising: culturing host cells containing nucleic acids encoding, in a form suitable for expression, any of the previously described anti-PDL1, anti-PD-1, or anti-PDL2 antibodies or antigen-binding fragments under conditions suitable for the production of such antibodies or fragments, and recovering the antibodies or fragments.

B.CTLA-4

Another immune checkpoint that can be targeted in the methods presented in this article is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when 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, and 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 stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells and is likely important for the function of regulatory T cells. T cell activation via T cell receptors and CD28 leads to increased expression of the inhibitory receptor CTLA-4 of the B7 molecule.

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, an immunoadhesin, a fusion protein, or an oligopeptide.

The anti-human CTLA-4 antibody (or derived from its VH and/or VL domains) suitable for the methods of this 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, the anti-CTLA-4 antibodies disclosed in the following literature can be used in the methods disclosed herein: US 8,119,129, WO 01/14424, WO 98/42752; WO 00/37504 (CP675,206, also known as tremelimumab; formerly known as ticilimumab), US 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. The teachings of each of the foregoing publications are hereby incorporated by reference. Antibodies that compete with any of these recognized antibodies in the art for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications Nos. WO2001014424 and WO2000037504 and U.S. Patent No. US8017114, all of which are incorporated herein by reference.

Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and) or its antigen-binding fragments and variants (see, for example, WOO 1/14424). 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 with the aforementioned antibody for binding to and/or binds to the same epitope on CTLA-4. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the aforementioned antibody (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

Other molecules used to modulate CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. US5844905, US5885796 and International Patent Application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference; and immunoadhesion, such as that described in U.S. Patent No. US8329867, which is incorporated herein by reference.

C. Killer immunoglobulin-like receptor (KIR)

Another immune checkpoint inhibitor used in this invention is an anti-KIR antibody. Suitable anti-human KIR antibodies (or VH/VL domains derived therefrom) for use in this invention can be produced using methods well known in the art.

Alternatively, anti-KIR antibodies recognized in the art may be used. Anti-KIR antibodies can cross-react with a variety of inhibitory KIR receptors and enhance the cytotoxicity of NK cells carrying one or more of these receptors. For example, anti-KIR antibodies can bind to each of KIR2D2DL1, KIR2DL2, and KIR2DL3 and enhance NK cell activity by reducing, neutralizing, and/or reversing the inhibition of NK cell cytotoxicity mediated by any or all of these KIRs. In some aspects, 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, as described in WO 2006/003179, the teachings of which are hereby incorporated by reference, may be used. Antibodies competing with any of these recognized antibodies for KIR binding may also be used. Additional anti-KIR antibodies that may be used include, for example, those disclosed in WO 2005/003168, WO 2005/009465, WO 2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/065939, WO 2012/071411 and WO/2012/160448.

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

VIII. Treatment Methods

This article provides a method for treating or delaying cancer progression in an individual, the method 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 oncolytic therapy - VirRx007). The therapy may also include at least one immune checkpoint inhibitor (e.g., a PD-1 axis binding antagonist and/or a CTLA-4 antibody).

In some embodiments, treatment contributes to a sustained response in an individual after treatment is discontinued. The methods described herein can be used to treat conditions requiring enhanced immunogenicity, such as increasing tumor immunogenicity for cancer treatment. This document also provides methods for enhancing the immune function of an individual, such as one with cancer, 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 mutant protein, and/or IL-15 mutant protein) and p53 and/or MDA-7 tumor suppressor gene therapy, or the viral oncolytic therapy VirRx007. The CD122/CD132 agonist may be an IL-15 mutant (e.g., IL-15N72D) that binds to an IL-15 receptor α/IgG1 Fc fusion protein (such as ALT-803). In some embodiments, the individual is a human.

In some aspects, the subject is further administered tumor-suppressive immunogene therapy (see PCT/US2016/060833, which is incorporated herein by reference in its entirety). In some aspects, the subject is further administered additional viral and nonviral gene therapy (PCT/US2017/065861; which is incorporated herein by reference in its entirety). In some aspects, the replicative and/or non-replicative viral and/or nonviral gene therapy can deliver one or more therapeutic genes, which may be tumor-suppressive genes or immunostimulatory genes.

Examples of cancers that are expected to be treated include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, precancerous lesions of the lungs, colon cancer, melanoma, and bladder cancer.

In some embodiments, an individual has cancer that is resistant to (has been proven to be resistant) to one or more anticancer therapies. In some embodiments, resistance to anticancer therapies includes cancer recurrence or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to anticancer therapies includes cancer progression during treatment with anticancer therapies. In some embodiments, the cancer is in an early or late stage.

In some embodiments, subjects are also treated with immune checkpoint inhibitors, such as PD-1 axis binding antagonists and/or anti-CTLA-4 antibodies. Individuals may have cancer expressing (e.g., as shown in diagnostic tests) the PD-L1 biomarker or have a high tumor mutational burden. In some embodiments, a patient's cancer expresses a low PD-L1 biomarker. In some embodiments, a patient's cancer expresses a high PD-L1 biomarker. PD-L1 biomarkers can be detected using methods selected from the group consisting of: FACS, Western blotting, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunoassay methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA sequencing, microarray analysis, SAGE, MassARRAY technology and FISH, and combinations thereof. Measurements of high mutational tumor burden can be determined by genome sequencing (e.g., Foundation One CDx assay).

In some embodiments, subjects are also treated with a histone deacetylase (HDAC) inhibitor (e.g., tractinostat, formerly known as CHR-3996 or VRx-3996, a class 1 selective histone deacetylase inhibitor administered orally).

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

In some embodiments of the methods disclosed herein, the cancer has a low level of T cell infiltration. In some embodiments, the cancer has no detectable T cell infiltration. In some embodiments, the cancer is a non-immunogenic cancer (e.g., non-immunogenic colorectal cancer and/or ovarian cancer). Without being bound by theory, combination therapy can increase the initiation, activation, and/or proliferation of T cells (e.g., CD4 ⁺ T cells, CD8 ⁺ T cells, memory T cells) relative to before the application of the combination.

In some embodiments of the methods disclosed herein, the activated CD4 and/or CD8 T cells in the individual are characterized by producing γ-IFN and/or exhibiting enhanced cytolytic activity relative to prior to the administration of the combination. γ-IFN can be measured by any means known in the art, including, for example, intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with an antibody against γ-IFN. Cytolytic activity can be measured by any means known in the art, for example, using cell killing techniques utilizing a mixture of effector cells and target cells.

This disclosure can be used with any human cells involved in an immune response, as a target of the immune system or as part of the immune system's response to foreign targets. The methods include ex vivo methods, in vivo methods, 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 regional injection into tumors.

A. Application

The combination therapies described herein include administration of a preferred 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 mutant protein, and/or IL-15 mutant protein) and p53, ADP, and/or MDA-7 gene therapy. The combination therapy can be administered in any suitable manner known in the art. For example, CD122/CD132 agonists (e.g., 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 mutant proteins, and/or IL-15 mutant proteins) and 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 in a composition different from p53, ADP, and/or MDA-7 gene therapies or their expression constructs. In some embodiments, the CD122/CD132 agonists are in the same composition as p53 and/or MDA-7 gene therapies. In some respects, nucleic acids encoding p53, ADP, and/or MDA-7 are administered to the subject before, simultaneously with, or after at least one CD122/CD132 agonist.

One or more CD122/CD132 agonists and p53, ADP, and/or MDA-7 gene therapies can be administered via the same route of administration or via different routes of administration. In some embodiments, CD122/CD132 agonists are administered intravenously, intramuscularly, subcutaneously, locally, orally, percutaneously, intraperitoneally, orbitally, via implantation, via inhalation, intrathecal, intracardiac, or intranasally. In some embodiments, p53, ADP, and/or MDA-7 gene therapies are administered intravenously, intramuscularly, subcutaneously, locally, orally, percutaneously, intraperitoneally, orbitally, via implantation, via inhalation, intrathecal, intracardiac, or intranasally. Effective amounts of CD122/CD132 agonists and p53, ADP, and/or MDA-7 gene therapies can be administered to prevent or treat disease. The appropriate dose of CD122/CD132 agonist and/or p53, ADP, and/or MDA-7 gene therapy can be determined based on the type of disease to be treated, the severity and duration of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the attending physician's judgment. In some embodiments, combination therapy using 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 mutant protein, and/or IL-15 mutant protein) and p53, ADP, and/or MDA-7 gene therapy is synergistic, thereby complementing the treatment of... Compared to single-agent treatment, the combination of single doses of p53, ADP, and/or MDA-7 gene therapy 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 mutant protein, and/or IL-15 mutant protein) has a supersum effect.

For example, therapeutically effective amounts of CD122/CD132 agonists (such as 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 mutants and/or IL-15 mutants) are administered via subcutaneous (SQ) or intravenous (IV) injection at intervals ranging from once a week to once every 2-4 weeks, at doses ranging from 5-100 μg/kg.

For example, when a therapeutically effective amount of one or more CD122/CD132 agonists, along with p53, ADP, and/or MDA-7 gene therapy, is further combined with an immune checkpoint inhibitor (such as an antibody), whether administered once or multiple times, the dosage will be in the range of about 0.01 mg/kg of patient body weight to about 50 mg/kg of patient body weight. In some embodiments, the antibody used is, for example, administered daily at doses of about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosing regimens may be useful. In one embodiment, the anti-PD-L1 antibody described herein is administered to humans on day 1 of a 21-day cycle at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg. This dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as multiple infusions. Progression of the therapy can be easily monitored using conventional techniques.

For the p53, ADP, and/or MDA-7 gene therapy components of combination therapy, intratumoral injection or injection into the tumor vascular system is specifically envisioned. Local, regional, or systemic administration may also be appropriate. For tumors >4 cm, the volume to be administered will be approximately 4–10 ml (specifically 10 ml), while for tumors <4 cm, a volume of approximately 1–3 ml (specifically 3 ml) will be used. Multiple injections as a single-dose delivery comprise volumes of approximately 0.1 ml to approximately 0.5 ml. For example, multiple injections administered to the tumor can advantageously expose the adenovirus particles.

Treatment regimens can also vary and often depend on the type of tumor, its location, disease progression, and the patient's health and age. Clearly, certain types of tumors will require more aggressive treatment, while some patients may not tolerate a more burdensome regimen. Clinicians will make such decisions based on the known efficacy and, if any, toxicities of the therapeutic agent.

In some embodiments, the treated tumor may be initially unresectable. Combination therapy can increase tumor resectability due to contraction at the margins or by eliminating certain particularly invasive portions. Resection is performed after combination therapy. Additional post-resection treatment is used to eliminate residual disease.

Treatment may include various “unit doses.” A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the capabilities of a person skilled in the clinical field. The unit dose need not be administered as a single injection, but may include continuous infusions over a set period of time. ^The unit dose of the present invention can be conveniently described in plaque-forming units (pfu) of the viral construct. Unit doses are in the range of ^10³ , ^10⁴ , ^10⁵ , ^10⁶ , ^10⁷ , ^10⁸ , ^10⁹ , ^10¹⁰ , 10¹¹, ^10¹² , ^10¹³ pfu and higher. Alternatively, depending on the type of virus and the achievable titer, 1 to 100, 10 to 50, 100-1000, or up to about 1× ^10⁴ , 1× ^10⁵ , 1× ^10⁶ , 1×10⁷, 1× ^10⁸ , 1× ^10⁹ , 1× ^10¹⁰ , 1× ^10¹¹ , 1× ^10¹² , 1× ^10¹³ , 1× ^10¹⁴ , or ¹ × ^10¹⁵ or higher infectious viral particles (VPs) may be delivered to the patient or patient cells.

B. Injectable compositions and formulations

One method of delivering an expression construct encoding one or more human p53, ADP, and MDA-7 proteins to overproliferating cells in this invention is via intratumoral injection, whereas CD122/CD132 agonists, immune checkpoint inhibitors, and HDAC inhibitors are administered systemically. However, as described in U.S. Patents 5,543,158, 5,641,515, and 5,399,363, the pharmaceutical compositions disclosed herein can alternatively be administered intratumorally, parenterally, intravenously, intradermally, intra-arterially, intramuscularly, percutaneously, or even intraperitoneally, all of which are incorporated herein by reference.

Nucleic acid constructs can be delivered via syringe or any other method for injecting solutions, as long as the expression construct can pass through a needle of a specific gauge required for injection. A novel needle-free injection system has been described (US Patent 5,846,233) having a nozzle defining an ampoule chamber for holding the solution and an energy device for propelling the solution from the nozzle to the delivery site. A syringe system for gene therapy has also been described that allows for the precise multiple injections of a predetermined amount of solution at any depth (US Patent 5,846,225). Another injection system that can be used is the QuadraFuse device, which includes a multipronged needle adjustable to different depths using an attached syringe.

Solutions of active compounds that are free bases or pharmacologically acceptable salts can be prepared by suitably mixing them with surfactants such as hydroxypropyl cellulose in water. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions (US Patent 5,466,468). In all cases, the form must be sterile and must be a fluid present to a degree that is easily injectable. It must be stable under manufacturing and storage conditions and must be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and/or vegetable oils. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Antimicrobial activity can be achieved through various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride, in the composition. The absorption of injectable compositions can be prolonged by using agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

For example, for parenteral administration in aqueous solutions, the solution should be appropriately buffered (if necessary) and the liquid diluent should first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this regard, those skilled in the art will know, based on this disclosure, the sterile aqueous media that can be used. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution, added to 1000 ml of subcutaneous infusion fluid, or injected at the recommended infusion site (see, for example, "Remington's Pharmaceutical Sciences," 22nd edition). Some variation in dosage will inevitably occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for the individual subject. Furthermore, for human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biologics Standards.

Sterile injectable solutions are prepared by incorporating the active compound in the desired amount along with the various other desired components listed above into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and the desired other components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce powders of the active ingredient and any additional desired components from a previously sterile filtered solution.

The compositions disclosed herein can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins) formed using inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). After formulation, the solution is administered in a dosage form compatible with therapeutic amounts. The formulation can be readily administered in various dosage forms (such as injectable solutions, drug-release capsules, etc.).

C. Additional anticancer therapy

To enhance the efficacy of p53, ADP, and/or MDA-7 nucleic acids, as well as at least one CD122/CD132 agonist, they can be combined with at least one additional agent effective in cancer treatment. More generally, these additional compositions will be provided in combined amounts that effectively kill or inhibit cell proliferation. The process may involve simultaneously contacting cells with an expression construct and one or more agents or factors. This can be achieved by contacting cells with a single composition or pharmacological formulation containing two agents, or by simultaneously contacting cells with two different compositions or formulations, one of which contains an expression construct and the other containing one or more second agents. Alternatively, the expression construct may contact proliferating cells, and the additional therapy may affect other cells in the immune system or tumor microenvironment to enhance antitumor immune responses and therapeutic efficacy. The at least one additional anticancer therapy may be, but is not limited to, surgical therapy, chemotherapy (e.g., administration of protein kinase inhibitors or EGFR-targeted therapy), radiotherapy, cryotherapy, thermotherapy, phototherapy, radioablation 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 biotherapy (such as monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozymes, or gene therapy). Unrestrictedly, biotherapy may be gene therapy, such as tumor suppressor gene therapy, cell death protein gene therapy, cell cycle regulation gene therapy, cytokine gene therapy, toxin gene therapy, immunogene therapy, suicide gene therapy, prodrug gene therapy, anti-cell proliferation gene therapy, enzyme gene therapy, or anti-angiogenic factor gene therapy.

Gene therapy can be administered with intervals ranging from minutes to weeks before or after other agent treatments. In embodiments where another agent and expression construct are administered to cells separately, it should generally be ensured that there is no significant time interval between each delivery, allowing the agent and expression construct to still exert a beneficial combined effect on the cells. In such cases, it is conceivable that cells may be exposed to each other in two ways for approximately 12–24 hours, and more preferably approximately 6–12 hours. In some cases, it may be necessary to significantly extend the treatment period; however, there may be a time elapsed between the respective administrations of 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). In some embodiments, one or more therapies can be continued with or without other therapies as maintenance therapy.

The following combinations can be used: gene therapy and CD122/CD132 agonists are "A", and an adjuvant agent, namely an immune checkpoint inhibitor, is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

1. Chemotherapy

Cancer therapy often includes various combination therapies with chemotherapy and radiation-based treatments. Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, dichloromethyldiethylamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, actinomycin D, daunorubicin, doxorubicin, bleomycin, procainoxam, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, paclitaxel, gemcitabine, vinorelbine, farnesyltransferase inhibitors, antiplatinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, temozolomide (the aqueous form of DTIC), or any of the aforementioned analogues or derivatives. The combination of chemotherapy and biological therapy is called biochemotherapy. Chemotherapy can also be administered in continuous low doses, a process known as rhythmic chemotherapy.

Further combination chemotherapy includes, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, indomethacin, and piperazine; aziridines such as benzoxotepa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, tratamiamine, triethylene ethylphosphamide, triethylene thiophosphamide, and trimethylolomelamine; polyacetyl (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); lichenin; and sponge polyacetyl (cal Lystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); nostocin (especially nostocin 1 and nostocin 8); sulphurin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, naphthylambucil, chlorophosphamide, estradiol, ifosfamide, dichloromethyldiethylamine, dichloromethyldiethylamine hydrochloride oxide, melphalan, and novobichin. Benzene mustard, cholesterol, prednisone, trofosfamide, uramustine; nitrosoureas, such as carmustine, chloramphenicol, formustine, lomustine, nimustine, and ramustine; antibiotics, such as endothymic antibiotics (e.g., calicheamicin, especially calicheamicin γI and calicheamicin ωI); dynemicin, including dynemicin A; bisphosphonates, such as clophosphonates; esperamicin; and new carcinogen chromophores and related chromogens, endothymic antibiotic chromophores, aclacinomysins, actinomycins, autramycin, diazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carbapenem, and carcinogens. Carzinophilin, chromomycinis, actinomycin D, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin (including morpholino-dxorubicin, cyanomorpholino-dxorubicin, 2-pyrrole-dxorubicin and deoxydxorubicin), epirubicin, epoxabicin, edabicin, ephedrine, mitomycin (such as mitomycin C), mycophenolic acid, nogalarnycin, oligomycin, pemycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, tuberculin, ubenimex, fentostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU). Folic acid analogs, such as folic acid, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, and fluorouridine; and androgens, such as calotestosterone. Drotahistamine propionate, cyclothiosterol, meandrosten, testosterone; anti-adrenergic drugs such as mitotane and tralostertan; folic acid supplements such as folinic acid; acetoglucuronolactone; aldehyde phosphoramide glycoside; aminolevulinic acid; enturacil; acridine; betrabucil; bismuth subcitrate; edatraxate; defofamine; dimethicone; diazinon Aziquone; elformithine; elifonitrile; epothilone; etogluconol; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids, such as maytansine and anthraquinone; mitoxanthraquinone; mitoxanthraquinone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; loxoanthraquinone; podophyllic acid; 2-acetylhydrazine; procarbazine; PSK polysaccharide complex; razoxin; rhizoxin; cizonan; germanium spiroamine; Alternaria ketoacid; triamine; 2,2′,2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A) A) Bacitracin A and anguidine; urethan; vincristine; dacarbazine; mannitol nitrogen mustard; dibromomannitol; dibromoeutherol; piperobromoethane; gacytosine; cytarabine (“Ara-C”); cyclophosphamide; taxanes, such as paclitaxel and docetaxel; 6-thioguanine; mercaptoside Primarily purines; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edaraxacin; donomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 20 00; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine; farnesyltransferase inhibitors, antiplatinum; and any pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the compositions provided herein may be used in combination with histone deacetylase inhibitors. In some embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, embodiments of the invention may be practiced in combination with glimepiride (e.g., glimepiride may be administered to a patient at a dose of about 400 to about 800 mg/day). In some embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.

2. Radiation therapy

Other factors that cause DNA damage and have been widely used include the targeted delivery of rays commonly referred to as gamma rays, X-rays, and/or radioactive isotopes to tumor cells. Other forms of DNA-damaging agents are also known, such as microwave and UV radiation. All of these factors are likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50 to 200 roentgens over long periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Radioactive isotope doses vary widely and depend on the isotope's half-life, the intensity and type of radiation emitted, and absorption by tumor cells.

3. Immunotherapy

Immunotherapy typically relies on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies that are specific to certain markers on the surface of tumor cells. An antibody alone can act as an effector of the therapy, or it can recruit other cells to actually achieve cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and used solely as a target. Alternatively, effectors can 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 engineered variants of these cell types modified to express chimeric antigen receptors. The transfer of the Mda-7 gene to tumor cells leads to tumor cell death and apoptosis. Apoptotic tumor cells are cleared by reticuloendothelial cells, including dendritic cells and macrophages, and presented to the immune system to generate anti-tumor immunity (Rovere et al., 1999; Steinman et al., 1999).

Those skilled in the field of cancer immunotherapy will recognize that other complementary immunotherapies can be added to the above regimens to further enhance their efficacy, including but not limited to adding GM-CSF to increase the number of myeloid-derived innate immune system cells, adding low doses of cyclophosphamide or PI3K inhibitors (e.g., PI3Kδ inhibitors) to eliminate T regulatory cells that suppress innate and acquired immunity, and adding 5FU (e.g., capecitabine), PI3K inhibitors, or histone deacetylase inhibitors to remove suppressive myeloid-derived suppressor cells. For example, PI3K inhibitors include, but are not limited to, LY294002, pirivoxetine, BKM120, duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, Palomida 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136. In some respects, PI3K inhibitors are PI3K δ inhibitors, such as, but not limited to, idelalisib, RP6530, TGR1202, and RP6503. Additional PI3K inhibitors are disclosed in US patent applications US20150291595, US20110190319 and international patent applications WO2012146667, WO2014164942, WO2012062748, and WO2015082376. Immunotherapy may also include the administration of interleukins (such as IL-2) or interferons (such as INFα).

Examples of immunotherapies that can be used in combination with p53, ADP, and/or MDA-7 gene therapy and CD122/CD132 agonists include immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (US Patent 5,801,005; US Patent 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy (e.g., interferon α, β, and γ; interleukins (IL-1, IL-2), GM-CSF, and TNF) (Bukowski et al.). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It has antitumor activity and has been approved for the treatment of malignant tumors (Dillman, 1999). Combination therapy with Herceptin and chemotherapy has been shown to be more effective than single therapy. Gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Patent 5,830,880 and US Patent 5,846,945) and monoclonal antibodies (e.g., antiganglioside GM2, antiHER-2, antip185) are also mentioned. Therefore, it is conceivable that one or more anticancer therapies could be used in conjunction with the p53, ADP, and/or MDA-7 gene therapies described herein.

Additional immunotherapies that can be combined with p53, ADP, and/or MDA-7 gene therapy and CD122/CD132 agonists include immune checkpoint inhibitors, co-stimulatory receptor agonists, innate immune cell stimulants, or innate immune activators. In some aspects, immune checkpoint inhibitors are CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, LAG-3 inhibitors, BTLA inhibitors, B7H3 inhibitors, B7H4 inhibitors, TIM3 inhibitors, KIR inhibitors, or A2aR inhibitors. In some aspects, at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some aspects, the anti-CTLA-4 antibody is trimelimumab or ipilimumab. In some aspects, at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. In some embodiments, the anti-KIR antibody is rilurumab. In some aspects, the PD-L1 inhibitor is devarulumab, atezolizumab, or avermab. In some cases, PD-L2 inhibitors are rHIgM12B7. In some cases, LAG3 inhibitors are IMP321 or BMS-986016. In some cases, A2aR inhibitors are PBF-509.

In some aspects, at least one immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) axis binding antagonist. In some aspects, 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 aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some aspects, the PD-1 binding antagonist inhibits the binding of PD-1 to PDL1 and/or PDL2. Specifically, 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, pilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224.

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

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, and Chi Lob 7/4), or anti-CD27 antibodies (e.g., Varlilumab, also known as CDX-1127). Stimulants of innate immune cells include, but are not limited to, KIR monoclonal antibodies (e.g., riluzumab), inhibitors of cytotoxic inhibitory receptors (e.g., NKG2A, also known as KLRC), and CD94, such as the monoclonal antibodies monalizumab and anti-CD96, also known as TACTILE), and toll-like receptor (TLR) agonists. TLR agonists can be BCG, TLR7 agonists (e.g., poly(OICLC) and imiquimod), TLR8 agonists (e.g., retinomod), 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 respects, immunotherapy is a stimulator of interferon gene (STING) agonists (Corrales et al., 2015).

Other immunotherapies contemplated for use with the methods of this disclosure include those described by Tchekmedyian et al., 2015, which are incorporated herein by reference. Immunotherapy may include suppressor T regulatory cells (Tregs), myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs). In some embodiments, the immunotherapy is a tumor vaccine (e.g., whole tumor cell vaccine, dendritic cell vaccine, DNA and/or RNA expression vaccine, peptide and recombinant tumor-associated antigen vaccine), or adoptive cell therapy (ACT) (e.g., T cells, natural killer cells, TILs, and LAK cells). T cells and/or natural killer cells can be engineered to specific tumor antigens using chimeric antigen receptors (CARs) or T cell receptors (TCRs). As used herein, a chimeric antigen receptor (or CAR) may refer to any engineered receptor specific to the antigen of interest, which, when expressed on T cells or natural killer cells, confers CAR specificity to the T cells or natural killer cells. Once T cells or natural killer cells expressing chimeric antigen receptors are created using standard molecular techniques, they can be introduced into a patient, such as using techniques like adoptive cell transfer. In some aspects, the T cells are activated CD4 and/or CD8 T cells in an individual, characterized by CD4 and/or CD8 T cells producing γ-IFN″ and/or enhanced cytolytic activity relative to prior to administration of the combination. CD4 and/or CD8 T cells may exhibit increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins. CD4 and/or CD8 T cells may be effector memory T cells. In some embodiments, CD4 and/or CD8 effector memory T cells are characterized by ^high CD44 and ^low CD62L expression.

In some respects, two or more immunotherapies can be combined with p53, ADP, and/or MDA-7 gene therapy and CD122/CD132 agonists, including combinations of additional immune checkpoint inhibitors with T-cell co-stimulatory receptor agonists, or combinations with TIL ACT. Other combinations include T-cell checkpoint blockade plus co-stimulatory receptor agonists, T-cell checkpoint blockade to improve the function of innate immune cells, checkpoint blockade plus IDO inhibition, or checkpoint blockade plus adoptive T-cell transfer. In some respects, immunotherapies include combinations of anti-PD-L1 immune checkpoint inhibitors (e.g., acimetidine), 4-1BB (CD-137) agonists (e.g., utomilumab), and OX40 (TNFRS4) agonists. Immunotherapy can be combined with histone deacetylase (HDAC) inhibitors (such as 5-azacytidine and entinostat).

Immunotherapy can be a cancer vaccine comprising one or more cancer antigens, particularly proteins or immunogenic fragments thereof; DNA or RNA encoding the cancer antigen, particularly the protein or immunogenic fragment thereof; cancer cell lysate; 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 compared to normal cells, or a protein with an abnormal structure due to mutation, can serve as a cancer antigen. In principle, a cancer antigen can be the product of a mutated or overexpressed oncogene and tumor suppressor gene, the product of other mutated genes, an overexpressed or aberrantly expressed cellular protein, a cancer antigen produced by a carcinogenic virus, an oncofetal antigen, altered cell surface glycolipids and glycoproteins, or a cell type-specific differentiation antigen. Examples of cancer antigens include aberrant or overexpressed products of the ras and p53 genes. Other examples include tissue differentiation antigens, mutant protein antigens, carcinogenic viral antigens, cancer-testis antigens, and vascular or matrix-specific antigens. Tissue differentiation antigens are those antigens specific to a particular type of tissue. Mutant protein antigens may be more specific to cancer cells because normal cells should not contain these proteins. Normal cells will display normal protein antigens on their MHC molecules, while cancer cells will display mutated forms. Some viral proteins are involved in cancer formation, and some viral antigens are also cancer antigens. Cancer-testis antigens are antigens primarily expressed in testicular germ cells, but also in embryonic ovarian and trophoblastic cells. Some cancer cells abnormally express these proteins and thus present these antigens, allowing them to be attacked by T cells specific to these antigens. Exemplary antigens of this type are CTAG1 B and MAGEA1, as well as Rindopepimut, a 14-mer intradermal injectable peptide vaccine targeting the epidermal growth factor receptor (EGFR) v111 variant. Rindopepimut is particularly suitable for the treatment of glioblastoma when used in combination with inhibitors of the CD95/CD95L signaling system described herein. Similarly, proteins that are normally produced in very low amounts but whose production increases dramatically in cancer cells can trigger an immune response. An example of such proteins is tyrosinase, which is essential for melanin production. Normally, tyrosinase is produced in extremely small amounts, but its levels are very high in melanoma cells. Carcinoembryonic antigens (CEA) are another important class of cancer antigens. Examples include alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). These proteins are typically produced early in embryonic development and disappear when the immune system is fully developed. Therefore, self-tolerance to these antigens does not develop. Cells infected with oncogenic viruses (such as EBV and HPV) also produce abnormal proteins. Cells infected with these viruses contain latent viral DNA, which is transcribed, and the resulting proteins produce an immune response. Cancer vaccines can include peptide cancer vaccines, which in some embodiments are personalized peptide vaccines. In some embodiments, peptide cancer vaccines are multivalent long peptide vaccines, polypeptide vaccines, peptide mixture vaccines, hybrid peptide vaccines, or peptide pulsed dendritic cell vaccines.

Immunotherapy can be an antibody, such as part of a polyclonal antibody preparation, or it can be a monoclonal antibody. The antibody can be a humanized antibody, a chimeric antibody, an antibody fragment, a bispecific antibody, or a single-chain antibody. The antibodies disclosed herein include antibody fragments such as, but not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdfv), and fragments containing VL or VH domains. In some aspects, the antibody or its fragments specifically bind to epidermal growth factor receptors (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 molecule, carcinoembryonic antigen, prostate-specific membrane antigen, mucin-1, CD30, CD33, or CD40.

Examples of monoclonal antibodies that can be used in combination with the compositions provided herein include, but are not limited to, 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); zalumumab (anti-EGFR mAb); nexituzumab (anti-EGFR mAb); 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); ofamumab (anti-CD20 mAb); tosimomab I131 (anti-CD20 mAb). 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 with IgG1 Fc); AMG386 (angiopoietin-1 and angiopoietin-2 binding peptides fused with IgG1 Fc); Dalotuzumab (anti-IGF-1R mAb); Gemtuzumab ozogamicin (anti-CD33 mAb); Alemumab (anti-Campath-1/CD52 mAb); Brentuximab Vedotin (anti-CD30 mAb); Catuxomab (a bispecific mAb targeting epithelial cell adhesion molecules and CD3); Naptumomab (anti-5T4 mAb); Girentuximab (anti-carbonic anhydrase ix); or Farletuzumab (anti-folate receptor). Other examples include antibodies such as: Panorex ^™ (17-1A) (mouse monoclonal antibody); Panorex (@(17-1A) (chimeric mouse monoclonal antibody); BEC2 (anti-idiotype mAb, mimicking GD epitopes) (using BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab, humanized 13′1 LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotype mouse mAb); 3622W94 mAb binding to the EGP40 (17-1A) pan-cancer antigen on adenocarcinoma; Zenapax (SMART anti-Tac (IL-2 receptor); SMART M195 Ab (humanized Ab, humanized Ab); NovoMAb-G2 (pan-cancer specific Ab); TNT (chimeric mAb against histone antigens); Gliomab-H (monoclonal-humanized Ab); GNI-250Mab; EMD-72000 (chimeric EGF antagonist); LymphoCide (humanized IL-12 antibody); and MDX-260, a bispecific GD-2-targeting ANA Ab, SMART IDIO Ab, SMART ABL 364Ab, or ImmuRAIT-CEA. Examples of antibodies include those described in U.S. Patent Nos. 5,736,167, 7,060,808, and 5,821,337.

Other examples of antibodies include zanulimumab (anti-CD4 mAb), keliximab (anti-CD4 mAb); ipilimumab (MDX-101; anti-CTLA-4 mAb); treemilimumab (anti-CTLA-4 mAb); daclizumab (anti-CD25/IL-2R mAb); basiliximab (anti-CD25/IL-2R mAb); MDX-1106 (anti-PD1 mAb); antibodies against GITR; GC1008 (anti-TGF-β antibody); metelimumab/CAT-192 (… Anti-TGF-β antibody; lerdelimumab/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-TGF-β antibody); NF); Etanercept (extracellular domain of TNFR fused with IgG1 Fc); Belatacept (extracellular domain of CTLA-4 fused with Fc); Abatacept (extracellular domain of CTLA-4 fused with Fc); Belimumab (anti-B lymphocyte stimulator); Muromonab-CD3 (anti-CD3 mAb); Otelixizumab (anti-CD3 mAb); Teplizumab (anti-CD3 mAb); Tocilizumab (anti-IL6R mAb) REGN88 (anti-IL6R mAb); Ustekinumab (anti-IL-12/23 mAb); Briakinumab (anti-IL-12/23 mAb); Natalizumab (anti-α4 integrin); Vedolizumab (anti-α4β7 integrin mAb); T1h (anti-CD6 mAb); Epratuzumab (anti-CD22 mAb); Efalizumab (anti-CD11a mAb); and Atacicept (extracellular domain of transmembrane activator and calcium-regulated ligand interactor fused to Fc).

a. Passive immunotherapy

There are various approaches to passive immunotherapy for cancer. These can be broadly categorized as follows: injecting antibodies alone; injecting antibodies conjugated to toxins or chemotherapy agents; injecting antibodies conjugated to radioactive isotopes; injecting anti-idiotype antibodies; and finally, eliminating tumor cells from the bone marrow.

Human monoclonal antibodies are preferably used in passive immunotherapy because they produce few or no side effects in patients. Human monoclonal antibodies against ganglioside antigens have been administered intralesionally to patients with recurrent cutaneous melanoma (Irie and Morton, 1986). Regression was observed in six out of ten patients after daily or weekly intralesional injections. In another study, moderate success was achieved by intralesional injection of two human monoclonal antibodies (Irie et al., 1989).

It may be advantageous to administer more than one monoclonal antibody targeting two different antigens, or even an antibody with multiple antigen specificities. Treatment regimens may also include the administration of lymphokines or other immune enhancers, as described by Bajorin et al. (1988). The development of human monoclonal antibodies is described in further detail elsewhere in the specification.

b. Active Immunotherapy

In active immunotherapy, antigenic peptides, polypeptides, or proteins, or compositions of autologous or allogeneic tumor cells, or “vaccines,” are typically administered with a unique bacterial adjuvant (Ravindranath and Morton, 1991; Morton and Ravindranath, 1996; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993). In melanoma immunotherapy, patients who elicit a high IgM response generally survive better than those who do not elicit or elicit low IgM antibodies (Morton et al., 1992). IgM antibodies tend to be transient, but exceptions to this rule appear to be antiganglioside or anticarbohydrate antibodies.

c. Adoptive immunotherapy

In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes from a patient are isolated in vitro, activated by 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 in combination with an adjuvant-incorporated antigen peptide composition as described herein. The activated lymphocytes are preferably the patient's own cells, previously isolated from blood or tumor samples and activated (or “expanded”) in vitro. This form of immunotherapy has yielded several cases of regression in melanoma and renal tumors, but the percentage of responders is small compared to non-responders. Recently, higher response rates have been observed when such adoptive cell therapies have incorporated genetically engineered T cells expressing chimeric antigen receptors (CARs) (referred to as CAR T-cell therapy). Similarly, autologous and allogeneic natural killer cells are isolated, expanded, and genetically modified to express receptors or ligands to promote their binding to and killing of tumor cells.

4. Other medicines

It is envisioned that other agents can be combined with the compositions provided herein to improve therapeutic efficacy. These additional agents include immunomodulators, agents affecting the upregulation of cell surface receptors and GAP binding, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of overproliferating cells to apoptosis inducers. Immunomodulators 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 is also envisioned that upregulation of cell surface receptors or their ligands (such as Fas/Fas ligands, DR4, or DR5/TRAIL) will enhance the apoptosis-inducing capacity of the compositions provided herein by establishing autocrine or paracrine effects on overproliferating cells. Increasing intercellular signaling by increasing GAP binding numbers will increase the anti-overproliferative effect on neighboring overproliferating cell populations. In other embodiments, cell growth inhibitors or differentiation agents may be combined with the compositions provided herein to improve the anti-overproliferative efficacy of the treatment. Cell adhesion inhibitors are expected to improve the efficacy of the invention. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin. It is also envisioned that other agents that increase the sensitivity of overproliferating cells to apoptosis (such as antibody C225) could be used in combination with the compositions provided herein to improve therapeutic efficacy.

In further embodiments, other agents may be one or more oncolytic viruses. These oncolytic viruses may be engineered to express p53 and/or IL24, and/or genes other than p53 and/or IL24, such as cytokines, ADP, or heat shock proteins. Examples of oncolytic viruses include single-stranded or double-stranded DNA viruses, RNA viruses, adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, herpesviruses, poxviruses, vaccinia virus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, Ebola virus, influenza virus and reovirus, myxoma virus, Malaba virus, rhabdovirus, enadenotucirev, or Coxsackie virus. In one particular embodiment, the other agent is talimogenelaherparepvec (T-VEC), which is a genetically engineered oncolytic herpes simplex virus expressing GM-CSF. Talimogene laherparepvec, HSV-1 [strain JS1] ICP34.5-/ICP47-/hGM-CSF (formerly known as OncoVEX ^{GM CSF} ), is an intratumorally delivered oncolytic immunotherapy containing immune-enhanced 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; these documents are incorporated herein by reference). In October 2015, the U.S. FDA approved T-VEC, branded under the name IMLYGIC ^™ , for the treatment of melanoma in patients with unresectable tumors. The characteristics and administration methods of T-VEC are described, for example, in the IMLYGIC ^™ packaging insert (Amgen, 2015) and U.S. Patent Publication No. US2015/0202290, both of which are incorporated herein by reference. For example, talimogene laherparepvec is typically administered intratumorally to injectable skin, subcutaneous, and lymph node tumors at a dose of up to 4.0 ml at ^10⁶ plaque-forming units/mL (PFU/mL) on day 1 of week 1, followed by intratumoral injection at a dose of up to 4.0 ml at ^10⁸ PFU/mL on day 1 of week 4, and then every 2 weeks (±3 days). The recommended volume of talimogene laherparepvec to be injected into one or more tumors depends on the size of those tumors and should be determined according to injection volume guidelines. Although T-VEC has shown clinical activity in melanoma patients, many cancer patients do not respond to or cease responding to T-VEC treatment. In one embodiment, p53, ADP, and/or MDA-7 nucleic acids, as well as at least one CD122/CD132 agonist, may be administered after, during, or before T-VEC therapy, such as to reverse treatment resistance. Exemplary oncolytic 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, Toca 511, Ankerui (H101), Onyx-015, H102, H103, and RIGVIR. Other exemplary oncolytic viruses are described in, for example, international patent publications WO2015/027163, WO2014/138314, WO2014/047350, and WO2016/009017; all of which are incorporated herein by reference.

In some embodiments, hormone therapy may also be used in conjunction with embodiments of the present invention, or in combination with any other cancer therapies previously described. The use of hormones can be used to treat certain cancers (such as breast cancer, prostate cancer, ovarian cancer, or cervical cancer) to lower the levels of certain hormones (such as testosterone or estrogen) or block their effects. This treatment is typically used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastasis.

In some respects, adjunctive anticancer agents are protein kinase inhibitors or monoclonal antibodies that inhibit receptors involved in protein kinase or 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 inhibitors of protein kinases or growth factor signaling pathways include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fotatinib, gefitinib, imatinib, lapatinib, lenvatinib, liritinib, nilotinib, panitumumab, pazopanib, pegatanib, lanibimab, ruxotinib, cicatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, vemurafenib, and MK. -2206, GSK690693, A-443654, VQD-002, Mitefocin, Perifocin, CAL101, PX-866, LY294002, Rapamycin, Tesirolimus, Everolimus, Desfolimus, Avozid, Gentianone, Selmetinib, AZD-6244, Vatalani, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

In some respects, PI3K inhibitors are selected from the group consisting of the following PI3K inhibitors: buparlisib, idelalisib, BYL-719, dactolisib, PF-05212384, pictilisib, copanlisib, copanlisib dihydrochloride, ZSTK-474, GSK-2636771, and duveriloxib. isib), GS-9820, PF-04691502, SAR-245408, SAR-245409, sonolisib, Archexin, GDC-0032, GDC-0980, apitolisib, pilaralisib, DLBS 1425, PX-866, voxtalisib, AZD-8186, BGT-226, DS-7423, GDC-0084, GSK-2126458, INK-11 17. SAR-260301, SF-1126, AMG-319, BAY-1082439, CH-5132799, GSK-2269557, P-7170, PWT-33597, CAL-263, RG-7603, LY-3023414, RP-5264, RV-1729, taselisib, TGR-1202, GSK-418, INCB-040093, Panulisib, GSK-1059615, CNX-135 1. AMG-511, PQR-309, 17β-hydroxywollamine, 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-311, PQR-316, RP-5090, VS-5584, X-480, AEZS-126, AS-60485 0. BAG-956, CAL-130, CZC-24758, ETP-46321, ETP-47187, GNE-317, GS-548202, HM-032, KAR-1139, LY-294002, PF-0497906 4. PI-620, PKI-402, PWT-143, RP-6530, 3-HOI-BA-01, AEZS-134, AS-041164, AS-252424, AS-605240, AS-605858, AS-606839 , BCCA-621C, CAY-10505, CH-5033855, CH-5108134, CUDC-908, CZC-19945, D-106669, D-87503, DPT-NX7, ETP-46444, ETP-46 992, GE-21, GNE-123, GNE-151, GNE-293, GNE-380, GNE-390, GNE-477, GNE-490, GNE-493, GNE-614, HMPL-518, HS-104, HS-10 6. HS-116, HS-173, HS-196, IC-486068, INK-055, KAR 1 141, KY-1 2420, Wolman penicillin, 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-2015 , SB-2312, SB-2343, SHBM-1009, SN 32976, SR-13179, SRX-2523, SRX-2558, SRX-2626, SRX-3636, SRX-5000, TGR-5237, TGX- 221, UB-5857, WAY-266175, WAY-266176, EI-201, AEZS-131, AQX-MN100, KCC-TGX, OXY-111A, PI-708, PX-2000, and WJD-008.

Envisioned additional cancer therapies could include antibodies, peptides, polypeptides, small molecule inhibitors, siRNAs, miRNAs, or gene therapies targeting, 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 receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; hepatic glycoside (EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE Receptor families, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) family; reticulin 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 (1L13Rα2), interleukin-6 (IL-6), IL-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (hemopoietin 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; cancer-testis (CT) antigen, lineage-specific antigen, differentiation antigen, α-actin-4, ARTC1, breakpoint clusters Bcr-abl fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), cell cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2), Ets variant gene 6/acute myeloid leukemia Blood disease 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB, low-density lipid receptor/GDP-L fucose:β-galactose 2-α-fucosyltransferase (LDLR/FUT) fusion protein, HLA-A2, arginine-to-isoleucine exchange at residue 170 of the α-helix in the α2-domain of the HLA-A2 gene (HLA-A*201-R170I), MLA-A11, heat shock protein 70 -2 mutant (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutants 1, 2, 3 (MUM-1, MUM-2, MUM-3), prostatic acid phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF 600, SIRT2, SNRPD1, SYT-SSX1 or SYT-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-acetylglucosamine transferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL recognition Melanoma epitope antigen (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, MAGE-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), Kallikrein 4, Mammary globulin White-A, OA1, prostate-specific antigen (PSA), prostate-specific membrane antigen, TRP-1/gp75, TRP-2, adipophilin, melanoma-deficient interferon-induced protein 2 (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), alpha-fetoprotein (AFP), M-CSF, mdm-2 (e.g., small molecule inhibitors of HDM2 (also known as MDM2) and/or HDM4, such as those used to reverse their inhibition of p53 activity, such as HDM201, cis-imidazolines (e.g., Nutlins), benzodiazepines (BDP), spiro-hydroxyindole), MUCI, p53 (TP53), PBF, FRAME, PSMA, RAGE-1, RNF43, RU2A S, SOX10, STEAP1, survivin (BIRCS), human telomerase reverse transcriptase (hTERT), telomerase, Wilmsoma 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, idiotype immunoglobulin, Benjohn's 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 1) 5-3), Cancer Antigen 27-29 (CA 27-29), Cancer Antigen 125 (CA 125), Cancer Antigen 19-9 (CA 19-9), β-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine-proline (707-AP), adenocarcinoma antigen recognized by T cells 4 The proteins contained in the following proteins: ART-4, carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel 2 (CLCA2), cyclophilic protein B (Cyp-B), human signet ring tumor-2 (HST-2), human papillomavirus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsid antigens, etc.), Ebola virus (EBV) proteins (EBV latent membrane proteins-LMP1, LMP2, etc.), hepatitis B or C virus proteins, and HIV proteins.

IX. Products or reagent kits

An article or kit is provided 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 mutant protein, and/or IL-15 mutant protein), and also provides nucleic acids encoding p53, ADP, and/or MDA-7 (e.g., ad-p53 and/or ad-MDA-7). The article or kit may also include a packaging insert containing instructions for use regarding the combined use of at least one CD122/CD132 agonist with tumor suppressor gene therapy to treat or delay cancer progression in an individual or to enhance the immune function of an individual with cancer. Any CD122/CD132 agonists described herein, as well as nucleic acids encoding p53, ADP, and/or MDA-7, may be included in the product or kit. The kit may additionally include extracellular matrix degradation proteins or expression constructs encoding extracellular matrix degradation proteins.

In some embodiments, at least one preferred 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 mutant protein and/or IL-15 mutant protein) and nucleic acids encoding p53, ADP and/or MDA-7 are contained in the same or separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. Containers can be formed from a variety of materials, such as glass, plastics (such as polyvinyl chloride or polyolefins), or metal alloys (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and instructions for use may be indicated on the container or on a label associated with the container. The product or kit may also contain other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts with instructions for use. In some embodiments, the product may also contain one or more other pharmaceutical agents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for one or more pharmaceutical agents include, for example, bottles, vials, bags, and syringes.

X. Example

The following examples illustrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that work well in the practice of the invention and can therefore be considered preferred embodiments of its practice. However, based on this disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention and still obtaining the same or similar results.

Example 1 - Combination of Ad-p53 and Ad-IL24 tumor suppressor and oncolytic virus (VirRx007) immunogene therapy to enhance local and systemic efficacy and reverse resistance to prior immunotherapies with one or more preferred CD122/132 agonists and immune checkpoint inhibitors.

In immunocompetent animal tumor models, the efficacy of combining CD122/CD132 agonists with tumor suppressor factors and viral oncolytic immunogene therapy to enhance local and systemic antitumor effects, including in tumors resistant to prior immunotherapy, has been demonstrated. The following treatment methods, dosages, and schedules were used:

Animals, Tumor Inoculation, and Measurement : Pathogen-free male C57BL/6 (B6) mice (6–8 weeks old, obtained from Charles River Labs) were used. B16F10 melanoma cells (ATCC, 5 × ^10⁵ cells/mouse, suspended in serum-free medium) were subcutaneously injected into the right abdomen of the animals to create a “primary tumor.” Treatment began when the tumor reached approximately 50 ^mm³ (this was designated Day 1 of treatment). Tumor growth was monitored by measuring the length (L) and width (w) of the tumor, and tumor volume was calculated using the following formula: Volume = 0.523L(w) ^² . Animals were monitored for up to 40 days and sacrificed when the tumor reached approximately 2000 ^mm³ .

Viral vectors : Replication-deficient human adenovirus type 5 (Ad5) encoding the p53 or IL24 tumor suppressor gene expression and replication-capable oncolytic adenovirus engineered to overexpress ADP (VirRx007) were used in these experiments. The construction, characterization, and purification of Ad5/CMV p53, IL24, and VirRx007 vectors have been reported elsewhere (Zhang 1994; Mhashilkar et al., 2001; US 7589069 B1). Three to 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). On day 21, additional intratumoral viral injections were administered in groups evaluating combinations of viral therapy with CD122/CD132 agonists and immune checkpoint inhibitors. Each viral dose contains 5 × ^10⁹ viral particles in a 50 μl volume.

CD122/CD132 agonist treatment: For the B16F10 model, mouse IL-2 (eBioscience or R&D Systems Minneapolis, MN) was mixed with S4B6-1 anti-mouse IL2 antibody (Bioxcell, West Lebanon, NH or BDBiosciences) at a 2:1 molar ratio to generate the preferred CD122/CD132 agonist immune complex. For studies involving human T cells, human IL-2 was mixed with MAB602 anti-human IL-2 antibody (R&D Systems). The IL-2/S4B6 or IL-2/MAB602 mAb immune complex was administered intraperitoneally (IP) at a dose of 2.5 μg IL2 on days 2, 6, and 10. Alternatively, the IL-2/S4B6 mAb immune complex (1.0 μg IL2/dose) was injected on days 2 through 6. An immune complex was prepared by incubating an anti-IL-2 monoclonal antibody with IL-2 for 15 minutes at room temperature.

In some rodent studies, the CD122/CD132 agonist was composed of recombinant mouse IL-15 (eBiosciences) and IL-15-Rα-Fc (R&D Systems). The immune complex was prepared by incubating these substances together at 37°C for 30 minutes, and this preferred CD122/CD132 agonist immune complex was administered intravenously (i.v.) for two consecutive days once the tumor became palpable. An alternative schedule was intraperitoneal (IP) administration of the IL-15 immune complex on days 3, 5, and 7 after the tumor became palpable. For IL-15 immune complex studies, recombinant mouse IL-15 (Peprotech, Rocky Hill, CT, USA) was administered intravenously once weekly at a dose of 2 μg per injection in in vivo. Recombinant mouse IL-15RαFc chimeric protein was obtained from R&D Systems (Minneapolis, MN) and administered at a dose equivalent to that of IL-15 cytokine (12 μg of IL-15-Ra-Fc per injection for every 2 μg of IL-15 protein in the immune complex).

Immune checkpoint inhibitors : To mimic the common clinical progression of tumors during immune checkpoint inhibitor therapy, anti-PD1 therapy was administered intraperitoneally at a dose of 200 μg/mouse starting on day 1 and every 3 days until day 30. In some studies, to evaluate the effect of a combination of tumor suppressor and oncolytic virus VirRx007 therapy with a preferred CD122/132 agonist and immune checkpoint inhibitor against tumors previously treated with immunotherapy, tumor suppressor therapy was initiated after tumor progression on anti-PD-1 therapy, with the first dose of tumor suppressor therapy administered 1 to 2 days after the start of anti-PD-1 therapy. B16F10 and B16 melanoma models are known to be highly resistant to immunotherapy. In these models, tumor progression during immune checkpoint inhibitor and preferred CD122/132 therapy was similar to that of control therapy using phosphate-buffered saline (PBS). The anti-mouse PD-1 antibody (CD279), specifically manufactured for in vivo use, was purchased from BioXcell (catalog number BE0146).

Reversal of resistance to prior immunotherapy : The ability to reverse resistance to prior immunotherapy was also demonstrated in combination with tumor suppressor or viral oncolytic therapy and preferential CD122/CD132 agonist and immune checkpoint inhibitor therapy. To simulate the common clinical situation of tumor progression during immune checkpoint inhibitor therapy, anti-PD-1 therapy was administered intraperitoneally at a dose of 10 mg/kg on day 1 and every 3 days until day 30. In some studies, to evaluate the effect of combination therapy with tumor suppressor or viral oncolytic therapy and CD122/CD132 therapy against tumors treated with prior immunotherapy, the combination therapy was initiated after tumor progression on anti-PD-1 therapy, where the first dose of tumor suppressor and CD122/CD132 therapy was administered 1 to 2 days after the initiation of anti-PD-1 therapy. These studies were performed in B16F10 and B16 melanoma models, which are known to have high resistance to immunotherapy. In these models, tumor progression during immune checkpoint inhibitor therapy was similar to that of control therapy using phosphate-buffered saline (PBS). The anti-mouse PD-1 antibody (CD279), specifically manufactured for in vivo use, was purchased from BioXcell (catalog number BE0146). Antibodies against PD-L1 and the immunomodulatory agent anti-LAG-3 were also purchased from BioXcell. The anti-mouse PD-L1 antibody (clone 9G2; Biolegend) and/or the anti-CTLA-4 antibody (clone UC10-4F10-11; Altor) were administered twice weekly via intraperitoneal (IP) injection of 100 μg each time for 2 weeks.

The efficacy of treatment and its synergistic interaction were demonstrated by measuring the tumor volume of the primary and contralateral tumors and statistically analyzing them using the T-test, analysis of variance (ANOVA), and Kruskal-Wallis ANOVA; and by comparing survival using the Kaplan-Mel test and the log-rank test.

Unexpectedly, these findings demonstrated a surprising and substantial synergistic effect between Ad-p53+CD122/132+anti-PD-1 and VirRx007+CD122/132+anti-PD-1 therapies, contributing to potentially curative treatment associated with complete tumor regression of both primary and contralateral tumors, and a significantly superior distant effect on distal tumors not treated with tumor suppressor therapy. These effects contributed to exceptionally long overall survival. For Ad-IL24+CD122/132+anti-PD-1 therapy, statistically significant improvements in tumor growth reduction and survival extension were also observed.

Ad-p53 plus CD122/132 agonist and checkpoint inhibitor immunotherapy: The therapeutic efficacy of the combination of Ad-p53 with CD122/132 agonist and anti-PD-1 therapy was evaluated by assessing tumor volume (in primary and contralateral tumors), complete tumor response rate, and survival. Regarding primary tumor volume, the diagram in Figure 4 illustrates the tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, CD122/132+anti-PD-1, Ad-p53, or combinations of Ad-p53+CD122/132, Ad-p53+anti-PD-1, and Ad-p53+CD122/132+anti-PD-1. Severe tumor progression occurred during CD122/132, anti-PD-1, and CD122/132+anti-PD-1 therapy, which was reversed by combination with Ad-p53 therapy. Compared to any of the individual therapies described, the efficacy of Ad-p53+CD122/132, Ad-p53+anti-PD-1, and Ad-p53+CD122/132+anti-PD-1 treatments was enhanced. By day 21, the mean tumor volume in the 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 combinations of 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 non-Ad-p53 therapy or Ad-p53 therapy alone. ANOVA comparison of tumor volume on day 21 confirmed a synergistic antitumor effect between 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 had exceeded 2,000 ^mm³ . Importantly, ANOVA comparison of tumor volume on day 30 confirmed that the synergistic antitumor effect was maintained only in the Ad-p53+CD122/132+anti-PD-1 treatment combination (overall p < 0.0001, and individual p < 0.0001 compared to each of the other treatment groups).

Ad-p53 Treatment Group: Assessment of Complete Tumor Response Rate. Complete tumor response to therapy is generally considered to be associated with significant treatment benefit and is essential for a curative outcome. As shown in Figure 5, for both the p53 treatment group and its controls, only Ad-p53+CD122/132+anti-PD-1 treatment resulted in complete tumor regression of both the primary and contralateral tumors. Complete tumor response to both the primary and contralateral tumors was observed in 60% of animals in the Ad-p53+CD122/132+anti-PD-1 treatment group, while no complete tumor response to either the primary or contralateral tumors was observed in the remaining 70 animals in the other treatment groups (p < 0.0001 by two-sided Fisher exact test comparing the Ad-p53+CD122/132+anti-PD-1 treatment group with all other treatment groups; p < 0.011 by two-sided Fisher exact test comparing the Ad-p53+CD122/132+anti-PD-1 treatment group with any other treatment group). Surprisingly, the complete tumor response was durable and maintained in 50% of the Ad-p53+CD122/132+ anti-PD-1 treatment group after 40 days, thus presumably curing these tumors in these animals.

Ad-p53 Treatment Group: Systemic/Distant Therapeutic Effects on Contralateral Tumor Growth. The systemic/distant effects of primary tumor treatment on contralateral implanted tumors were assessed in rodents whose primary tumors had been treated with one of the Ad-p53 therapies, and the results are shown in Figure 6. Consistent with the unexpectedly and significantly enhanced synergistic effect of Ad-p53+CD122/132+anti-PD-1 treatment on primary tumor growth and complete regression, we also observed a surprisingly strong and statistically significant distant 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 was eliminated in 90% (9 out of 10 animals) of the animals receiving Ad-p53+CD122/132+anti-PD-1 primary tumor treatment. In contrast, contralateral tumor growth was observed in 62.5% to 100% of animals in other Ad-p53 treatment groups. The difference in contralateral tumor growth was statistically significant (p = 0.0004 by chi-square analysis of all treatment groups; p < 0.0430 by two-sided Fisher exact test comparing the Ad-p53+CD122/132+anti-PD-1 treatment group with any other treatment group). Figure 6B depicts a graph showing the contralateral tumor volume over time in rodents receiving the three most effective primary tumor treatments: Ad-p53+CD122/132, Ad-p53+anti-PD-1, or Ad-p53+CD122/132+anti-PD-1 combination. ANOVA comparison of contralateral tumor volumes on day 22 confirmed the synergistic antitumor effect of Ad-p53+CD122/132+anti-PD-1 therapy (overall p = 0.0435). 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 = 0.0360). In summary, these findings suggest that among all Ad-p53 therapies, only the triple combination Ad-p53+CD122/132+anti-PD-1 therapy produced curative efficacy by inducing robust local and systemic antitumor immunity that mediates substantial distant effects.

Ad-p53 treatment group: Therapeutic efficacy contributed to prolonged survival. Kaplan-Mel survival curves for mice treated with PBS, CD122/132+ anti-PD-1, Ad-p53, or the combination of Ad-p53+CD122/132, Ad-p53+ anti-PD-1, and Ad-p53+CD122/132+ anti-PD-1 are shown in Figure 7. These survival curves were statistically significant by log-rank test (overall p <0.0001; p < 0.0003 for the Ad-p53+CD122/132+ anti-PD-1 treatment group compared to any other treatment group). The results also demonstrated an unexpectedly substantial synergistic effect of Ad-p53+CD122/132+ anti-PD-1 therapy. The median survival in the Ad-p53+CD122/132+ anti-PD-1 therapy group was not reached after 40 days, and 80% of those in this group remained alive. In stark contrast, 98% (49/50) of the animals in the other treatment groups died by day 30, with a median survival ranging from 10 to 28 days.

VirRx007 plus CD122/132 agonist and checkpoint inhibitor immunotherapy: The equally impressive and unexpected therapeutic efficacy of the combination of VirRx007 with CD122/132 agonist and anti-PD-1 therapy was also observed by assessing tumor volume (in primary and contralateral tumors), intact tumor response rate, and survival. Regarding primary tumor volume, the diagram in Figure 8 illustrates the tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, CD122/132 + anti-PD-1, VirRx007, or combinations of VirRx007 + CD122/132, VirRx007 + anti-PD-1, and VirRx007 + CD122/132 + anti-PD-1. Severe tumor progression occurred during CD122/132, anti-PD-1, and CD122/132+anti-PD-1 therapy, which was reversed by combination with VirRx007 therapy. Results showed enhanced efficacy of VirRx007+anti-PD-1 and VirRx007+CD122/132+anti-PD-1 therapy compared to any of these therapies alone. Contrary to findings with Ad-p53, VirRx007 did not demonstrate 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 VirRx007 + anti-PD-1 and VirRx007 + CD122/132 + anti-PD-1 combination therapies induced significant reductions in tumor volume compared to either non-VirRx007 therapy alone or VirRx007 therapy alone. ANOVA comparisons of tumor volume at day 30 determined a synergistic effect of VirRx007 + anti-PD-1 and VirRx007 + CD122/132 + anti-PD-1 therapy (p < 0.0001 for the overall or individual treatments compared to VirRx007). VirRx007 + CD122/132 + anti-PD-1 therapy was superior to VirRx007 + anti-PD-1 (p = 0.0002). Surprisingly, although the combination therapy of VirRx007+CD122/132 did not show a significant benefit compared to VirRx007 monotherapy, it demonstrated the synergistic effect of triple therapy of VirRx007+CD122/132+anti-PD-1.

Assessment of complete tumor response rate in the VirRx007 treatment group. Complete tumor response to therapy is generally considered to be associated with significant treatment benefit and is essential for a cure. As shown in Figure 9, for both the VirRx007 treatment group and its control group, only VirRx007 + CD122/132 + anti-PD-1 therapy resulted in complete tumor regression of both the primary and contralateral tumors. Complete tumor responses to both primary and contralateral tumors were observed in 60% of animals in the VirRx007+CD122/132+ antiPD-1 treatment group, while no complete tumor response to either primary or contralateral tumors was observed in 70 animals in the other treatment groups (p < 0.0001 by two-sided Fisher exact test compared to animals in the VirRx007+CD122/132+ antiPD-1 treatment group with all other treatment groups; p < 0.011 by two-sided Fisher exact test compared to animals in the VirRx007+CD122/132+ antiPD-1 treatment group with any other treatment group). Unexpectedly, the complete tumor response was durable and maintained in 50% of animals in the VirRx007+CD122/132+ antiPD-1 treatment group after 40 days, presumably curing these tumors in these animals.

VirRx007 Treatment Group – Systemic/Distant Therapeutic Effects on Contralateral Tumor Growth . The systemic/distant effects of primary tumor treatment on contralateral implanted tumors were assessed in rodents whose primary tumors had been treated with one of the VirRx007 therapies, and the results are shown in Figure 10. Consistent with the unexpectedly and significantly enhanced synergistic effect of VirRx007+CD122/132+anti-PD-1 treatment on primary tumor growth and complete regression, we also observed a surprisingly strong and statistically highly significant distant effect of VirRx007+CD122/132+anti-PD-1 treatment compared to other VirRx007 treatment groups. As shown in Figure 10A, contralateral tumor growth was eliminated in 80% of animals that received VirRx007+CD122/132+anti-PD-1 primary tumor treatment. In contrast, contralateral tumor growth was observed in 80% to 100% of animals in other VirRx007 treatment groups. The difference in contralateral tumor growth was statistically significant (p = 0.0002 by chi-square analysis comparing all treatment groups; p < 0.0230 by two-sided Fisher exact test comparing the VirRx007+CD122/132+anti-PD-1 treatment group with any other treatment group). These findings suggest that the combination of VirRx007+CD122/132+anti-PD-1 induces robust systemic anti-tumor immunity and mediates substantial distant effects with potential curative efficacy. Figure 10B depicts a plot showing the contralateral tumor volume over time in rodents treated with the three most effective combinations of primary tumors: VirRx007+CD122/132, VirRx007+anti-PD-1, or VirRx007+CD122/132+anti-PD-1. ANOVA comparison of contralateral tumor volumes on day 22 confirmed the synergistic antitumor effect of VirRx007+CD122/132+antiPD-1 therapy (overall p = 0.0171). Only the VirRx007+CD122/132+antiPD-1 group showed a statistically significant reduction in contralateral tumor growth compared to the VirRx007+antiPD-1 group (p = 0.0115). In summary, these findings suggest that among all VirRx007 therapies, only the triple combination VirRx007+CD122/132+antiPD-1 therapy produced curative efficacy by inducing robust local and systemic antitumor immunity mediating substantial distant effects.

VirRx007 Treatment Group – Treatment Efficacy Contributes to Extended Survival. Kaplan-Mel survival curves for mice treated with PBS, CD122/132+ anti-PD-1, VirRx007, or the combination of VirRx007+CD122/132, VirRx007+ anti-PD-1, and VirRx007+CD122/132+ anti-PD-1 are shown in Figure 11. These survival curves were statistically significant by log-rank test (overall p <0.0001; p < 0.0005 for the VirRx007+CD122/132+ anti-PD-1 treatment group compared to any other treatment group). The results also demonstrate an unexpectedly substantial synergistic effect of VirRx007+CD122/132+ anti-PD-1 therapy. The median survival of the VirRx007+CD122/132+anti-PD-1 therapy group had not been reached after 40 days, and 90% of animals in this group remained alive. In stark contrast, 98% (49/50) of the animals in the other treatment groups died by day 40, with median survival ranging from 10 to 33 days. Remarkably, although the combination therapy of VirRx007+CD122/132 did not demonstrate a significant survival benefit compared to VirRx007 monotherapy, it did demonstrate the synergistic effect of the triple therapy of VirRx007+CD122/132+anti-PD-1.

Ad-IL24 plus CD122/132 agonist and checkpoint inhibitor immunotherapy: Similar superior therapeutic efficacy of the combination of Ad-IL24 with CD122/132 agonist and anti-PD-1 therapy was also observed by assessing primary tumor volume and survival. Regarding primary tumor volume, the diagram in Figure 12 illustrates the tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, CD122/132+anti-PD-1, Ad-IL24, or the combination of Ad-IL24+CD122/132 and Ad-IL24+CD122/132+anti-PD-1. Severe tumor progression was observed during CD122/132, anti-PD-1, and CD122/132+anti-PD-1 therapy, which was reversed by combination with Ad-IL24 therapy. Compared to any of the individual therapies described, the efficacy of Ad-IL24+CD122/132+anti-PD-1 therapy was enhanced. By day 16, the mean tumor volume in the groups treated with PBS, CD122/132, anti-PD-1, CD122/132+anti-PD-1, and Ad-IL24 all exceeded 2,000 ^mm³ . In contrast, the combination therapy of Ad-IL24+CD122/132+anti-PD-1 induced a significant reduction in tumor volume compared to either non-Ad-IL24 therapy alone or any of the Ad-IL24 therapies. ANOVA comparisons of tumor volumes at day 16 confirmed the synergistic antitumor effect of Ad-IL24+CD122/132+anti-PD-1 therapy (p < 0.0001). Compared with 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.

Ad-IL24 Treatment Group – Treatment Efficacy Facilitates Extended Survival. Kaplan-Mel survival curves for mice treated with PBS, CD122/132+ anti-PD-1, Ad-IL24, or the combination of Ad-IL24+CD122/132+ anti-PD-1 are shown in Figure 13. These survival curves were statistically significant (p < 0.0001) by log-rank test. The results demonstrate an unexpectedly substantial synergistic effect of Ad-IL24+CD122/132+ anti-PD-1 therapy. Median survival was synergistically improved in the Ad-IL24+CD122/132+ anti-PD-1 therapy group. All animals in the PBS, CD122/132+ anti-PD-1, and IL24 treatment groups died on day 16, while 50% of the animals in the Ad-IL24+CD122/132+ anti-PD-1 therapy group survived to day 19. Compared with Ad-IL24 alone (p = 0.0003) or CD122/132+ anti-PD-1 therapy alone (p < 0.0001), the Ad-IL24+CD122/132+ anti-PD-1 therapy group showed a statistically significant prolonged survival. Interestingly, the Ad-IL24+CD122/132 dual therapy showed a surprising superior efficacy compared with the CD122/132+ anti-PD-1 dual therapy (p = 0.0002 by log-rank test, data not shown).

Negative control with Ad-luciferase (Ad-Luc). Ad-Luc control + CD122/132 agonist + anti-PD-1: tumor volume. The diagram in Figure 14 depicts the primary tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132, anti-PD-1, CD122/132 + anti-PD-1, Ad-Luc control, or combinations of Ad-Luc control + CD122/132, Ad-Luc control + anti-PD-1, and Ad-Luc control + CD122/132 + anti-PD-1. There was no significant increase in efficacy when Ad-Luc was combined with anti-PD-1, CD122/132, or CD122/132 + anti-PD-1 treatment compared to treatment with Ad-p53, VirRx007, and Ad-IL24. By day 16, the mean tumor volume in all groups exceeded 2,000 ^mm³ . The comparison of tumor volume by ANOVA on day 16 was not statistically significant (p = 0.1212; mean tumor volume was not statistically significant between any treatment groups).

Compared to the Ad-Luc negative control group using CD122/132+ anti-PD-1, the "triple therapy" combining Ad-p53, VirRx007, and Ad-IL24 with CD122/132+ anti-PD-1 showed significant advantages. Regarding survival, Figure 15 reveals that the "triple therapy" combining Ad-p53, VirRx007, and Ad-IL24 with CD122/132+ anti-PD-1, respectively, resulted in statistically significant prolonged survival compared to treatment with Ad-Luc + CD122/132+ anti-PD-1. These survival curves were statistically significant (p < 0.0001) according to the log-rank test. Compared with the triple therapy of Ad-Luc + CD122/132 + anti-PD-1 (p < 0.0001 for both Ad-p53 and VirRx007 with CD122/132 + anti-PD-1, and p < 0.015 for Ad-IL24 with CD122/132 + anti-PD-1), the triple therapy of each of Ad-p53, VirRx007 and Ad-IL24 with CD122/132 + anti-PD-1 showed a statistically significant increase in survival.

The CD122/CD132 agonist was prepared by incubating these agents together at 37°C for 30 minutes and then administering the resulting immune complexes intraperitoneally (IP) on days 3, 5, and 7 after tumor palpability.

Ad-p53 plus CD122/132 (IL15) agonist and checkpoint inhibitor immunotherapy – Tumor volume: The therapeutic efficacy of Ad-p53 combined with IL15-based CD122/132 agonists and anti-PD-1 therapy was evaluated by assessing tumor volume (in primary and contralateral tumors) and survival. Regarding primary tumor volume, the diagram in Figure 16 illustrates the tumor volume over time in rodents receiving phosphate-buffered saline (PBS) control, CD122/132 + anti-PD-1, Ad-p53 alone, or the combination of Ad-p53 + CD122/132 (IL15) + anti-PD-1. Severe tumor progression occurred during PBS, CD122/132 + anti-PD-1, and Ad-p53 therapy. Consistent with the earlier results of the Ad-p53 combination therapy described above, the efficacy of Ad-p53 + CD122/132(IL15) + anti-PD-1 therapy was significantly improved compared to any of the aforementioned therapies. 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) comparing tumor volumes confirmed the synergistic antitumor effect of Ad-p53 + CD122/132(IL15) + anti-PD-1 therapy (overall p < 0.0001, and individual p < 0.0001 compared to each of the other treatment groups).

Ad-p53 plus CD122/132 (IL15) agonist and checkpoint inhibitor immunotherapy— systemic/distant therapeutic effects on contralateral tumor growth. The systemic/distant effects of primary tumor treatment on contralateral implanted tumors were assessed in rodents whose primary tumors had been treated with one of the Ad-p53 therapies, and the results are shown in Figure 17. Consistent with the unexpectedly substantial synergistic effect of Ad-p53+CD122/132(IL15)+anti-PD-1 therapy on primary tumor growth shown in Figure 16, we also observed a surprisingly strong and statistically significant distant effect of Ad-p53+CD122/132(IL15)+anti-PD-1 therapy compared to other Ad-p53 treatment groups. Figure 17 depicts a graph showing the contralateral tumor volume over time in rodents receiving primary tumor treatment with the combination of Ad-p53+CD122/132, Ad-p53+anti-PD-1, or Ad-p53+CD122/132(IL15)+anti-PD-1. ANOVA comparison of these contralateral tumor volumes at day 22 determined the synergistic antitumor effect of Ad-p53+CD122/132(IL15)+anti-PD-1 treatment (overall p = 0.0433). 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 = 0.0359).

Ad-p53 combined with CD122/132 (IL15) agonist and checkpoint inhibitor immunotherapy—therapeutic efficacy contributed to prolonged survival. Kaplan-Mel survival curves for mice treated with PBS, CD122/132+anti-PD-1, Ad-Luc+CD122/132+anti-PD-1 control, Ad-p53, or the combination of Ad-p53+CD122/132(IL15)+anti-PD-1 are shown in Figure 18. These survival curves were statistically significant by log-rank test (overall p <0.0001; p < 0.0001 for Ad-p53+CD122/132(IL15)+anti-PD-1 treatment group compared to any other treatment group). The results also demonstrated an unexpectedly substantial synergistic effect of Ad-p53+CD122/132(IL15)+anti-PD-1 therapy. In the Ad-p53+CD122/132(IL15)+anti-PD-1 therapy group, 50% of the animals were alive by day 36. In stark contrast, all animals in the other treatment groups died by day 22, with a median survival ranging from 10 to 18 days.

Example 2 - Application using a vaccinia virus vector engineered to have N1L deletion and IL12 expression, combined with a preferred CD122/CD132 agonist and a PI3Kδ/γ inhibitor for local and systemic administration.

In another embodiment of this treatment method, a novel oncolytic vaccinia virus called VVL 15-N1L-IL12 is used as an adjunct therapeutic virus to enhance the efficacy of the method described in Example 1 above. Several oncolytic vaccinia virus strains have been reported, such as the Western Reserve, Wyeth, and Lister strains. Various deletion mutants of each of these strains have been created. Wang et al. (Patent WO2015/150809A1) have developed a TK-deficient vaccinia virus strain with an inactivated N1L gene, which exhibits enhanced selectivity and antitumor efficacy. N1L is believed to inhibit apoptosis and NF-κB activation in infected cells. N1L gene deletion has been shown to increase pro-inflammatory antiviral cytokines controlled by NF-κB, in addition to regulating natural killer (NK) cell responses. N1L deletion derivatives are described in Wang et al., 2015 (Patent WO2015/150809A1). To enhance the antitumor efficacy of VVL 15N1L, GM-CSF, IL-12, IL-21, tumor suppressor factors, and other therapeutic genes were inserted into the N1L region of the VVL 15N1L vector. These therapeutically "armed" VVL 15N1L vectors were then used in combination with single or multiple therapies as described in Example 1 above to enhance both local and distant therapeutic effects.

In addition to evaluating the methods described in Examples 1 and 2 above, the viral vector was also combined with a PI3K inhibitor. Examples of intravenous administration of the viral vector incorporating a PI3Kδ or PI3Kγ/δ inhibitor to enhance its efficacy are described. Animals received IC87114 (a PI3Kδ inhibitor) at a concentration of 75 mg ^kg⁻¹ , followed three hours later by intravenous injection of the VVL 15N1L vector via the tail vein at a concentration of 1 × ^10⁸ PFU/mouse in 100 μl PBS. This treatment was administered at least three times on days 0, 3, and 5. These treatments were combined with the same therapies described above. Tumor size and animal survival were measured, and the data were analyzed as described above, demonstrating increased efficacy of the treatment combined with Ad-p53 and/or Ad-IL24 and/or the VVL 15N1L vector, a CD122/CD132 agonist, an immune checkpoint inhibitor, and a PI3K inhibitor.

Example 3 - Combination therapy using intratumoral Ad-p53, CD122/CD132 agonists, and anti-PD-1 therapy in patients whose disease progressed with prior treatment, including immunotherapy.

Each vial supplies 2 mL of Ad-p53; each mL contains 1 × ^10¹² viral particles (vp). It is provided as a sterile viral suspension in phosphate-buffered saline (PBS) containing 10% (v/v) glycerol as a stabilizer. Prior to administration, Ad-p53 is diluted and filtered according to the protocol description. Anti-PD-1 therapy is administered according to the FDA-approved package insert instructions. CD122/CD132 agonist therapy (e.g., 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 mutant protein, and/or IL-15 mutant protein) is administered via SQ or IV at intervals ranging from once weekly to every 2–4 weeks at doses between 5–100 μg/kg. CD122/CD132 agonists can be IL-15 mutants (e.g., IL-15N72D) that bind to IL-15 receptor α/IgG1 Fc fusion proteins (such as ALT-803).

This treatment was designed to improve the prognosis of patients with advanced HNSCC, for example, by using Ad-p53, a preferred CD122/CD132 agonist, and an anti-PD-1 antibody. Clinical efficacy of the combination therapy was assessed including overall response rate [ORR = partial response (PR) + complete response (CR)], complete remission rate (CRR), durable response rate (DRR = PR + CR maintained for at least 6 months); rate and duration of visceral organ metastasis; progression-free survival (PFS); and overall survival (OS). The effects of the investigational drug on lymphocyte phenotype and serum cytokines, disease-related biomarkers, antibody responses to selected antigens, and humoral and cellular responses to tumor antigens were also evaluated. In the exploratory analysis, efficacy endpoints were correlated with PD-L1, PD-L2, immune cell infiltration, and tumor mutational burden biomarkers.

Every 28 days, patients receive intratumoral injections of Ad-p53 on days 1, 2, and 3; starting from day 5, nivolumab infusions are administered every 2 weeks, and preferred CD122/CD132 agonist therapy (e.g., IL2/anti-IL2 immune complexes and/or IL15/anti-IL15 immune complexes and/or IL15/IL15 receptor α-IgG1-Fc (IL15/IL15Rα-IgG1-Fc) immune complexes and/or PEGylated IL2 and/or PEGylated IL15 and/or IL2 mutant protein and/or IL15 mutant protein) is administered at intervals ranging from weekly to every 2-4 weeks via SQ or IV at doses ranging from 5-100 μg/kg.

The Ad-p53 injection dose (mL) and method of administration should be determined according to the tumor lesion diameters listed in Table 3: Table 3 should be used to determine the Ad-p53 injection dose (mL) for each tumor lesion, corresponding to the two-dimensional lesion diameter measured during CT or MRI scans. The Ad-p53 injection dose, in mL, should be dispensed from the provided 2 mL vials containing 1 × ^10¹² viral particles (vp)/mL. The Ad-p53 injection dose (mL) corresponding to the listed lesion diameters will ensure that each tumor lesion receives at least 1 × ^10¹¹ viral particles (vp)/ ^cm³ of tumor volume of Ad-p53. This treatment dose was determined based on tumor response, survival, and safety data from previous Ad-p53 clinical trials.

All tumor lesions should be treated. However, the total Ad-p53 injection dose (mL) must be less than 25 mL because the MTD of Ad-p53 is 2.5 × ^10¹³ vp/treatment day. The table below lists the corresponding Ad-p53 injection methods for each lesion based on the Ad-p53 injection dose (mL). Based on the amount of Ad-p53 injection dose (mL), use the Ad-p53 injection methods listed in the table below for each lesion. Ad-p53 injection doses less than or equal to 2 mL should be administered using a fine-needle syringe technique, while Ad-p53 injection doses equal to or greater than 4 mL should be administered using the Quadra-Fuse device shown below, according to the instructions for use on its packaging insert.

Table 3: Ad-p53 injection dose (mL) and Ad-p53 injection method based on lesion diameter.

a: Lesion diameter - L is the longer diameter, and W is the shorter diameter (in cm) (rounded to the nearest integer). b: The Ad-p53 injection dose in mL from vials containing 1 × ^10¹² vp (viral particles)/mL will result in each tumor lesion receiving approximately 1 × ^10¹¹ viral particles (vp)/ ^cm³ tumor volume of Ad-p53. The total Ad-p53 injection dose (mL) must be less than 25 mL because the MTD of Ad-p53 is 2.5 × ^10¹³ vp/treatment day. c: Ad-p53 injection method.

Ad-p53 Injection Methods: Based on the Ad-p53 injection dose (mL), the Ad-p53 injection methods for each lesion are as listed in Table 3. Ad-p53 injection doses (mL) less than or equal to 2 mL should be administered using a fine-needle syringe technique, while Ad-p53 injection doses (mL) greater than 2 mL should be administered using a Quadra-Fuse device as described.

Fine-needle syringes for Ad-p53 injection doses (mL) less than or equal to 2 mL: For lesions requiring an Ad-p53 injection dose (mL) less than or equal to 2 mL, a standard 1 mL syringe with a 27-gauge needle should be used to deliver the Ad-p53 injection dose (1 or 2 mL). One-quarter of the total Ad-p53 injection dose (mL) should be injected into each quadrant of the tumor lesion, positioning the needle during injection to maximize distribution within each quadrant.

Quadra-Fuse device for Ad-p53 injection doses (mL) greater than 2 mL: For lesions requiring an Ad-p53 injection dose (mL) greater than 2 mL, the Quadra-Fuse delivery device (Rex Medical, PA) should be used to deliver the Ad-p53 injection dose. The Quadra-Fuse device (FDA Class 1 medical device) consists of a central cannula from which three tips extend radially in an adjustable 1-5 cm diameter (Figure 3).

The Quadra-Fuse device allows for precise and diffuse drug delivery to multiple areas of the lesion simultaneously, as described below.

1. Draw the Ad-p53 injection dose (mL) from Table 3, which is matched to the two-dimensional diameter of the tumor lesion, into a standard syringe and attach it to the extension tube of the Quadra-Fuse device.

2. First, treat the lower half of the tumor lesion. Using the device's depth markers, position the tip of the Quadra-Fuse central cannula at the bottom of the longest diameter (L) of the lesion to match the longest diameter of the lesion under CT or ultrasound guidance. Adjust the Quadra-Fuse device's tip array treatment diameter to be 1 cm smaller than the shorter width diameter (W) of the tumor lesion (tip array treatment diameter = width of tumor diameter - 1 cm). After adjusting the Quadra-Fuse device to the tip array treatment diameter, open the tip and deliver one-quarter of the Ad-p53 injection dose (mL) at this position. (Note: Use the same tip array treatment diameter for all four Ad-p53 injection doses for each lesion. Retract the tip and rotate the device 60 degrees at the same depth. Then reopen the tip and deliver the second-quarter of the Ad-p53 injection dose (mL) at this position. These steps effectively deliver half of the Ad-p53 treatment dose to the lower half of the tumor lesion.)

3. To treat the upper portion of the tumor lesion, retract the tip again and move the central cannula tip upwards to the midpoint of the longest L-diameter of the tumor lesion. Extend the tip again to the tip array treatment diameter = (width of tumor diameter - 1 cm), and deliver the third quarter of the Ad-p53 injection dose (mL) at this location. Retract the tip and rotate the device 60 degrees at the same midpoint tumor depth. Then reopen the tip and deliver the final quarter of the Ad-p53 injection dose (mL) at this location.

In this way, a total of 48 sites within each tumor lesion will receive Ad-p53. There are a total of 12 simultaneous drug infusion sites for each tip array deployment, and each tip has two through-holes (four fluid outlets) (4 tip array deployments × 12 = 48 Ad-p53 delivery sites).

Treatment of 5×4cm tumor lesions: As listed in Table 3, tumors with a diameter L (longest diameter) of 5cm and a diameter W (shortest diameter) of 4cm will be treated by Quadra-Fuse Ad-p53 injection, using a dose of 4mL of Ad-p53.

Under CT or US guidance, the initial placement of the Quadra-Fuse central cannula tip should be at the base of the longer tumor diameter (L = 5 cm). The tip should be extended to a 3 cm tip array treatment diameter (4 cm - 1 cm = 3 cm wide of the tumor diameter) and the first quarter of the treatment dose (1 mL) should be infused. (Note: Use the same tip array treatment diameter for all four Ad-p53 injection doses for this lesion. Retract the tip and rotate the device 60 degrees at the same base of the tumor, 5 cm deep. Extend the tip back to a 3 cm tip array treatment diameter and infuse the second quarter of the Ad-p53 dose (1 mL). These procedures have distributed half of the Ad-p53 injection dose to the lower half of the tumor.)

To treat the upper part of the tumor lesion, the Quadra-Fuse central cannula is advanced to the midpoint of the longest lesion diameter (2.5 cm). The tip is then extended again to a 3 cm treatment diameter using the tip array, and the third quarter dose (1 mL) is infused. Maintaining the same intratumoral depth of 2.5 cm along the longest tumor diameter, the tip is retracted, the central cannula is rotated 60°, and the tip is re-extended to a 3 cm treatment diameter using the tip array, and the fourth and final quarter dose (1 mL of Ad-p53) is infused. These procedures have distributed half of the Ad-p53 injection dose to the upper part of the tumor. In total, these procedures deliver 4 mL of Ad-p53 to 48 infusion points within the tumor lesion.

Treatment duration: One treatment cycle lasts 28 days (4 weeks). The first day of the treatment cycle will be the first day of treatment administration. The treatment protocol is as follows:

Treatment regimen: The planned treatment days will be Ad-p53 administered on days 1, 2, and 3, every 28 days. Nivolumab will be administered every 2 weeks starting on day 5. Anti-PD-1 therapy will be administered according to the FDA-approved package insert instructions. Preferred CD122/CD132 agonist therapy (e.g., IL2/anti-IL2 immune complexes and/or IL15/anti-IL15 immune complexes and/or IL15/IL15 receptor α-IgG1-Fc (IL15/IL15Rα-IgG1-Fc) immune complexes and/or PEGylated IL2 and/or PEGylated IL15 and/or IL2 mutant protein and/or IL15 mutant protein) will be administered at intervals ranging from weekly to every 2-4 weeks via SQ or IV at doses ranging from 5-100 μg/kg.

On day 28 or 29 of the study, examinations to assess one or more tumor locations and measurements will be completed before the start of a new treatment cycle.

Unless there is local disease progression (excluding new treatable lesions) or unacceptable adverse events, the patient will be treated for three or more cycles.

Efficacy evaluation criteria:

1. Monitor tumor size using CT or MRI. If using CT or MRI, measurements will be performed on day 28 or 29 of the study, prior to injection on day 1 of the third cycle, with scans every 8 weeks. RECIST 1.1 criteria will apply.

2. The response duration is defined as the time elapsed from the response date to the progress time.

3. Progression-free survival is defined as the time elapsed from the date of randomization to the date of progress recording.

4. Overall survival is defined as the time elapsed from the date of randomization to death.

5. In the exploratory analysis, the efficacy endpoint was associated with biomarkers of PD-L1, PD-L2, immune cell infiltration, and tumor mutation burden.

Security assessment:

1. Adverse event reporting.

2. Physical examination, vital signs, laboratory tests including CBC, biochemical and urinalysis.

3. Biodistribution of glandular vectors using antibody testing.

Beneficial biomarkers for Ad-p53 efficacy are required for inclusion criteria in treatment and research patients and include wild-type p53 gene sequences or less than 20% p53-positive tumor cells obtained by immunohistochemical methods as described in Sobol et al., 2012.

In light of the recent breakthrough designation and accelerated approval of anti-PD-1 therapy in relapsed HNSCC, we conducted a meta-analysis of Ad-p53 treatment data in patients with relapsed HNSCC to identify treatment doses and schedules with the potential to improve published anti-PD-1 outcomes. The meta-analysis included 54 patients (n=54) with favorable p53 biomarker profiles, most of whom had previously received surgery, radiation, and platinum-based chemotherapy. The highest response rates were observed in clinical trials in which Ad-p53 was administered as a regimen of three intratumoral injections per week for three consecutive days during the first week or every other day for the first two weeks of a monthly treatment cycle. All responders (defined by RECIST 1.1 criteria) received an Ad-p53 dose greater than 7 × ^10¹⁰ viral particles/ ^cm³ tumor volume.

As shown in the table below, there was a statistically significant difference in tumor response between patients treated with a dose greater than 7 × ^10¹⁰ viral particles/ ^cm³ and those treated with a lower dose of Ad-p53 (the tumor response rate was 31 ^% (9/29) for Ad-p53 > 7 × 10¹⁰ viral particles/ ^cm³ , compared to 0% (0/25) for Ad-p53 < 7 × ^10¹⁰ viral particles/ ^cm³ ; p = 0.0023).

Table 6. RECIST target lesion response rate obtained by HNSCC with Ad-p53 dose.

Figure 1 shows a waterfall plot of tumor response in a subgroup of patients treated with Ad-p53 > 7 × ^10¹⁰ viral particles/ ^cm³ (left inset) compared to those treated with Ad-p53 doses ≤ 7 × ^10¹⁰ viral particles/ ^cm³ (right inset).

A more detailed examination of the Ad-p53 responders revealed that the majority of responders (7/9 patients) had received an Ad-p53 dose close to or greater than 1 × ^10¹¹ vp/ ^cm³ (range 7.81 to 333.2 × ^10¹⁰ vp/ ^cm³ ).

One-year survival and overall survival of patients with favorable Ad-p53 biomarker profiles treated with Ad-p53 doses ≥7 × ^10¹⁰ viral particles/ ^cm³ were compared with those of patients with favorable Ad-p53 biomarker profiles treated with methotrexate from a previous phase 3 clinical trial of relapsed HNSCC. Results are shown in Figure 2 and demonstrate a statistically significant improvement in overall survival with both Ad-p53 biomarker and dose-optimized Ad-p53 treatment compared to methotrexate (median survival of 11.5 months with Ad-p53 treatment vs. 4.5 months with methotrexate; p < 0.016, HR 1.9767).

As shown in Table 4, the best Ad-p53 treatment for recurrent HNSCC from meta-analysis data was more favorable than standard of care (SOC) chemotherapy and anti-PD-1 therapy reported by Ferris et al., 2016, in terms of efficacy endpoints of tumor response, 1-year survival, and median overall survival.

Table 4: Comparison of efficacy endpoints of Ad-p53, Standard of Care (SOC), and anti-PD-1 in recurrent head and neck squamous cell carcinoma (HNSCC).

*N=30; Predictive p53 biomarkers and dose optimization population

Therefore, based on this data, a dose of Ad-p53 (1 × ^10¹¹ vp/ ^cm³ tumor volume) was selected for combination therapy with preferred CD122/CD132 agonists and anti-PD-1 therapy.

Example 4 - Combination therapy using Ad-MDA7 (IL24), CD122/CD132 agonists, and anti-PD1 antibodies

Anti-PD-1 therapy has become the accepted treatment for patients with advanced, unresectable melanoma. Although anti-PD-1 represents a breakthrough treatment that benefits many patients, clinical data from multiple studies indicate that most patients do not respond to this therapy.

This therapy was designed to improve the prognosis of patients with advanced melanoma by using Ad-MDA-7 (note that Ad-MDA-7 = Ad-IL24) along with a CD122/CD132 agonist and an anti-PD-1 antibody. Clinical efficacy of the combination therapy was assessed including overall response rate [ORR = partial response (PR) + complete response (CR)], complete remission rate (CRR), durable response rate (DRR = PR + CR maintained for at least 6 months); rate and duration of visceral organ metastasis; progression-free survival (PFS); and overall survival (OS). The effects of the investigational drug on lymphocyte phenotype and serum cytokines, disease-related biomarkers, antibody response to selected antigens, and humoral and cellular responses to tumor antigens were also evaluated.

In addition, examine the pathological association of clinical activity in tumor samples, including (but not limited to) the abundance and characteristics of inflammatory infiltrates (e.g., expression of CD8 and CD4 cells, as well as programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) on lymphocytes and tumor cells, respectively) and tumor mutational burden.

If the patient responds at that time, treatment can be continued for up to 12 months or up to 18 months. Patients who respond at 12 months (CR or PR) should continue treatment until 18 months or until clinically relevant progressive disease (PDr) develops, whichever is earlier.

Because immunotherapy may delay tumor response and is associated with tumor inflammation that is mistaken for tumor progression, three types of progressive disease (PD) are defined. Non-clinically relevant progressive disease (PDn) is defined as PD in patients who do not experience a decline in performance status and/or do not require alternative therapy according to their physician's opinion. Patients exhibiting PDn should continue treatment. Clinically relevant progressive disease (PDr) is defined as PD associated with a decline in performance status and/or the need for alternative therapy according to their physician's opinion. Patients with PDr are allowed to continue treatment for up to 24 weeks unless their physician deems additional treatment necessary. CNS progressive disease (PDcns) is defined as progression in the central nervous system (brain).

Ad-IL24 treatment is provided as a frozen vial suspension (2.0 mL/vial) at a concentration of 1 × ^10¹² vp/mL in a neutral buffer containing saline and 10% glycerol. There is no minimum size of tumor mass required for injection. Skin lesions should be included in the first group of tumors to be treated to enhance the immune response of the therapy mediated by skin antigen-presenting cells.

Individual patients may have up to 20 lesions, with the longest diameter of any single lesion not exceeding 5 cm. The goal is to eventually treat all lesions with at least one cycle of Ad-IL24 therapy (intratumoral injection twice weekly for 3 weeks). Each patient's lesions are grouped into Ad-IL24 treatment groups, with the number of lesions in each group determined by tumor diameter and dose escalation cohort, ensuring that the Ad-IL24 delivered on each treatment day does not exceed the total volume dose allowed per treatment day as specified in the dose escalation protocol in Table 4. The total dose (volume) delivered to one or more tumors will not exceed the volume specified in Table 4, and the amount injected into each individual tumor within a treatment group depends on the size of one or more tumor nodules and is determined according to the following algorithm:

• For tumors with a maximum length of 0.5 cm, the maximum is 0.1 mL.

• For tumors with a maximum size of 0.5 to 1.5 cm, the maximum dose is 0.5 mL.

• For tumors with a maximum size of 1.5 to 2.5 cm, the maximum dose is 1.0 mL.

• For tumors with a maximum size of 2.5 to 5 cm, the maximum volume is 2.0 mL.

The maximum volume injected into any single lesion is 2 mL. The maximum dose on any treatment day is 2 mL, 4 mL, or 6 mL, depending on the treatment dose escalation cohort specified in the table below.

Table 4: Treatment schedule.

Patients treated with anti-PD-1 therapy become refractory to anti-PD-1 therapy.

Table 5: Dosage escalation design.

The treatment regimens listed above will be combined with preferred CD122/CD132 agonists (such as IL2/anti-IL2 immune complexes and/or IL15/anti-IL15 immune complexes and/or IL15/IL15 receptor α-IgG1-Fc (IL15/IL15Rα-IgG1-Fc) immune complexes and/or PEGylated IL2 and/or PEGylated IL15 and/or IL2 mutants and/or IL15 mutants), administered at intervals ranging from weekly to every 2-4 weeks at doses between 5-100 ug/kg via SQ or IV.

In summary, the animal study populations described in the examples used highly aggressive forms of cancer known to be resistant to immunotherapy. Surprisingly, the combination of localized tumor suppressor and oncolytic virus therapy with preferential CD122/CD132 therapy reversed resistance to systemic immune checkpoint inhibitor therapy, demonstrating an unexpected synergistic effect with immune checkpoint inhibitor therapy. Furthermore, the combination therapy induced a superior distant effect on distal tumors not treated with tumor suppressor or oncolytic virus therapy. In highly immunotherapy-resistant cancers, these treatments remarkably contributed to complete tumor regression and curative outcomes.

In light of this disclosure, all methods disclosed and claimed herein can be performed and carried out without excessive experimentation. Although the compositions and methods of the invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be made to the methods described herein, as well as the steps or order of the steps, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

***

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Claims (124)

1. The use of (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 PD-1 or PD-L1 immune checkpoint inhibitor in a medicament for preparing a method for treating a subject with melanoma, the method comprising administering to the subject an effective amount of (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, 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 mutant protein, IL-15 mutant protein and/or IL-15 mutants that bind to IL-15 receptor α/IgG1 Fc fusion protein, wherein at least one CD122/CD132 agonist preferentially binds to CD122/CD132. 2. The use according to claim 1, wherein the nucleic acid encoding p53 is administered to the subject. 3. The use according to claim 1, wherein the subject is given a nucleic acid encoding MDA7. 4. The use according to claim 1, wherein the subject is given a nucleic acid encoding p53 and a nucleic acid encoding MDA7. 5. The use according to claim 1, wherein the IL-15 is pre-complexed with IL-15Rα to preferentially bind to CD122/CD132. 6. The use according to claim 1, wherein one, two, three, or four CD122/CD132 agonists are administered to the subject. 7. The use according to claim 1, wherein the at least one CD122 agonist and/or CD132 agonist is not F42K. 8. The use according to claim 1, wherein the melanoma is metastatic. 9. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is in an expression cassette. 10. The use according to claim 9, wherein p53 and MDA-7 are under the control of a single promoter. 11. The use according to claim 10, wherein the promoter is cytomegalovirus (CMV), SV40, or PGK. 12. The use according to claim 9, wherein the expression cassette is in a viral vector. 13. The use according to claim 12, 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. 14. The use according to claim 12, wherein the viral vector is an adenovirus vector or a vaccinia virus vector. 15. The use according to claim 14, wherein the vaccinia virus vector is further defined as a vaccinia virus vector lacking NIL. 16. The use according to claim 14, wherein the adenovirus vector is further defined as an adenovirus vector having increased ADP expression. 17. The use according to claim 12, wherein the viral vector is applied with viral particles of between ^10³ and ^10¹³ . 18. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is administered by a non-viral method. 19. The use according to claim 1, wherein the method comprises restoring and/or amplifying p53 and/or MDA-7 function through gene editing. 20. The use according to claim 19, wherein gene editing comprises expressing p53 and/or MDA-7 using a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a clustered regularly spaced short palindromic repeat sequence (CRISPR). 21. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is administered via a viral vector and gene editing. 22. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 are administered to the subject intravascularly, intrapleurally, intraperitoneally, intratracheally, intratumorally, intrathecally, intramuscularly, intramyoscopically, intralesionally, percutaneously, subcutaneously, locally, stereotactically, or by direct injection or infusion. 23. The use according to claim 22, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is administered intravenously to the subject. 24. The use according to claim 22, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is administered intra-arterially to the subject. 25. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is administered intratumorally to the subject. 26. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 is administered to the subject more than once. 27. The use according to claim 1, wherein the subject is administered the at least one CD122/CD132 agonist more than once. 28. The use according to claim 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. 29. The use according to claim 1, wherein the nucleic acid encoding p53 and/or the nucleic acid encoding MDA-7 are administered to the subject in the form of a liposome complex. 30. The use according to claim 29, wherein the liposome complex comprises DOTAP and at least one cholesterol, cholesterol derivative, or mixture of cholesterol. 31. The use according to claim 1, wherein the application includes local or regional injection. 32. The use according to claim 1, wherein the administration is carried out via continuous infusion, intratumoral injection or intravenous injection. 33. The use according to claim 1, wherein the subject is a human. 34. The use according to claim 1, further comprising administering at least one additional anticancer treatment. 35. The use according to claim 34, wherein the at least one additional anticancer treatment is surgical therapy, chemotherapy, radiotherapy, small molecule therapy, cryotherapy, or biotherapy. 36. The use according to claim 34, wherein the at least one additional anticancer treatment is radioablation. 37. The use according to claim 34, wherein the at least one additional anticancer treatment is hormone therapy. 38. The use according to claim 34, wherein the at least one additional anticancer treatment is immunotherapy. 39. The use according to claim 34, wherein the at least one additional anticancer treatment is cytokine therapy. 40. The use according to claim 34, wherein the at least one additional anticancer treatment is a receptor kinase inhibitor therapy or an anti-angiogenic therapy. 41. The use according to claim 1, wherein the at least one checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. 42. The use according to claim 41, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224. 43. The use according to claim 41, wherein the anti-PD-L1 antibody is durvalumab, atezolizumab, or avermectin. 44. The use according to claim 41, wherein the anti-PD-L2 antibody is rHIgM12B7. 45. The use according to claim 1, wherein more than one checkpoint inhibitor is applied. 46. The use according to claim 1, wherein the immune checkpoint inhibitor is administered systemically. 47. The use according to claim 34, wherein the at least one additional anticancer treatment is a histone deacetylase (HDAC) inhibitor. 48. The use according to claim 47, wherein the HDAC inhibitor is tractinostat. 49. The use according to claim 1, further comprising providing extracellular matrix degradation proteins. 50. The use according to claim 49, wherein the extracellular matrix degrading protein is relaxin, hyaluronidase, or core proteoglycan. 51. The use according to claim 35, wherein the biotherapy is a monoclonal antibody, siRNA, miRNA, antisense oligonucleotide, ribozyme, or gene therapy. 52. The use according to claim 34, wherein the at least one additional anticancer treatment is an oncolytic virus. 53. The use according to claim 52, wherein the oncolytic virus is engineered to express p53, MDA-7, IL-12, at least one heat shock protein, a TGF-β inhibitor and/or an IL-10 inhibitor. 54. The use according to claim 52, wherein the oncolytic virus is an adeno-associated virus, retrovirus, lentivirus, herpesvirus, poxvirus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, Ebstein-Barr virus, influenza virus, reovirus, myxoma virus, Malaba virus, rhabdovirus, enadenotucirev, Coxsackie virus, or an E1b-deficient adenovirus. 55. The use according to claim 52, wherein the oncolytic virus is a herpes simplex virus. 56. The use according to claim 52, wherein the oncolytic virus is a single-stranded or double-stranded DNA virus or an RNA virus. 57. The use according to claim 52, wherein the oncolytic virus is a vaccinia virus. 58. The use according to claim 52, wherein the oncolytic virus is an adenovirus. 59. The use according to claim 52, wherein the oncolytic virus is engineered to express cytokines. 60. The use according to claim 59, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL12. 61. The use according to claim 52, wherein the oncolytic virus is further defined as talimogenelaherparepvec (T-VEC). 62. The use according to claim 34, wherein the at least one additional anticancer treatment is a protein kinase or a growth factor signaling pathway inhibitor. 63. The use according to claim 62, wherein the protein kinase or growth factor signaling pathway inhibitor is afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fotatinib, gefitinib, imatinib, lapatinib, lenvatinib, lilotinib, nilotinib, panitumumab, pazopanib, pilgatanib, lanibimumab, ruxotinib, zicatinib, or sorafil. Nitrosamine, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vermuprin, CAL101, PX-866, LY294002, Rapamycin, Tesirolimus, Everolimus, Desfolimus, Avozidil, Gentianaerum, Selmetinib, AZD-6244, Vatalani, P1446A-05, AG-024322, ZD1839, P276-00, or GW572016. 64. The use according to claim 62, wherein the protein kinase inhibitor is a PI3K inhibitor. 65. The use according to claim 64, wherein the PI3K inhibitor is a PI3K δ inhibitor. 66. The use according to claim 38, wherein the immunotherapy comprises cytokines. 67. The use according to claim 66, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL12. 68. The use according to claim 67, wherein the cytokine is interleukin and/or interferon. 69. The use according to claim 68, wherein the interleukin is IL-2. 70. The use according to claim 68, wherein the interferon is IFNα. 71. The use according to claim 38, wherein the immunotherapy comprises a co-stimulatory receptor agonist, an innate immune cell stimulator, or an innate immune activator. 72. The use according to claim 71, wherein the co-stimulatory receptor agonist is an anti-OX40 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-CD40 antibody, or an anti-CD27 antibody. 73. The use according to claim 71, wherein the immune cell stimulant is an inhibitor of cytotoxic inhibitory receptors or an agonist of immune-stimulating toll-like receptors (TLRs). 74. The use according to claim 73, wherein the cytotoxic inhibitory receptor is an inhibitor of NKG2A/CD94 or CD96 TACTILE. 75. The use according to claim 73, wherein the TLR agonist is a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist. 76. The use according to claim 38, wherein the immunotherapy comprises a combination of a PD-L1 inhibitor, a 4-1BB agonist, and an OX40 agonist. 77. The use according to claim 38, wherein the immunotherapy comprises an interferon gene-stimulating factor (STING) agonist. 78. The use according to claim 71, wherein the innate immune activator is an IDO inhibitor, a TGFβ inhibitor, or an IL-10 inhibitor. 79. The use according to claim 35, wherein the chemotherapy comprises a DNA damaging agent. 80. The use according to claim 79, wherein the DNA damaging agent is gamma radiation, X-ray, UV radiation, microwave, electron emission, doxorubicin, 5-fluorouracil (5FU), capecitabine, etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), or hydrogen peroxide. 81. Use in a medicament of a method for preparing a method of treating a subject with melanoma, comprising at least one oncolytic virus, a nucleic acid encoding p53 and/or a nucleic acid encoding MDA-7, at least one CD122/CD132 agonist and at least one PD-1 or PD-L1 immune checkpoint inhibitor, wherein the at least one CD122/CD132 agonist is selected from the group consisting of: 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 mutant protein, IL-15 mutant protein and/or IL-15 mutant bound to IL-15 receptor α/IgG1 Fc fusion protein. 82. The use according to claim 81, wherein the at least one oncolytic virus is engineered to express p53, MDA-7, cytokines and/or immunostimulatory genes. 83. The use according to claim 82, wherein the cytokine is GM-CSF or IL-12. 84. The use according to claim 82, wherein the immunostimulatory gene is a TGFβ inhibitor, an IL-10 inhibitor, or a heat shock protein. 85. The use according to claim 81, wherein the at least one oncolytic virus is selected from the group consisting of: adeno-associated virus, retrovirus, lentivirus, herpesvirus, poxvirus, vesicular stomatitis virus, poliovirus, Newcastle disease virus, Ebola virus, influenza virus, reovirus, myxoma virus, Malaba virus, rhabdovirus, enadenotucirev and Coxsackie virus. 86. The use according to claim 81, wherein the oncolytic virus is a single-stranded or double-stranded DNA virus or an RNA virus. 87. The use according to claim 81, wherein the oncolytic virus is a vaccinia virus. 88. The use according to claim 81, wherein the oncolytic virus is an adenovirus. 89. The use according to claim 81, wherein the at least one oncolytic virus is Ad5-yCD/mutTKSR39rep-hIL12, CAVATAK™, CG0070, DNX-2401, G207, HF10, IMLYGIC™, JX-594, MG1-MA3, MV-NIS, OBP-301, REOLYSIN®, Toca 511, Ankerui (H101), H102, H103, RI GVIR, adenovirus overexpressing adenoviral death protein (ADP), T-VEC, N1L-deficient vaccinia virus, E1b-deficient adenovirus, adenovirus regulated by the alpha-fetoprotein (AFP) promoter Ad E1a gene, modified TERT promoter oncolytic adenovirus, HRE-E2F-TERT hybrid promoter oncolytic adenovirus, and/or adenovirus with a Pea3 binding site E1a regulatory sequence deletion and an Elb-19K clonal insertion site. 90. The use according to claim 89, wherein the adenovirus overexpressing ADP is ViRx007. 91. The use according to claim 89, wherein the N1L-deficient vaccinia virus is engineered to express IL-12. 92. The use according to claim 81, wherein one, two, three, or four CD122/CD132 agonists are administered to the subject. 93. The use according to claim 81, wherein the at least one CD122/CD132 agonist is not F42K. 94. The use according to claim 81, wherein the subject is administered the at least one CD122/CD132 agonist more than once. 95. The use according to claim 81, wherein the oncolytic virus is administered to the subject before, simultaneously with or after the at least one CD122/CD132 agonist. 96. The use according to any one of claims 92, 94 and 95, wherein the application comprises local or regional injection. 97. The use according to any one of claims 92, 94 and 95, wherein the administration is carried out via continuous infusion, intratumoral injection or intravenous injection. 98. The use according to any one of claims 92, 94 and 95, wherein the administration is performed by intratumoral injection. 99. The use according to claim 81, wherein the subject is a human. 100. The method according to claim 81, further comprising administering at least one additional anticancer treatment. 101. The use according to claim 100, wherein the at least one additional anticancer treatment is surgical therapy, chemotherapy, radiotherapy, small molecule therapy, cryotherapy, or biological therapy. 102. The use according to claim 100, wherein the at least one additional anticancer treatment is hormone therapy. 103. The use according to claim 100, wherein the at least one additional anticancer treatment is immunotherapy. 104. The use according to claim 100, wherein the at least one additional anticancer treatment is cytokine therapy. 105. The use according to claim 100, wherein the at least one additional anticancer treatment is a receptor kinase inhibitor therapy or an anti-angiogenic therapy. 106. The use according to claim 100, wherein the at least one additional anticancer treatment is a dendritic cell vaccine. 107. The use according to claim 106, wherein the dendritic cell vaccine is engineered to express p53 as a tumor-associated antigen. 108. The use according to claim 81, wherein the at least one checkpoint inhibitor is selected from CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, LAG3 inhibitors, BTLA inhibitors, B7H3 inhibitors, B7H4 inhibitors, TIM3 inhibitors, KIR inhibitors, or A2aR inhibitors. 109. The use according to claim 108, 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. 110. The use according to claim 109, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab, pilizumab, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA or AMP-224. 111. The use according to claim 109, wherein the anti-PD-L1 antibody is durvalumab, atezolizumab, or avermectin. 112. The use according to claim 109, wherein the anti-PD-L2 antibody is rHIgM12B7. 113. The use according to claim 108, wherein the LAG3 inhibitor is IMP321 or BMS-986016. 114. The use according to claim 108, wherein the A2aR inhibitor is PBF-509. 115. The use according to claim 109, wherein the anti-CTLA-4 antibody is trimelimumab or ipilimumab. 116. The use according to claim 109, wherein the anti-KIR antibody is riluzumab. 117. The use according to claim 81, wherein more than one checkpoint inhibitor is applied. 118. The use according to claim 81, wherein the immune checkpoint inhibitor is administered systemically. 119. The use according to claim 100, wherein the at least one additional anticancer treatment is a histone deacetylase (HDAC) inhibitor. 120. The use according to claim 119, wherein the HDAC inhibitor is tractinostat. 121. The use according to claim 81, wherein the method further comprises providing extracellular matrix degradation proteins. 122. The use according to claim 121, wherein the extracellular matrix degrading protein is relaxin, hyaluronidase, or core proteoglycan. 123. 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 PD-1 or PD-L1 immune checkpoint inhibitor, wherein the at least one CD122/CD132 agonist is selected from the group consisting of: 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 mutant protein, IL-15 mutant protein and/or IL-15 mutant protein bound to IL-15 receptor α/IgG1 Fc fusion protein. 124. A pharmaceutical composition comprising (a) an oncolytic virus; (b) a nucleic acid encoding p53 and/or a nucleic acid encoding MDA-7; (c) at least one CD122/CD132 agonist; and (d) at least one PD-1 or PD-L1 immune checkpoint inhibitor, wherein the at least one CD122/CD132 agonist is selected from the group consisting of: 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 mutant protein, IL-15 mutant protein and/or IL-15 mutant protein bound to IL-15 receptor α/IgG1 Fc fusion protein.