Methods and compositions for TUSC2 immunotherapy

Abstract

Methods of treating a subject with cancer are provided. The method includes administering TUSC2 therapy, a tumor suppressor therapy, to a subject, wherein the subject has been treated or is currently being treated with at least one immune checkpoint inhibitor, preferably an anti-PD1 agent.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 407,329, filed October 12, 2016, which is incorporated herein by reference in its entirety.

[0002] 1. Field of the Invention The embodiments provided herein relate generally to the fields of molecular biology, immunology, and cancer therapy. [Background technology]

[0003] 2. Description of Related Art As the molecular and genetic mechanisms of tumorigenesis become more clearly understood, the focus of cancer treatment has shifted from the tissue to the genetic level (Bishop, 1991). Mutations in two major classes of genes, oncogenes and tumor suppressor genes (TSGs), play a central role in the carcinogenic process. TSGs appear to require homozygous deletion or mutation for inactivation, and restoration of TSG expression is possible in human tumors (Lowe et al., 2004; Roth, 2006). Homozygous deletions in the 3p21.3 region in lung cancer cell lines and primary lung tumors have identified multiple genes with tumor suppressor activity from this region (Lerman et al., 2000). These deletions have led to the development of targeted anticancer therapies. However, methods that can enhance the efficacy of such therapies remain unclear. Summary of the Invention

[0004] In a first aspect, a method for treating a subject with cancer is provided, comprising administering a tumor suppressor therapy (e.g., TUSC2 therapy) in combination with an immune checkpoint inhibitor. Thus, a method for treating a subject with cancer who is currently being treated with (or has previously been treated with) at least one immune checkpoint inhibitor is provided, comprising administering a tumor suppressor therapy, such as TUSC2 therapy, to the subject. For example, a subject to be treated with TUSC2 therapy may have been administered an immune checkpoint inhibitor less than 1 hour, 6 hours, 12 hours, 1 day, 3 days, 1 week, or 2 weeks prior to the administration of TUSC2 therapy. As used herein, TUSC2 therapy can be any type of therapy that provides or induces the expression of a TUSC2 polypeptide in cancer cells (see, for example, U.S. Patent No. 7,902,441, incorporated herein by reference). For example, TUSC2 therapy can include delivery of a TUSC2 polypeptide or a TUSC2 expression vector to cancer cells. The therapy can be delivered, for example, via nanoparticles or, in the case of a nucleic acid expression vector, through the use of a viral vector.

[0005] In some embodiments, a method for treating a subject with cancer is provided, comprising administering a TUSC2 therapy to the subject in combination with at least one immune checkpoint inhibitor.For example, the TUSC2 therapy can be administered before, after, or essentially simultaneously with at least one immune checkpoint inhibitor.Thus, in some embodiments, a composition is provided comprising a TUSC2 therapeutic agent and an immune checkpoint inhibitor in a therapeutically effective amount for treating cancer.

[0006] In some aspects, the at least one checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In some aspects, the 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 a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist. 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. In certain aspects, the PD-1 binding antagonist is a monoclonal antibody or an antigen-binding fragment thereof. In certain aspects, the PD-1 binding antagonist is nivolumab, pembrolizumab, pidilizumab, KEYTRUDA®, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224. In some aspects, the at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. In certain aspects, the anti-CTLA-4 antibody is tremelimumab, YERVOY®, or ipilimumab. In some aspects, the at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. In some aspects, the anti-KIR antibody is lirilumab. In some aspects, the subject has previously received or is currently receiving two or more immune checkpoint inhibitors, such as an anti-PD1 antibody and an anti-CTLA4 antibody.

[0007] In some embodiments, administration of TUSC2 therapy involves administration of a TUSC2 expression vector, such as a DNA plasmid encoding TUSC2. Expression vectors for use in the embodiments provided herein generally include control elements for expression of the TUSC2 coding sequence. For example, the vector may include promoter and enhancer elements effective for expression in the cancer cells of interest. In some aspects, for example, TUSC2 expression is driven by a CMV promoter or a recombinant version thereof, such as the CMV promoter construct described in U.S. Patent Application Publication No. 20070092968, incorporated herein by reference. In some embodiments, the vectors provided herein include a modified CMV promoter. In some embodiments, the vectors provided herein include a mini-CMV promoter. Additional expression control elements may also be included, such as, for example, introns, drug response elements, RNA stabilization or destabilization sequences, cellular localization signals, polyadenylation signal sequences, and / or optimized translation initiation codons. Plasmid DNA vectors may also include sequences that help facilitate DNA production, bacterial origins of replication, and / or drug resistance markers. In one particular aspect, the TUSC2 expression vector is the pLJ143 / KGB2 / FUS1 plasmid.

[0008] Methods for delivery of expression vectors to cells (e.g., in vivo delivery) are well known in the art and include, but are not limited to, nanoparticles (e.g., liposomal nanoparticles), lipid conjugates, and viral vectors. In one aspect, the TUSC2 expression vector is administered in nanoparticles, such as N-[1-(2,3-(dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol liposome nanoparticles. One of skill in the art will recognize that various properties of liposomes can be tailored to optimize vector delivery. For example, liposomes may be tailored to have a particular size range and / or a particular ratio of DNA to lipid; DNA to cholesterol; or lipid to cholesterol. For example, in the case of DOTAP:cholesterol liposomes, the DOTAP:cholesterol ratio can be defined as about 1.5:1 to 1:1.5, e.g., about 10:9. In a further aspect, the TUSC2 expression vector is provided in liposome nanoparticles comprising an average particle size of about 50 to about 500 nm (e.g., 200-500 nm). In yet a further aspect, the TUSC2 nanoparticle formulation has an OD of about 0.65 to 0.95. 400 It can be defined by its optical density (OD), such as having

[0009] In yet further embodiments, TUSC2 therapy may include administration of a TUSC2 polypeptide. Methods for administering TUSC2 polypeptides are described, for example, in U.S. Patent Application Publication Nos. 20060251726 and 20090023207, which are incorporated herein by reference. TUSC2 polypeptides may be modified to enhance their activity and / or ability to enter cancer cells. For example, the polypeptide can be modified with a lipid moiety (e.g., myristoylated). In some aspects, TUSC2 is provided as nanoparticles (e.g., lipid-based nanoparticles), such as superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, or nanotubes.

[0010] The TUSC2 therapy and / or immune checkpoint inhibitor according to the embodiments provided herein is typically formulated in a pharmaceutically acceptable carrier. The therapy according to the embodiments may be delivered, for example, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, or by infusion, and the route of delivery may depend on the type of cancer to be treated. For example, a TUSC2 expression vector complexed with DOTAP:cholesterol liposomes can be administered via intravenous infusion. In certain aspects, the TUSC2 therapy is administered intravenously at a dose of about 0.01 mg / kg to about 0.10 mg / kg, for example, about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10 mg / kg. In further aspects, the TUSC2 therapy can be administered two or more times (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 times). The timing between doses of such therapy can vary, including, but not limited to, about 1, 2, or 3 days, about 1, 2, or 3 weeks, or more than one month between doses.

[0011] In yet another embodiment, a method for treating a subject with cancer is provided, comprising administering TUSC2 therapy to the subject in combination with at least one immune checkpoint inhibitor and / or in combination with one or more anti-inflammatory agents.For example, the anti-inflammatory agent can be administered before, after, or during TUSC2 therapy.In a further aspect, two or more anti-inflammatory agents are administered, such as administering an antihistamine and a corticosteroid.Thus, in certain aspects, the anti-inflammatory agent for use in combination with TUSC2 therapy is diphenhydramine and / or dexamethasone.

[0012] In a further embodiment, the method provided herein further comprises administering an additional anti-cancer treatment.The additional anti-cancer treatment may be, but is not limited to, surgical therapy, chemotherapy (for example, administration of protein kinase inhibitors or EGFR targeting therapy), radiation therapy, cryotherapy, hyperthermia, phototherapy, radioablation therapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, or biological therapy, such as monoclonal antibody, siRNA, antisense oligonucleotide, ribozyme or gene therapy.But is not limited to, biological therapy may be gene therapy, such as tumor suppressor gene therapy, cell death protein gene therapy, cell cycle regulator gene therapy, cytokine gene therapy, toxin gene therapy, immune gene therapy, suicide gene therapy, prodrug gene therapy, anti-cell proliferation gene therapy, enzyme gene therapy, or anti-angiogenic factor gene therapy.

[0013] Therefore, in yet another aspect, the present invention provides compositions, therapies and methods for treating cancer patients, comprising administering to the patient TUSC2 therapy (for example, TUSC2 polypeptide or TUSC2 expression vector) in combination with immune checkpoint inhibitors and additional anti-cancer agents, for example, chemotherapeutic agents.For example, the chemotherapeutic agent can be a protein kinase inhibitor, such as Src or Akt kinase inhibitor.In some aspects, the chemotherapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor.

[0014] Therefore, in some embodiments, a method for treating a subject with cancer is provided, comprising administering a TUSC2 therapy to the subject in combination with at least one immune checkpoint inhibitor and optionally a protein kinase inhibitor.For example, the TUSC2 therapy and / or the immune checkpoint inhibitor can be administered before, after, or essentially simultaneously with the protein kinase inhibitor.Thus, in some embodiments, a composition is provided comprising a TUSC2 therapeutic agent, an immune checkpoint inhibitor, and a protein kinase inhibitor in a therapeutically effective amount for treating cancer.Protein kinase inhibitors for use in this embodiment include, but are not limited to, inhibitors 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. For example, protein kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, and trastuzumab. The protein kinase inhibitor may be tuzumab, vandetanib, AP23451, vemurafenib, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or a mixture thereof. In some aspects, the protein kinase inhibitor is an AKT inhibitor (e.g., MK-2206, GSK690693, A-443654, VQD-002, miltefosine, or perifosine).

[0015] EGFR-targeted therapies for use in accordance with this embodiment include, but are not limited to, inhibitors of EGFR / ErbB1 / HER, ErbB2 / Neu / HER2, ErbB3 / HER3, and / or ErbB4 / HER4. A wide variety of such inhibitors are known, including, but not limited to, tyrosine kinase inhibitors active against the receptor and EGFR-binding antibodies or aptamers. For example, the EGFR inhibitor may be gefitinib, erlotinib, cetuximab, matuzumab, panitumumab, AEE788; CI-1033, HKI-272, HKI-357, or EKB-569. In some embodiments, the compositions and therapies provided herein are administered systemically or locally. In one embodiment, the compositions and therapies provided herein are administered systemically. In some aspects, the EGFR inhibitor is administered to the patient before, after, or essentially simultaneously with TUSC2 therapy. For example, the therapies may be administered simultaneously, such as by co-administration via intravenous infusion. In some embodiments, the TUSC2 inhibitor and the EGFR inhibitor can be administered in any amount effective to treat cancer. In some embodiments, the compositions, therapies, and methods provided herein include administering the TUSC2 inhibitor and the EGFR inhibitor at a lower dose than either composition administered alone. In some embodiments, the compositions, therapies, and methods include administering the TUSC2 inhibitor and the EGFR inhibitor at a lower dose that reduces side effects. In some embodiments, the compositions, therapies, and methods include administering the TUSC2 therapy, the immune checkpoint inhibitor, and the EGFR inhibitor at doses effective to provide an additive, cooperative, or synergistic effect greater than that provided by either composition administered alone. In some aspects, the cancer for treatment with such therapy can be any of the cancers described herein, such as lung cancer (e.g., non-small cell lung cancer). In some preferred aspects, the cancer for treatment with combination therapy is an EGFR-expressing cancer.In some embodiments, an EGFR-expressing cancer contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of tumor cells that express EGFR.

[0016] In yet another embodiment, provided herein is a method for treating a subject with cancer previously determined to express EGFR, comprising administering TUSC2 therapy to the subject in combination with an immune checkpoint inhibitor and an EGFR inhibitor.In one embodiment, provided herein is a method for treating a subject with cancer, comprising determining whether the cancer expresses EGFR, and administering TUSC2, an immune checkpoint inhibitor, and an EGFR inhibitor to the subject.Methods for assessing the EGFR expression status of cancer are described, for example, in US Patent Application Publication No. 20110052570, which is incorporated herein by reference.In one aspect, the cancer that expresses EGFR can be a cancer that expresses mutant EGFR, such as a cancer that expresses EGFR with L858R and / or T790M mutation. In certain embodiments, the compositions and therapies provided herein are administered to patients with EGFR-expressing cancers that contain at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of tumor cells that express EGFR. In yet a further aspect, the subject for treatment has a cancer that has previously been determined to express EGFR and in which at least 10% of the cells of the cancer are apoptotic. In some embodiments, the methods provided herein further include determining whether at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the cancer that express EGFR are apoptotic.

[0017] In some embodiments, the cancer for treatment or evaluation may be present as a tumor, such as a primary tumor or a metastatic tumor.Cancer may be an early stage cancer, or a metastatic or late stage cancer.In some aspects, cancer may be oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, blood cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, prostate cancer, pheochromocytoma, pancreatic islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, The cancer may be parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and type II, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In a further aspect, the cancer may be defined as a cancer that is resistant to one or more anti-cancer treatments, such as a chemotherapy-resistant cancer. For example, the cancer may be a cancer that is resistant to platinum-based chemotherapy agents, such as cisplatin.

[0018] In some aspects of the above embodiments, administering TUSC2 therapy and at least one immune checkpoint inhibitor increases NK cell and / or CD8 + This results in an increase in the density of T cells. In certain cases, CD8 + The density of T cells is increased by at least 3-fold, e.g., 4, 5, 6, 7, 8, 9, or 10-fold. In some aspects, administering TUSC2 therapy and at least one immune checkpoint inhibitor results in CcL3, CcL4, CcL21a, and / or CcL19 serum levels.

[0019] In yet a further aspect, the present invention provides a kit comprising a TUSC2 therapeutic agent and at least one immune checkpoint inhibitor.For example, in some aspects, the kit provided herein comprises a TUSC2 therapeutic agent, at least one immune checkpoint inhibitor, and a reagent for testing a subject to determine its response to the TUSC2 therapeutic agent and / or immune checkpoint inhibitor.For example, the reagent for testing a subject to determine its response to the TUSC2 therapeutic agent can be a reagent for determining the level of apoptosis in the subject's cancer cells.In a further aspect, the kit further comprises one or more anti-inflammatory agents or kinase inhibitors.In yet a further aspect, the kit may comprise one or more additional components, including, but not limited to, a pharmaceutically acceptable diluent, a syringe, an infusion bag, an infusion line, and / or a set of instructions for using the kit.

[0020] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Similarly, aspects of the present embodiments discussed in the context of a method for treating a subject are equally applicable to a method for predicting a response in a subject, and vice versa.

[0021] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the words "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0022] As used herein, "essentially free" of specific components means that none of the specific components are intentionally formulated in the composition and / or only exist as contaminants or in trace amounts.Therefore, the total amount of specific components that result from any unintentional contamination of the composition is far less than 0.01%.Most preferably, the composition is one in which any amount of specific components cannot be detected by standard analytical methods.

[0023] As used herein and in the claims, "a" or "an" can mean one or more. As used herein and in the claims, "a" or "an," when used in conjunction with the word "comprise," can mean one or more than one. As used herein and in the claims, "another" or "further" can mean at least a second or more.

[0024] As used in this specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among test subjects.

[0025] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, the detailed description and specific examples, while indicating certain aspects of the invention, are intended for purposes of illustration only, and 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. [The present invention 1001] A method of treating a subject having cancer, comprising administering TUSC2 therapy to the subject, wherein the subject has been treated or is currently being treated with at least one immune checkpoint inhibitor. [The present invention 1002] 1001. The method of claim 1001, further comprising administering to the subject at least one immune checkpoint inhibitor. [The present invention 1003] The method of claim 1002, wherein the step of administering at least one immune checkpoint inhibitor to the subject comprises administering at least one immune checkpoint inhibitor prior to TUSC2 therapy. [The present invention 1004] The method of claim 1002, wherein the step of administering at least one immune checkpoint inhibitor to the subject comprises administering at least one immune checkpoint inhibitor after or simultaneously with TUSC2 therapy. [The present invention 1005] The method of claim 1001, wherein the subject has been administered at least one immune checkpoint inhibitor within two weeks prior to TUSC2 therapy. [The present invention 1006] 1001. The method of claim 1001, wherein the at least one immune checkpoint inhibitor comprises an anti-PD1 agent. [The present invention 1007] 1006. The method of claim 10, wherein the anti-PD1 agent comprises an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-PDL2 antibody. [The present invention 1008] The method of claim 1006, wherein the anti-PD1 agent is nivolumab, pembrolizumab, pidilizumab, KEYTRUDA®, AMP-514, REGN2810, CT-011, BMS 936559, MPDL328OA, or AMP-224. [The present invention 1009] 1001. The method of claim 1001, wherein the at least one immune checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. [The present invention 1010] 1001. The method of claim 1001, wherein the at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody. [The present invention 1011] The method of claim 1010, wherein the anti-CTLA-4 antibody is tremelimumab, YERVOY®, or ipilimumab. [The present invention 1012] 1001. The method of claim 1001, wherein the at least one immune checkpoint inhibitor is an anti-killer cell immunoglobulin-like receptor (KIR) antibody. [The present invention 1013] The method of claim 1012, wherein the anti-KIR antibody is lirilumab. [The present invention 1014] The method of claim 1001, wherein the subject has been treated or is currently being treated with two immune checkpoint inhibitors. [The present invention 1015] The method of the present invention 1014, wherein the two immune checkpoint inhibitors are an anti-PD1 antibody and an anti-CTL4 antibody. [The present invention 1016] 1001. The method of claim 1001, wherein the TUSC2 therapy comprises administration of a TUSC2 expression vector. [The present invention 1017] The method of claim 1016, wherein the TUSC2 expression vector is a plasmid DNA. [The present invention 1018] 1017. The method of claim 1017, wherein the plasmid is pLJ143 / KGB2 / FUS1. [The present invention 1019] The method of claim 1016, wherein the TUSC2 expression vector is provided in a liposome. [The present invention 1020] 1019. The method of claim 1019, wherein the liposome is a DOTAP:cholesterol liposome. [The present invention 1021] The method of claim 1020, wherein the DOTAP:cholesterol ratio is about 1.5:1 to 1:1.5. [The present invention 1022] The method of claim 1020, wherein the DOTAP:cholesterol ratio is about 10:9. [The present invention 1023] The method of claim 1020, wherein the TUSC2 expression vector and DOTAP:cholesterol liposomes are administered at a dose of about 0.01 mg / kg to about 0.10 mg / kg. [The present invention 1024] The method of claim 1001, wherein the TUSC2 therapy is administered more than once. [The present invention 1025] 1001. The method of claim 1001, further comprising the step of administering an anti-inflammatory agent. [The present invention 1026] 1001. The method of claim 1001, wherein the TUSC2 therapy comprises administration of a TUSC2 polypeptide. [The present invention 1027] The method of claim 1026, wherein the TUSC2 polypeptide is myristoylated. [The present invention 1028] The method of claim 1026, wherein the TUSC2 polypeptide is contained in a nanoparticle. [The present invention 1029] The method of claim 1028, wherein the nanoparticle is a lipid-based nanoparticle, a superparamagnetic nanoparticle, a nanoshell, a semiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle, a metal-based nanoparticle, a fullerene, or a nanotube. [The present invention 1030] The method of claim 1001, further comprising the step of administering an additional anti-cancer treatment to the subject. [The present invention 1031] The method of claim 1030, wherein the additional anti-cancer treatment is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or cytokine therapy. [The present invention 1032] The method of claim 1031, wherein the further anti-cancer treatment is an EGFR inhibitor. [The present invention 1033] 1001. The method of claim 1001, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer, or blood cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1034] The method of claim 1033, wherein the cancer is lung cancer. [This invention 1035] The method of claim 1034, wherein the lung cancer is non-small cell lung cancer. [The present invention 1036] The method of claim 1034, wherein the cancer is metastatic lung cancer. [This invention 1037] 1001. The method of claim 1001, wherein the cancer is resistant to at least a first chemotherapy. [The present invention 1038] The method of claim 1037, wherein the cancer is resistant to platinum-based chemotherapy. [This invention 1039] Administering TUSC2 therapy and at least one immune checkpoint inhibitor may result in the development of NK cells and / or CD8 + The method of claim 1002, which results in an increase in T cell density. [The present invention 1040] CD8 + 1039. The method of claim 1039, wherein the T cell density is increased by at least three-fold. [This invention 1041] The method of claim 1002, wherein administering TUSC2 therapy and at least one immune checkpoint inhibitor results in elevated CcL3, CcL4, CcL21a, and / or CcL19 serum levels. [The present invention 1042] A kit comprising a TUSC2 therapeutic agent and an immune checkpoint inhibitor. [This invention 1043] The kit of the present invention 1042, wherein the immune checkpoint inhibitor is an anti-PD1 antibody. [This invention 1044] The method of claim 1032, wherein the EGFR inhibitor is a tyrosine kinase inhibitor. [This invention 1045] The method of claim 1032, wherein the EGFR inhibitor is an EGFR-binding antibody or aptamer. [The present invention 1046] 1032. The method of claim 1032, wherein the EGFR inhibitor is gefitinib, erlotinib, cetuximab, matuzumab, panitumumab, AEE788, CI-1033, HKI-272, HKI-357, or EKB-569. [This invention 1047] A composition comprising a TUSC2 therapeutic agent and an immune checkpoint inhibitor in therapeutically effective amounts to treat cancer. [Brief explanation of the drawings]

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1A]Combined treatment of TUSC2 and anti-PD1 in the CMT167 subcutaneous model demonstrated enhanced antitumor activity. (A) Sequential treatment strategy showing tumor inoculation, treatment schedule and dose, blood and spleen collection for immune cell analysis, tumor harvest for immunohistochemistry, and RNA isolation. (B) Surface expression levels of PD-L1 on CMT167-luc cells were determined by flow cytometry. (C), (D) Tumor growth curves for four different treatment groups (n=10 mice per group) were determined based on tumor volume and bioluminescence intensity obtained from small animal imaging with IVIS 200. The control group was treated with nanovesicles loaded with an empty (TUSC2 gene-less) vector. TUSC2+PD1 therapy resulted in the greatest inhibition of tumor growth, followed by TUSC2, PD1, and control. (E) Representative images of tumor-bearing mice from each treatment group with bioluminescence signals captured by IVIS 200 imaging. Data are representative of four independent experiments. Image intensities between treatment groups were compared using the CONTRAST statement in the PROC MIXED procedure of SAS. All analysis calculations were performed using SAS version 9.4 and S-Plus version 8.04. Unless otherwise stated, statistical values ​​are presented at a significance level of p<0.05. *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2A]The combination of TUSC2 and anti-PD1 up-regulates natural killer cells and cytotoxic T cells and down-regulates regulatory cells. TUSC2 + anti-PD1 treatment altered immune cell populations in peripheral blood and spleen. The control group was treated with nanovesicles loaded with an empty (TUSC2 gene-less) vector. (A) The effect of TUSC2 on NK, T, and B cells in tumor-free mice. Pooled samples from n=3 mice per group were used for flow cytometry analysis. In vivo uptake of TUSC2 nanovesicles was determined based on exogenous TUSC2 expression. 24 hours after intravenous injection of TUSC2 nanovesicles, four distinct immune populations (T cells, B cells, NK cells, and Lin-negative cells) were sorted from the spleen, and RT-PCR was performed to determine TUSC2 expression. (B) The effects of TUSC2 treatment and TUSC2 + anti-PD1 treatment on natural killer (NK) cells, T cells, and B cells at 2 weeks after tumor implantation. (C) TUSC2 treatment altered MDSC status. Monocytic and granulocytic MDSCs were determined using the following gating strategy: CD45+>CD3->MHCII low>CD11b+>Gr-1+. CD11b+ Gr-1 high were considered granulocytic MDSCs, and CD11b+ Gr-1 low were considered monocytic MDSCs. Data are shown as mean percentages ± SD, n = 5. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) The effect of treatment on Tregs in peripheral blood and spleen cells. CD4+CD25+ double-positive T lymphocyte populations were considered Tregs. Data are presented as mean percentages ± SD, n = 5; **, P < 0.01; *** P < 0.001. (E) Surface expression of PD1, CTLA4, and Tim-3 on T lymphocytes. Data are presented as mean percentages ± SD, n = 5; *, P < 0.05; **, P < 0.01; *** P < 0.001. (F) The effect of TUSC2 and anti-PD1 treatment is shown as the ratio of NK / MDSC cells and CD8 T effector / Treg cells among peripheral blood leukocytes. Data are presented as mean ± SD, n = 5.Statistical analysis of flow data was performed by the general linear regression model and the CONTRAST statement in the PROC GENMOD procedure of SAS. *, P<0.05; **, P<0.01; *** P<0.001. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 3A]Combining TUSC2 with anti-PD1 increased the infiltration of NK cells and CD8 T cells and prevented the infiltration of MDSCs and Tregs. (A) Subcutaneous tumors were treated with nanovesicles loaded with an empty (TUSC2 gene-less) vector (control), TUSC2 nanovesicles, anti-PD1, and TUSC2 + anti-PD1. Formalin-fixed resected tumors were immunostained with anti-CD8 and anti-NKp46 for activated NK cells, anti-Gr-1 for MDSCs, and anti-Foxp3 for Tregs. High-resolution images (20x) were captured using a Vectra automated imaging system, imaging 25% of the tumor area. N = 5 tumor sections were imaged per group. Approximately 100 images per treatment group were analyzed by InForm software for H-score calculation. A generalized linear regression model was used for statistical analysis of H-scores between treatment groups. A compound symmetric covariance structure was used to account for inter-mouse variability and repeated measurements in the data. H-scores were compared between each pair of treatment groups using the ESTIMATE statement in the PROC MIXED procedure in SAS. *, P<0.05; **, P<0.01; ***, P<0.001. (B) RNA was extracted from freshly resected tumors from each treatment group (n=3 tumors per treatment group), and chemokine gene expression was determined using NanoString technology. Data were normalized, and fold changes in expression were analyzed using nCounter analysis software. Fold changes were compared to control samples. Bars indicate the mean fold change (n=3). (C) Serum CCL4 and CCL5 chemokine levels induced by TUSC2 treatment are shown. Subcutaneous tumor-bearing mice were treated with TUSC2 according to the protocol described in the Methods section. Serum was collected 10 days after treatment, and Luminex multiplex ELISA was performed. Chemokines were compared between treatment groups using a linear regression model. Chemokines were compared between each pair of treatment groups using the ESTIMATE statement in the PROC MIXED procedure of SAS. Calculations for all analyses were performed using SAS version 9.4 and S-Plus version 8.04. Data: Mean ± SD; N = 3; ***, P < 0.001. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4A] The antitumor activity of TUSC2 depends on natural killer cells, which generate Th1-mediated immune responses. The cytokines IL-15 and IL-18 are involved in natural killer cell regulation. (A) NK cell depletion suppressed treatment efficacy and antitumor immune responses. A graph of tumor bioluminescence intensity shows the signal derived from tumors treated with TUSC2 and the combination in NK-depleted and non-depleted mice. NK1.1 antibody was injected five times every three days for NK cell depletion. The control group was treated with nanovesicles loaded with an empty (TUSC2 gene-less) vector. *, P<0.05; **, P<0.01. (B) The antitumor activity of TUSC2 + anti-PD1 treatment was affected by CD8 T cell depletion, as shown in the tumor intensity graph (n=5 mice / group). (C) Ten days after treatment, serum levels of IFN-γ and IL-4 cytokines were determined by Luminex assay in NK-depleted and non-depleted mice. Bars represent the ratio of IFN-γ (Th1) and IL-4 (Th2). Data are shown as mean ± SD, n=3. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. (D) Levels of IL-18 and IL-15 cytokines change after treatment in NK-depleted and non-depleted mice. Serum cytokines were measured using Luminex assay. Data are shown as mean pg / ml ± SD, n=3. *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001. (E) Fold expression of IL-15Ra and IL-18R1 in sorted NK cells from mice treated with TUSC2 compared to controls. Data are shown as mean ± SD, n=3; *, P<0.05, **, P<0.01, as determined by multiple t-test. (F) NanoString analysis of TUSC2- and TUSC2+PD1-treated tumors to compare fold change in IL-15Ra and IL-18R1 mRNA expression. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 5A]Combined treatment with TUSC2 and anti-PD1 significantly improved survival and recruited natural killer cells to tumor-bearing lungs in a mouse model of KRAS-mutated lung metastasis. (A) 344SQ-luc cells, carrying the KrasG12D allele and the Trp53R172HΔG allele, were used in this experimental metastasis model. PD-L1 expression levels were determined by flow cytometry and compared with those of CMT167-luc cells. (B) Sequential treatment with checkpoint blockade (anti-PD1 and anti-CTLA4) and TUSC2 is illustrated. (C) Kaplan-Meier curves show survival after treatment. 344SQ cells were intravenously injected, and mice were treated with TUSC2 and checkpoint blockade alone or in combination (indicated groups). A control group was treated with nanovesicles loaded with an empty (TUSC2 gene-less) vector, and survival time was recorded (n = 10 mice per group). A univariate Cox model was used for statistical analysis to compare overall survival between treatment groups. The highest survival rate was observed in the TUSC2+PD1 group (left), followed by TUSC2, PD1, and control. Similarly, the highest survival rate was observed in the TUSC2+PD1+CTL4 group (right), followed by TUSC2, PD1+CTLA4, and control. (D) Bioluminescence images of tumor-bearing mice captured by IVIS 200 demonstrated lung-specific colonization of tumor cells. Signal intensity levels are shown among treatment groups. A representative image from three independent experiments is shown. (E) Images of dissected lungs showing tumor nodule status 2 weeks after tumor implantation. (F) (G) (H) and (I) Single cells were prepared from metastatic lungs from different groups according to the protocol described in the Methods, and CD49b+ NK cells, CD4+CD25+ Tregs, Gr-1+ MDSCs, and PD-L-1 and PD-L2(+) leukocyte infiltration were determined by flow cytometry. Data were normalized based on per gram of tumor tissue. NK cells were gated from CD45+CD3-CD19-; MDSCs were gated as follows: CD45+>CD3->MHCII low>CD11b+>GR-1+. PD+CT indicates anti-PD1 and anti-CTLA4 treatment. Data are shown as gram of cells / tissue ± SD, n=5. *, P<0.05; **, P<0.01; ***, P<0.001.Flow data were compared for statistical analysis using the CONTRAST statement in the PROC GENMOD method of SAS. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 5G] See legend to Figure 5A. [Figure 5H] See legend to Figure 5A. [Figure 5I] See legend to Figure 5A. [Figure 6-1]Combination treatment with TUSC2 and anti-PD1 altered immune gene expression profiles in the tumor microenvironment. Gene expression analysis of the 776-gene NanoString pan-cancer immune panel was performed on 12 samples (n = 3 / treatment group) from four treatment groups (empty vector nanovesicles, TUSC2, anti-PD1, and combination). Data generated by the nCounter system were normalized before use to quantify gene profiles and statistical analysis. Positive controls, housekeeping genes, and negative controls were used to adjust for sample preparation variation, background noise, and RNA abundance variation. Linear models were used to assess overall treatment effects, and contrasts were used for pairwise comparisons of interest. A beta-uniform mixture (BUM) model was used to model resulting p-values, determine false discovery rate (FDR) cutoffs, and identify significantly differentially expressed genes. (A) Heatmap shows overall significant genes between treatment groups. 33 genes were significantly altered by treatment. (B) A pairwise comparison between TUSC2 + anti-PD1 and anti-PD1 identified another set of 13 genes. Volcano plots show the separation of genes up- and downregulated by treatment. Statistically significant genes are color-coded. (C) Selected genes known for their anti-tumor immune response were upregulated at least two-fold in the combination treatment group compared to single-agent treatment. (D), (E), and (F) show the fold change in CD8, INF-γ, and transcription factors (Tbx21, Gata3) expression with TUSC2, anti-PD1, and combination treatment compared to control, respectively. All gene expression data shown here was generated by NanoString technology. [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 7] A gating strategy for peripheral blood leukocytes and splenocytes was shown to determine immune subpopulations in multicolor flow cytometry assays. [Figure 8A]Effect of NK-depleting antibody (NK1.1) on other immune cells. Five intraperitoneal injections of NK1.1 were performed according to the protocol described in the methods. The effect of NK depletion was evaluated 3 days after the final injection. Splenocytes were analyzed by flow cytometry for T cells, B cells, and NK cells. (A) Gating strategy used in the analysis. (B) N=3 mouse samples were pooled together for CD45, CD3, CD19, and CD49b antibody staining; the percentage of each population is shown in a bar graph. A representative scatter plot shows the efficacy of NK depletion. [Figure 8B] See legend to Figure 8A. [Figure 9] Effect of CD8 T cell depletion on other immune cells. Five intraperitoneal injections of CD8 T cell-depleting antibodies were performed according to the protocol described in the Methods. The effect of NK cell depletion was evaluated 3 days after the final injection. Splenocytes were analyzed by flow cytometry for T cells, B cells, and NK cells. Representative scatter plots show the effectiveness of CD8 T cell depletion without affecting other cells. [Figure 10] NanoString gene expression analysis in the tumor microenvironment. Pairwise comparisons between PD1 and TUSC2+PD1 combination treatments revealed 13 significantly altered genes. P-values ​​and fold changes for all 13 genes are listed. A linear model was used to assess overall treatment effects, and contrasts were used to perform pairwise comparisons of interest. A beta uniform mixture (BUM) model was used to model the resulting p-values, determine false discovery rate (FDR) cutoffs, and identify significantly differentially expressed genes. DETAILED DESCRIPTION OF THE INVENTION

[0027] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Cancer development involves the deregulation of several cellular pathways that control normal cell growth. Healthy cells express several tumor suppressor genes, which function as molecular gatekeepers and prevent uncontrolled cell division. Therefore, a key step in cancer cell development is the disruption of tumor suppressor signaling pathways. In light of this, one promising avenue for cancer treatment involves the expression of tumor suppressor genes in cancer cells to restore normal cell growth control. However, to date, it is unknown which types function to enhance the efficacy of tumor suppressor therapies, such as TUSC2 therapy.

[0028] The research in this patent application demonstrates for the first time that TUSC2 therapy is particularly effective when administered in combination with immune checkpoint inhibitors. Immune checkpoint inhibitors, such as anti-PD1 therapy, work by enhancing an individual's own immune cells to inhibit tumor growth. In contrast, therapeutic treatment of tumors with tumor suppressors, such as TUSC2 therapy, aims to reverse the transformed phenotype of cancer cells. Because the latter therapy tends to render tumor cells "less transformed" and as immunogenic as possible, it has previously been counterintuitive to attempt to use TUSC2 therapy together with immune checkpoint inhibitors. Nevertheless, the research presented herein demonstrates that the antitumor effects of immune checkpoint inhibitors (e.g., anti-PD1 and / or CTLA4) are indeed significantly enhanced when the therapy is combined with TUSC2 therapy (see, e.g., Figure 1).

[0029] Specifically, in this study, anti-PD1 therapy showed limited efficacy in suppressing tumor growth and prolonging survival in syngeneic mouse models of lung adenocarcinoma with two Kras mutations, G12V and G12D, which had varying levels of PDL-1 expression. However, when combined with TUSC2 gene restoration, the effects on tumor regression and survival were much greater. TUSC2 altered both innate and adaptive immune cell populations. This was evidenced by a significant increase in circulating NK cells and CD8+ T cells, and a decrease in myeloid-derived suppressor cells (MDCs), regulatory T cells (Tregs), B cells, T cell checkpoint receptors PD1 and T lymphocyte-associated protein 4 (CTLA-4), and mucin domain-3 (TIM-3). Tumor-infiltrating NK cell and CD8+ T cell populations were induced by the TUSC2-anti-PD1 combination. In vivo depletion of NK cells or CD8+ T cells completely and partially attenuated the efficacy of this combination, respectively, suggesting that CD8+ T cells may contribute to TUSC2-enhanced sensitivity to anti-PD1 and that NK cells are required for this synergy. Cytokine levels of interferon gamma (IFNγ) and interleukin 15 and 18 (IL15 and IL18) significantly increased after TUSC2 restoration, which also enhanced survival with dual checkpoint blockade, anti-PD1 plus anti-CTLA-4. Gene expression profiling analysis demonstrated changes in the tumor microenvironment following TUSC2-anti-PD1 combination treatment. These data indicate that this novel combination therapy may be a potential strategy for treating Kras-mutated lung adenocarcinoma.

[0030] Thus, the methods detailed herein provide the first effective method for treating cancer through the combined use of an immune checkpoint inhibitor and a tumor suppressor (e.g., TUSC2 therapy).

[0031] I. Immune Checkpoint Blockade The term "immune checkpoint" refers to a component of the immune system that provides an inhibitory signal to that component to regulate 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. The pathway involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR is recognized in the art as constituting an immune checkpoint pathway, similar to the CTLA-4 and PD-1-dependent pathway (see, e.g., Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489).

[0032] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, with the result that T cell function is restored or enhanced (e.g., proliferation, cytokine production, target cell killing), resolving T cell dysfunction resulting from signaling to the PD-1 signaling axis. The term "PD-1 axis" refers to any component of the PD-1 immune checkpoint (e.g., PD-1, PD-L1, and PD-L2). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0033] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or prevents signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. A PD-1 binding antagonist may be a molecule that inhibits the binding of PD-1 with one or more of its binding partners. In certain aspects, a PD-1 binding antagonist inhibits the binding of PD-1 with PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or prevent signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. An exemplary PD-1 binding antagonist is an anti-PD-1 antibody. For example, the PD-1 binding antagonist is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), or AMP-224.

[0034] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or prevents signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. For example, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 with its binding partners. In certain aspects, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. PD-L1 binding antagonists can include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or prevent signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. For example, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediate signaling by PD-L1, such that dysfunction of dysfunctional T cells is reduced (e.g., enhancing the effector response to antigen recognition). In one example, the PD-L1 binding antagonist is an anti-PD-L1 antibody. The anti-PD-L1 antibody may be YW243.55.S70, MDX-1105, MPDL3280A, or MEDI4736.

[0035] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or prevents signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. A PD-L2 binding antagonist can be a molecule that inhibits the binding of PD-L2 with one or more of its binding partners. For example, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1, and such PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or prevent signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1.

[0036] "Immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduction and complete blockage of function. Specifically, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is specifically an inhibitor of a human immune checkpoint protein.

[0037] Therefore, the present disclosure provides a method for enhancing the effects of immune checkpoint blockade by administering a tumor suppressor, such as TUSC2 therapy. As described above, immune checkpoints either increase signals (e.g., costimulatory molecules) or decrease signals. Inhibitory immune 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 attenuator (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 V domain Ig suppressor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0038] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or antibodies such as human antibodies (see, e.g., International Patent Publication No. WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012, both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, and in particular, chimeric, humanized, or human forms of antibodies may be used. Those skilled in the art will understand that alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable within the context of the present invention. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0039] It is contemplated that any immune checkpoint inhibitor known in the art to stimulate immune responses can be used. This includes inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. These immune checkpoint inhibitors include, but are not limited to, agents that target immune checkpoint proteins and pathways, including 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, incorporated herein by reference), and mouse or humanized antibodies that block human LAG3 (such as IMP701, disclosed in WO2008132601, incorporated herein by reference), or fully human antibodies that block human LAG3 (such as those disclosed in EP 2320940, incorporated herein by reference). Another example is provided by the use of blocking agents against BTLA, including, but not limited to, antibodies that block the interaction of human BTLA with its ligands (such as 4C7, disclosed in WO2011014438, incorporated herein by reference). Yet another example is provided by the use of agents that neutralize B7H4, including, but not limited to, antibodies against human B7H4 (such as those disclosed in WO2013025779 and WO2013067492, each of which is incorporated herein by reference), or soluble recombinant forms of B7H4 (such as those disclosed in US20120177645, incorporated herein by reference). Yet another example is provided by agents that neutralize B7-H3, including, but not limited to, antibodies that neutralize human B7-H3 (e.g., MGA271, disclosed as BRCA84D and derivatives in US20120294796, incorporated herein by reference).Yet another example is provided by agents that target TIM3, including, but not limited to, antibodies that target human TIM3 (e.g., antibodies such as those disclosed in WO 2013006490 A2, the anti-human TIM3 blocking antibody F38-2E2 disclosed by Jones et al., J Exp Med. 2008;205(12):2763-79, each of which is incorporated herein by reference).

[0040] A. PD-1 axis antagonists T cell dysfunction or anergy is accompanied by the inducible and persistent expression of the inhibitory receptor, programmed cell death 1 polypeptide (PD-1). Therefore, therapeutic targeting of PD-1 and other molecules that signal through interaction with PD-1, such as programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2), is provided herein. PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813). Therefore, an improved method for treating cancer is provided by inhibiting PD-L1 / PD-1 interaction in combination with the administration of tumor suppressors, such as TUSC2 therapy.

[0041] 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.

[0042] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0043] 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 the extracellular portion or PD-1-binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab is an anti-PD-1 antibody described in WO2006 / 121168, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®. Pembrolizumab is an anti-PD-1 antibody described in WO2009 / 114335, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475. CT-011 is an anti-PD-1 antibody described in WO2009 / 101611, also known as hBAT or hBAT-1. AMP-224 is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342, also known as B7-DCIg. Additional PD-1 binding antagonists include pidilizumab, also known as CT-011, MEDI0680, also known as AMP-514, and REGN2810.

[0044] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, or avelumab, also known as MSB00010118C. In some aspects, the immune checkpoint inhibitor is a PD-L2 antagonist, such as rHIgM12B7. In some aspects, the immune checkpoint inhibitor is a LAG-3 antagonist, such as, but not limited to, IMP321 and BMS-986016. The immune checkpoint inhibitor may also be an adenosine A2a receptor (A2aR) antagonist, such as PBF-509.

[0045] In some embodiments, an antibody described herein (such as an anti-PD-1 antibody, anti-PDL1 antibody, or anti-PDL2 antibody) further comprises a human or mouse constant region. In even further aspects, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In even further particular aspects, the human constant region is IgG1. In even further particular aspects, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In even further particular aspects, the antibody has reduced or minimal effector function. In even further particular aspects, minimal effector function is achieved by production in prokaryotic cells. In even further particular aspects, minimal effector function is achieved by an "effector-less Fc mutation" or aglycosylation.

[0046] Thus, antibodies used herein may be non-glycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a 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 hydroxylated amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Removal of glycosylation sites from an antibody is conveniently accomplished by altering the amino acid sequence to remove one of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may be made by substitution of an asparagine, serine, or threonine residue within a glycosylation site with another amino acid residue (eg, glycine, alanine, or a conservative substitution).

[0047] Antibodies or antigen-binding fragments thereof may be produced using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the aforementioned anti-PDL1, anti-PD-1, or anti-PDL2 antibodies, or antigen-binding fragments, in a form suitable for expression, under conditions suitable for producing such antibodies or fragments, and recovering the antibodies or fragments.

[0048] B. CTLA-4 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and functions as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell costimulatory protein CD28; both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0049] 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), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligonupeptide.

[0050] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in 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 can be used in the method disclosed herein. The teachings of each of the above-mentioned publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications WO2001014424, WO2000037504, and US Pat. No. US8017114, all of which are incorporated herein by reference.

[0051] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-described antibody (eg, at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0052] Other molecules for modulating CTLA-4 include soluble CTLA-4 ligands and receptors such as those described in U.S. Pat. Nos. US5844905, US5885796, and International Patent Applications WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesins such as those described in U.S. Pat. No. US8329867, which is incorporated herein by reference.

[0053] C. Killer Immunoglobulin-Like Receptors (KIRs) Another immune checkpoint inhibitor for use in the present disclosure is an anti-KIR antibody. Anti-human KIR antibodies (or VH / VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.

[0054] Alternatively, art-recognized anti-KIR antibodies can be used. Anti-KIR antibodies can be cross-reactive with multiple inhibitory KIR receptors, enhancing the cytotoxicity of NK cells bearing one or more of these receptors. For example, an anti-KIR antibody can bind to each of KIR2D2DL1, KIR2DL2, and KIR2DL3, thereby enhancing 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, the anti-KIR antibody does not bind to KIR2DS4 and / or KIR2DS3. For example, monoclonal antibodies 1-7F9 (also known as IPH2101), 14F1, 1-6F1, and 1-6F5, described in WO 2006 / 003179, the teachings of which are incorporated herein by reference, can be used. Antibodies that compete with any of these art-recognized antibodies for binding to KIR can also be used. Additional art-recognized anti-KIR antibodies that can 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, all of which are incorporated herein by reference.

[0055] An exemplary anti-KIR antibody is lirilumab (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 lirilumab. Thus, in one embodiment, the antibody comprises the CDR1 domain, CDR2 domain, and CDR3 domain of the heavy chain variable (VH) region of lirilumab, and the CDR1 domain, CDR2 domain, and CDR3 domain of the light chain variable (VL) region of lirilumab. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with lirilumab.

[0056] II. Tumor suppressor therapy In some aspects, the present invention relates to compositions and methods for delivering nucleic acids or polypeptides to cells. Specifically, the present invention provides nanoparticle-nucleic acid complexes or nanoparticle-polypeptide complexes, and methods for administering such complexes to subjects. The complexes comprise TUSC2 polypeptides and / or nucleic acids associated with nanoparticles. As used herein, "association" refers to physical association, chemical association, or both. For example, association can include covalent bonding, hydrophobic interaction, encapsulation, or surface adsorption.

[0057] Polypeptides and nucleic acids typically have difficulty crossing cell membranes. Both types of molecules contain charged residues that hinder membrane binding and transport into cells. The present embodiment overcomes this difficulty by providing nanoparticle conjugates that facilitate cellular uptake.

[0058] According to this embodiment, polypeptide and / or nucleic acid can be associated with nanoparticle to form nanoparticle complex.In some embodiments, nanoparticle is liposome or other lipid-based nanoparticle, for example, lipid-based vesicle (for example, DOTAP: cholesterol vesicle).Liposome used in cancer treatment utilizes the increased fenestrations in cancer neovasculature to enhance liposome concentration at tumor site.

[0059] In other embodiments, the nanoparticles are nonlipid nanoparticles, such as iron oxide-based superparamagnetic nanoparticles. Superparamagnetic nanoparticles, ranging in diameter from approximately 10 to 100 nm, are small enough to avoid splenic sequestration and large enough to avoid clearance by the liver. Particles of this size can penetrate very small capillaries and distribute efficiently throughout body tissues. Superparamagnetic nanoparticle conjugates can be used as MRI contrast agents to identify and track cells that take up the therapeutic conjugate. In some embodiments, the nanoparticles are semiconductor nanocrystals or semiconductor quantum dots, both of which can be used in optical imaging. In further embodiments, the nanoparticles can be nanoshells, comprising a gold layer over a silica core. One advantage of nanoshells is that polypeptides or nucleic acids can be conjugated to the gold layer using standard chemistry. In other embodiments, the nanoparticles can be fullerenes or nanotubes (Gupta et al., 2005).

[0060] According to this embodiment, the nanoparticle conjugate can target specific tissues and cells. This can be achieved by conjugating a cell-targeting moiety to the nanoparticle. The targeting moiety can be, but is not limited to, a protein, peptide, lipid, steroid, sugar, carbohydrate, or synthetic compound. A cell-targeting moiety, such as a ligand, recognizes and binds to its cognate receptor on the surface of the cell. Similarly, an antibody can function as a cell-targeting moiety by recognizing its cognate antigen on the cell surface. In some embodiments, the targeted nanoparticle conjugates provided herein can enhance the specificity of disease treatment and increase the amount of therapeutic agent that enters the target cell.

[0061] A. Nanoparticles As used herein, the term "nanoparticle" refers to any substance having a dimension in the range of 1 to 1,000 nm. In some embodiments, the nanoparticle has a dimension in the range of 50 to 500 nm. Nanoparticles used in this embodiment include nanoscale substances such as lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes (Ferrari, 2005). Conjugation of polypeptides or nucleic acids to nanoparticles provides structures with potential applications in targeted delivery, controlled release, enhanced cellular uptake and trafficking, and molecular imaging of therapeutic peptides in vitro and in vivo (West, 2004; Stayton et al., 2000; Ballou et al., 2004; Frangioni, 2003; Dubertret et al., 2002; Michalet et al., 2005; Dwarakanath et al., 2004).

[0062] 1. Lipid-based nanoparticles Lipid-based nanoparticles include liposomes, lipid preparations, and lipid-based vesicles (e.g., DOTAP:cholesterol vesicles). Lipid-based nanoparticles can be positively charged, negatively charged, or neutral. In some embodiments, lipid-based nanoparticles are neutrally charged (e.g., DOPC liposomes).

[0063] "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the creation of an enclosed lipid bilayer or aggregate. Liposomes can be characterized as having a vesicular structure with a bilayer generally comprising phospholipids and an internal medium generally comprising an aqueous composition. The liposomes provided herein include unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. The liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In some embodiments, the liposomes are neutrally charged.

[0064] Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when lipids, including phospholipids, are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Lipophilic molecules, or molecules with lipophilic regions, can also be dissolved in or associated with the lipid bilayer.

[0065] In certain aspects, the polypeptide or nucleic acid may be, for example, encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the polypeptide / nucleic acid, encapsulated within the liposome, or complexed with the liposome, etc.

[0066] Liposomes used in this embodiment can be prepared by various methods known to those skilled in the art. For example, phospholipids such as the neutral phospholipid dioleoylphosphatidylcholine (DOPC) (Avanti Polar Lipids, Alabaster, AL) are dissolved in tert-butanol. The lipids are then mixed with polypeptides, nucleic acids, and / or other components. Tween 20 is added to the lipid mixture to approximately 5% of the composition's weight. Excess tert-butanol is added to this mixture so that the volume of tert-butanol is at least 95%. The mixture is vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparation can be stored at -20°C and used for up to 3 months. When needed, the lyophilized liposomes are reconstituted with 0.9% saline.

[0067] Alternatively, liposomes can be prepared by mixing lipids with a solvent in a container, such as a glass pear-shaped flask. The container should have a volume greater than 10 times the volume of the expected liposome suspension. The solvent is removed using a rotary evaporator under negative pressure at approximately 40°C. The solvent is usually removed within about 5 minutes to 2 hours, depending on the desired liposome volume. The composition can be further dried in a desiccator under vacuum. Dried lipids are generally discarded after about one week due to their tendency to deteriorate over time.

[0068] The dried lipids can be hydrated to approximately 25-50 mM phospholipid with sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous liposomes can then be divided into aliquots, each placed in a vial, lyophilized, and sealed under vacuum.

[0069] The dried lipids or lyophilized liposomes prepared as described above can be hydrated or reconstituted with a protein or peptide solution and diluted to an appropriate concentration with a suitable solvent, such as DPBS. The mixture is then vigorously shaken in a vortex mixer. Any unencapsulated additional substances, including but not limited to hormones, drugs, and nucleic acid constructs, are removed by centrifugation at 29,000 x g, and the liposome pellet is washed. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., approximately 50-200 mM. The amount of encapsulated additional substance or active agent can be determined by standard methods. After determining the amount of additional substance or active agent encapsulated in the liposome preparation, the liposomes can be diluted to an appropriate concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically contain a sterile pharmaceutically acceptable carrier or diluent, such as water or saline solution.

[0070] In other alternative methods, liposomes can be prepared according to other known experimental methods (see, for example, Bangham et al., 1965; Gregoriadis, 1979; Deamer and Uster, 1983; Szoka and Papahadjopoulos, 1978, each of which is incorporated herein by reference in relevant parts).Additional liposomes that can be useful in this embodiment include, for example, cationic liposomes as described in WO 02 / 100435A1, U.S. Patent No. 5,962,016, U.S. Patent Application No. 2004 / 0208921, WO 03 / 015757A1, WO 04029213A2, U.S. Patent No. 5,030,453, and U.S. Patent No. 6,680,068, all of which are incorporated herein by reference in their entirety without any proviso. The process of making liposomes is also described in WO 04 / 002453A1. Neutral lipids can be incorporated into cationic liposomes (e.g., Farhood et al., 1995). Various neutral liposomes that can be used in some embodiments are disclosed in U.S. Patent No. 5,855,911, which is incorporated herein by reference. These methods differ in their respective capacities to capture aqueous substances and their respective aqueous space-to-lipid ratios.

[0071] The size of liposomes varies depending on the method of synthesis. Liposomes in this embodiment can be of various sizes. In some embodiments, the liposomes are small, e.g., less than about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or about 50 nm in outer diameter. For example, generally, prior to nucleic acid uptake, DOTAP:cholesterol liposomes for use in this embodiment comprise a size of about 50-500 nm. Such liposome formulations may also be defined by particle charge (zeta potential) and / or optical density (OD). For example, DOTAP:cholesterol liposome formulations typically have an OD of less than 0.45 prior to nucleic acid uptake. 400Similarly, the overall charge of such particles in solution can be defined by a zeta potential of approximately 50-80 mV.

[0072] In preparing such liposomes, any of the protocols described herein or known to those skilled in the art may be used. Further non-limiting examples of preparing liposomes are described in U.S. Pat. Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; International Patent Applications PCT / US85 / 01161 and PCT / US89 / 05040; UK Patent Application GB 2193095A; Mayer et al., 1986; Hope et al., 1985; Mayhew et al., 1987; Mayhew et al., 1984; Cheng et al., 1987; and Liposome Technology, 1984, each of which is incorporated herein by reference.

[0073] In some embodiments, the lipid-based nanoparticle is a neutral liposome (e.g., DOPC liposome). As used herein, "neutral liposome" or "uncharged liposome" is defined as a liposome having one or more lipid components that result in an essentially neutral net charge (substantially uncharged). "Essentially neutral" or "essentially uncharged" means that only a small fraction, if any, of the lipid components in a given population (e.g., a population of liposomes) contain a charge that is not counteracted by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1% of the components contain a charge that is not counteracted). In some embodiments, neutral liposomes may primarily contain lipids and / or phospholipids that are themselves neutral under physiological conditions (i.e., at about pH 7).

[0074] The liposomes and / or lipid-based nanoparticles of this embodiment may contain phospholipids. In some embodiments, a single type of phospholipid may be used to prepare the liposomes (e.g., a neutral phospholipid such as DOPC may be used to prepare a neutral liposome). In other embodiments, two or more types of phospholipids may be used to prepare the liposomes.

[0075] Phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine, because phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at about pH 7), and these compounds may be particularly useful for producing neutral liposomes.In some embodiments, phospholipid DOPC is used to produce uncharged liposomes.In some embodiments, lipids other than phospholipids (e.g., cholesterol) may also be used.

[0076] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoylphosphatidylcholine ("MPPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoylphosphatidylcholine ("MPPC"), 1-palmitoylphosphatidylcholine ("DP ... 1-Stearoyl-2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol dioleoylphosphatidylglycerol ("DOPG"), distearoyl sphingomyelin ("DSSP"), distearoyl phosphatidylethanolamine ("DSPE"), dioleoyl phosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl dipalmitoylphosphatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoylsphingomyelin ("DPSP"), dimyristylphosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyl eoylphosphatidylcholine ("POPC"), palmitoyl eoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0077] Phospholipid can be derived from natural or synthetic sources.However, the phospholipid from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin, and plant or bacterial phosphatidylethanolamine, may cause the resulting liposome to be unstable and prone to leak, so in some embodiments, it is not used as the main phosphatide (i.e., it constitutes more than 50% of the total phosphatide composition).

[0078] 2. DOTAP: Cholesterol Nanoparticles In some embodiments, lipid-based vesicle is DOTAP:cholesterol nanoparticle.DOTAP:cholesterol nanoparticle is prepared by mixing cationic lipid DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane) with cholesterol.The vesicle prepared with DNA can form a structure (called "sandwich") in which DNA appears to be condensed between two lipid bilayers (US Pat. No. 6,770,291 and US Pat. No. 6,413,544).

[0079] DOTAP:cholesterol-nucleic acid complexes can be prepared as follows, non-limitingly: DOTAP:cholesterol (DC) nanoparticles (50-500 nm in size) are synthesized as previously described (U.S. Patent Nos. 6,770,291 and 6,413,544; Templeton, 1997). Briefly, 420 mg of DOTAP and 208 mg of cholesterol are measured and mixed with 30 ml of chloroform. The mixture is then dried on a rotary evaporator for 30 minutes and lyophilized for 15 minutes. The dried mixture is reconstituted in 30 ml of D5W by swirling at 50°C for 45 minutes and at 37°C for 10 minutes. The mixture is then subjected to low-frequency sonication for 5 minutes to form liposomes. The DOTAP:cholesterol liposomes are then heated to 50°C and filtered sequentially through 1.0, 0.45, 0.2, and 0.1 μm sterile Whatman filters. The synthesized nanoparticles are stored at 4 °C and used to prepare nanoparticle complexes. The formulated DOTAP:cholesterol liposomes have a particle size of 50-250 nm and an OD of less than 0.45. 400 , and should be uniformly dispersed with a zeta potential of 50-80 mV. Residual CHCl3 levels should be less than 60 ppm.

[0080] To prepare DOTAP:cholesterol-nucleic acid nanoparticles, dilute 240 μl of liposomes (see above) with 360 μl D5W at room temperature. Add DNA (approximately 5 mg / ml) to the mixture up to a total volume of 600 μl. Pipette the mixture up and down to mix. Once settled, the mixture should reach an OD of 0.65-0.95. 400 The liposome complex should have a particle size of 200-500 nm and be confirmed as Gram-stain negative. The liposome complex should be stored at 3°C-28°C and agitated as little as possible.

[0081] B. Nanoparticle Targeting Targeted delivery is achieved by adding a ligand without impairing the nanoparticle's ability to deliver its payload. This is intended to enable delivery to specific cells, tissues, and organs. The target specificity of ligand-based delivery systems is based on the distribution of ligand receptors on various cell types. Targeting ligands may be non-covalently or covalently associated with nanoparticles and can be conjugated to nanoparticles by various methods, as described herein.

[0082] Examples of proteins or peptides that can be used to target nanoparticles include transferrin, lactoferrin, TGF-α, nerve growth factor, albumin, HIV Tat peptide, RGD peptide, and insulin (Gupta et al., 2005; Ferrari, 2005).

[0083] C. TUSC2 expression vector The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where the nucleic acid sequence can be replicated. The nucleic acid sequence can be "exogenous," meaning that the nucleic acid sequence is foreign to the cell into which the vector is inserted, or that the sequence is homologous to a sequence in the cell but is located in a location within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art are fully capable of constructing vectors using standard recombinant techniques (see, for example, Maniatis et al., 1989 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0084] The term "expression vector" refers to any type of genetic construct containing a nucleic acid encoding an RNA capable of being transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences required for the transcription, and possibly translation, of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions and are described below.

[0085] In some embodiments, the use of nucleic acid TUSC2-encoding sequences is provided herein. For example, such vectors can be used for recombinant production of TUSC2 polypeptides and / or for in vivo expression of TUSC2 in a subject. The sequence can be modified to allow for a single amino acid to be coded for by multiple different codons while still encoding the same protein or polypeptide. Optimization of codon selection can also be performed in light of the specific organism used in recombinant expression, or can be optimized for maximum expression in human cells (e.g., cancer cells). Vectors for use in this embodiment further include elements that control gene expression and / or aid in vector production and purification.

[0086] 1. Promoters and enhancers A "promoter" is a regulatory sequence, a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of a nucleic acid sequence. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" refer to the promoter being in the correct functional location and / or orientation relative to a nucleic acid sequence to control the initiation and / or expression of the transcription of that sequence.

[0087] Promoters generally contain sequences that function to position the start point of RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoters for mammalian terminal deoxynucleotidyl transferase genes and the SV40 late genes, separate elements surrounding the start point itself help fix the position of initiation. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start point, but some promoters have been shown to contain functional elements downstream of the start point as well. To bring a coding sequence "under the control" of a promoter, the 5' end of the transcriptional reading frame's transcription start site is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates DNA transcription and promotes expression of the encoding RNA.

[0088] The spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. It is clear that, depending on the promoter, individual elements can function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0089] A promoter may be a promoter naturally associated with a nucleic acid sequence, such as may be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon. Such promoters may be referred to as "endogenous" or "homologous." Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the nucleic acid sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant exogenous or heterologous promoter, which refers to a promoter not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include viral promoters and enhancers, such as the CMV promoter.

[0090] Naturally, it is important to utilize a promoter and / or enhancer that effectively directs the expression of a DNA segment in the organelle, cell, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology generally understand the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter utilized may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, such as those advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0091] Additionally, any promoter / enhancer combination (such as, for example, the Eukaryotic Promoter Database EPDB, www.epd.isb-sib.ch / ) 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 an appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0092] 2. Translation initiation signal Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may also need to be provided. One of ordinary skill in the art can readily determine this and provide the necessary signals. It is well known that the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0093] 3. Multiple cloning sites A vector can include a multiple cloning site (MCS), a nucleic acid region containing multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant techniques to digest the vector (see, e.g., Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, which are incorporated herein by reference). "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific locations in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, vectors are linearized or fragmented using a restriction enzyme that cuts within the MCS, allowing exogenous sequences to be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments that may or may not be adjacent to each other. Restriction enzymes and the techniques involved in ligation reactions are well known to those skilled in the art of recombinant technology.

[0094] 4. Splicing Sites Many transcribed eukaryotic RNA molecules undergo RNA splicing, removing introns from the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure proper processing of the transcript for protein expression (see, e.g., Chandler et al., 1997, incorporated herein by reference). Inclusion of such splice sites can also enhance expression by preventing nonsense-mediated decay of the resulting RNA transcript.

[0095] 5. Termination Signal The vector or construct of this embodiment generally contains at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in some embodiments, a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve a desired message level.

[0096] Terminators contemplated for use in this embodiment include any known transcription terminator described herein or known to one of skill in the art, including, but not limited to, a gene termination sequence, e.g., the bovine growth hormone terminator, or a viral termination sequence, e.g., the SV40 terminator, etc. In some embodiments, the termination signal may lack transcribable or translatable sequence, such as due to sequence truncation.

[0097] 6. Polyadenylation Signal In expression, particularly in eukaryotic expression, a polyadenylation signal is typically included to ensure proper polyadenylation of the transcript. The nature of the polyadenylation signal is not considered critical to the successful implementation of this embodiment, and any such sequence may be utilized. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, which are convenient and known to function satisfactorily in a variety of target cells. Polyadenylation may increase the stability of the transcript or facilitate cytoplasmic transport.

[0098] 7. Origin of replication To propagate a vector in a host cell, it may contain one or more origin of replication sites (often called "ori"), which are specific nucleic acid sequences at which replication is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0099] 8. Selectable and Screenable Markers In some embodiments, cells containing the nucleic acid constructs provided herein can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers a identifiable change to the cell, allowing for easy identification of cells containing the expression vector. Generally, a selectable marker is a marker that confers a selectable characteristic. A selectable positive marker is a marker whose presence allows its selection, and a selectable negative marker is a marker whose presence prevents its selection. An example of a selectable positive marker is a drug resistance marker.

[0100] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers. In addition to markers that confer phenotypes that allow for the identification of transformants based on the implementation of conditions, other types of markers are also contemplated, including screenable markers, such as GFP, whose basis is colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art will also know how to utilize immunomarkers, perhaps in conjunction with FACS analysis. The marker used is not believed to be critical, so long as it is capable of being expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0101] 9. Plasmid Vectors In some embodiments, a plasmid vector is intended for use in transforming a host cell. Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector usually carries a replication site and marking sequences capable of providing phenotypic selection in transformed cells. In a non-limiting example, Escherichia coli (E. coli) is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance, thus providing an easy means for identifying transformed cells. pBR plasmids, or other microbial plasmids or phages, must also contain, or be modified to contain, promoters that can be used by the microorganism, for example, for expression of its own proteins.

[0102] In addition, phage vectors containing replicon and control sequences compatible with host microorganisms can also be used as transforming vectors in connection with these hosts. For example, phage λGEM™-11 can be utilized in generating recombinant phage vectors that can be used to transform host cells such as, for example, E. coli LE392.

[0103] Additional useful plasmid vectors include the pIN vector (Inouye et al., 1985) and the pGEX vector, for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins include those with β-galactosidase, ubiquitin, and the like.

[0104] Bacterial host cells, e.g., E. coli, containing the expression vector are grown in any of several suitable media, e.g., LB. Expression of the recombinant protein in a particular vector can be induced by contacting the host cells with an agent specific for a particular promoter, as will be understood by those skilled in the art, for example, by adding IPTG to the medium or by switching the incubation to a higher temperature. After culturing the bacteria for an additional period, generally 2 to 24 hours, the cells are harvested by centrifugation and washed to remove residual medium.

[0105] 10. Viral Vectors 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).Therefore, viruses encoding and expressing TUSC2 may be utilized.Non-limiting examples of viral vectors that can be used to deliver TUSC2 nucleic acids are described below.

[0106] Adenoviral Vectors. A particular method for nucleic acid delivery involves the use of adenoviral expression vectors. Adenoviral vectors are known to have a low ability to integrate into genomic DNA, but this feature is offset by the high efficiency of gene transfer afforded by these vectors. "Adenoviral expression vector" is meant to include constructs containing sufficient adenoviral sequences to (a) support packaging of the construct and (b) ultimately express tissue- or cell-specific constructs cloned therein. Knowledge of the genetic organization, or adenovirus, a 36 kb linear, double-stranded DNA virus, allows for the replacement of large segments of adenoviral DNA with foreign sequences up to 7 kb in size (Grunhaus and Horwitz, 1992).

[0107] AAV vector. Nucleic acid may be introduced into cells using adenovirus-mediated transfection. Increased transfection efficiency has been reported in cell lines using adenovirus conjugation systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) has a high integration rate and can infect non-dividing cells, making it useful for gene delivery into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a wide host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details regarding the production and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0108] Retroviral Vectors. Retroviruses have the ability to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide spectrum of species and cell types, and be packaged in specialized cell lines (Miller, 1992). To construct a retroviral vector, a nucleic acid (e.g., a nucleic acid encoding a protein of interest) is inserted into the viral genome in place of a specific viral sequence, resulting in a virus that is replication-deficient. To produce virions, a packaging cell line is constructed that contains the gag, pol, and env genes but lacks the LTRs and packaging components (Mann et al., 1983). The recombinant plasmid containing the cDNA together with the retroviral LTR and packaging sequence is introduced into a special cell line (for example, by calcium phosphate precipitation), and 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 Rubinstein, 1988; Temin, 1986; Mann et al., 1983).The 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 the division of host cells (Paskind et al., 1975).

[0109] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2), and simian immunodeficiency viruses (SIV). Lentiviral vectors have been created by multiple attenuation of HIV pathogenic genes, for example, deleting the genes env, vif, vpr, vpu, and nef, making the vector biologically safe.

[0110] Other viral vectors. Other viral vectors may be used as vaccine constructs in this embodiment. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus may be used. They offer multiple attractive features in a variety of mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0111] Modified virus. The nucleic acid to be delivered may be contained within an infectious virus that has been engineered to express a specific binding ligand. Thus, the viral particle specifically binds to the cognate receptor of the target cell and delivers its contents to the cell. A novel approach designed to enable specific targeting of retroviral vectors has been developed based on chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification allows specific infection of hepatocytes via sialoglycoprotein receptors.

[0112] Another approach to targeting recombinant retroviruses was designed, in which biotinylated antibodies against retroviral envelope proteins and specific cellular receptors were used. The antibodies were conjugated via the biotin moiety using streptavidin (Roux et al., 1989). Antigens against major histocompatibility complex class I and class II antigens were used to demonstrate in vitro infection of a variety of human cells bearing the surface antigens of ecotropic viruses (Roux et al., 1989).

[0113] III. Pharmaceutical Preparations The pharmaceutical compositions provided herein comprise an effective amount of one or more TUSC2 therapeutic agents and / or immune checkpoint inhibitors, and optionally additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse drug reactions, allergic reactions, or other untoward reactions when administered to an animal, such as a human, as appropriate. The preparation of pharmaceutical compositions containing at least a TUSC2 nucleic acid, peptide, or nanoparticle complex, or additional active ingredients, is known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0114] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and any of the like substances and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0115] In certain embodiments, the pharmaceutical composition may contain various types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether the route of administration, such as injection, requires sterility. In certain embodiments, the pharmaceutical compositions provided herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion directly bathing target cells, via a catheter, via irrigation, in a cream, in a lipid composition (e.g., liposomes), or by other methods known to those skilled in the art or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0116] In some embodiments, the pharmaceutical composition is administered intraperitoneally. In further embodiments, the pharmaceutical composition is administered intraperitoneally to treat cancer (e.g., cancerous tumor). For example, the pharmaceutical composition may be administered intraperitoneally to treat gastrointestinal cancer. In some embodiments, it may be desirable to administer the pharmaceutical composition into or near the tumor.

[0117] In certain preferred embodiments, the pharmaceutical compositions are administered orally to treat cancer (eg, gastrointestinal cancer).

[0118] In certain embodiments, the actual dosage of a composition administered to a patient can be determined by physical and physiological factors, such as body weight, severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in the composition and appropriate dose for the individual subject.

[0119] In certain embodiments, pharmaceutical compositions may contain, for example, at least about 0.1% of the active compound, while in other embodiments the active compound may comprise from about 2% to about 75% of the weight of the unit, or from about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, dosages may also include about 1 microgram / kg / body weight, about 5 micrograms / kg / body weight, about 10 micrograms / kg / body weight, about 15 micrograms / kg / body weight, about 20 micrograms / kg / body weight, about 25 micrograms / kg / body weight, about 30 micrograms / kg / body weight, about 35 micrograms / kg / body weight, about 0.04 milligrams / kg / body weight, about 0.05 milligrams / kg / body weight, about 0.06 milligrams / kg / body weight, about 0.07 milligrams / kg / body weight, about 0.08 milligrams / kg / body weight, about 0.09 milligrams / kg / body weight, about 0.1 milligrams / kg / body weight, about 0.2 milligrams / kg / body weight to about 0.5 mg / kg / body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derived from the numbers recited herein, ranges such as about 0.01 mg / kg / body weight to about 0.1 mg / kg / body weight, about 0.04 micrograms / kg / body weight to about 0.08 milligrams / kg / body weight, etc., can be administered based on the above numerical values.

[0120] In any case, the compositions may include various antioxidants to retard the oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0121] One or more peptides, nanoparticle complexes, or additional agents may be formulated in the composition in free base form, neutral form, or salt form.Pharmaceutically acceptable salts include acid addition salts, such as those formed by the free amino group of proteinaceous compositions, or those formed by inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid.The salt formed by free carboxyl group can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0122] In an embodiment where the composition is in liquid form, the carrier can be a solvent or dispersion medium, including, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipid (e.g., triglyceride, vegetable oil, liposome), and combinations thereof. The appropriate fluidity can be maintained, for example, by using a coating such as lecithin; by maintaining the required particle size by dispersing in a carrier such as liquid polyol or lipid; by using a surfactant such as hydroxypropyl cellulose; or by a combination of such methods. In many cases, it is preferable to include an isotonic agent, for example, sugar, sodium chloride, or a combination thereof.

[0123] In other embodiments, eye drops, nasal drops or sprays, aerosols, or inhalants may be used in this embodiment. Such compositions are generally designed to be compatible with the type of target tissue. In a non-limiting example, nasal solutions are typically aqueous solutions designed to be administered to the nasal cavity as drops or sprays. Nasal solutions are formulated to resemble nasal secretions in many respects, so that normal ciliary action is maintained. Thus, in preferred embodiments, aqueous nasal solutions are typically isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives similar to those used in ophthalmic preparations and drugs, or appropriate drug stabilizers, may be included in the formulation, if necessary. For example, a variety of commercially available nasal solution preparations are known, including drugs such as antibiotics or antihistamines.

[0124] In some embodiments, one or more polypeptides, nucleic acids, or nanoparticle complexes are prepared for administration by routes such as oral ingestion. In these embodiments, solid compositions may include, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft-shell gelatin capsules), sustained-release formulations, buccal compositions, lozenges, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be taken directly with food during the meal. Preferred carriers for oral administration include inert diluents, assimilable edible carriers, or combinations thereof. In other aspects, oral compositions may be prepared as syrups or elixirs. Syrups or elixirs may, for example, contain at least one active agent, a sweetener, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

[0125] In some preferred embodiments, oral compositions may contain one or more binders, excipients, disintegrants, lubricants, flavoring agents, and combinations thereof. In some embodiments, the compositions may contain one or more of the following: binders, such as tragacanth gum, acacia, corn starch, gelatin, or combinations thereof; excipients, such as dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, such as corn starch, potato starch, alginic acid, or combinations thereof; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose, saccharin, or combinations thereof; flavoring agents, such as peppermint, wintergreen oil, cherry flavor, orange flavor, or combinations thereof; or combinations of the above. When the dosage unit is a capsule, in addition to the above-mentioned materials, it may contain a carrier such as a liquid carrier. Various other materials may be present as coating agents or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.

[0126] Additional formulations suitable for other administration modes include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the cavity fluid. In general, in suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In some embodiments, suppositories may be formed from a mixture containing, for example, about 0.5% to about 10%, preferably about 1% to about 2% of the active ingredient.

[0127] Sterile injectable solutions are prepared by incorporating the active compound in the required amount into a suitable solvent together with various other ingredients listed above, and then, if necessary, filter sterilization.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients.For sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which obtains a powder of the active ingredient from the liquid medium that has been previously sterile-filtered, plus any additional desired ingredients.The liquid medium should be appropriately buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose before injection.The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as a solvent is expected to result in extremely rapid penetration, allowing high concentrations of the active ingredient to be delivered to a narrow area.

[0128] The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a minimum at a safe level, e.g., less than 0.5 ng / mg protein.

[0129] In certain embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

[0130] IV. Combination Therapy To increase the effectiveness of the nucleic acid, polypeptide, or nanoparticle complexes of the present embodiments, it may be desirable to combine these compositions with other agents effective in treating the disease of interest.

[0131] As a non-limiting example, cancer treatment may be performed with the TUSC2 therapeutic agent and / or immune checkpoint inhibitor of this embodiment in conjunction with other anti-cancer agents. An "anti-cancer" agent can negatively affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, suppressing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions will be provided in a combined amount effective to kill or inhibit the growth of the cells. This process may involve simultaneously contacting the cells with the anti-cancer peptide or nanoparticle conjugate and the agent or multiple factors. This may be accomplished by contacting the cells with a single composition or pharmaceutical formulation containing both agents, or by simultaneously contacting the cells with two different compositions or formulations, one containing the anti-cancer peptide or nanoparticle conjugate and the other containing a second agent. In certain embodiments, an anti-cancer peptide can be one agent and an anti-cancer nanoparticle conjugate can be the other agent.

[0132] Treatment with the anti-cancer peptide or nanoparticle conjugate may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the anti-cancer peptide or nanoparticle conjugate are applied to cells separately, generally, no significant time period will elapse between delivery times, so that the agent and the anti-cancer peptide or nanoparticle conjugate can still exert their beneficial combined effect on the cells. In such instances, it is contemplated that cells may be contacted with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period, so that 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) elapse between each administration.

[0133] Similarly, in some aspects, TUSC2 therapy is administered in combination with an immune checkpoint inhibitor. Various combinations may be used, where TUSC2 therapy is "A" and the immune checkpoint inhibitor is "B." TIFF0007825465000001.tif17128

[0134] In some embodiments, the administration of the TUSC2 therapy and / or immune checkpoint inhibitor of this embodiment to a patient follows the general protocol for administering chemotherapy drugs, taking into account the toxicity of the vector, if any. It is expected that treatment cycles will be repeated as necessary. It is also contemplated that various standard therapies and surgical interventions may be applied in combination with the described hyperproliferative cell therapy.

[0135] a. Chemotherapy Cancer treatment also includes various combination therapies. In some aspects, the TUSC2 therapeutic agent and / or immune checkpoint inhibitor of this embodiment is administered (or formulated) in combination with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor, such as 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. Non-limiting examples of protein kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, and dasatinib. , erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, vemurafenib, MK-2206, GSK690693, A-443654, V QD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0136] Still further combination chemotherapy includes, for example, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocalcin lumycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω11); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, australamycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicinone), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; florinic acid Folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatlaxate, defofamine, demecolcine, diaziquone, elflormitin, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxinSchizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel Included are mucitabine; 6-thioguanine; mercaptopurine; platinum complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycinone; aminopterin; xeloda; ibandronate; irinotecan (for example, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitor, transplatinum, and the pharmaceutically acceptable salt, acid or derivative of any of the above.In some embodiments, the compositions provided herein can be used in combination with gefitinib. In other embodiments, this embodiment may be practiced in combination with Gleevec (e.g., about 400 to about 800 mg / day of Gleevec may be administered to the patient). In certain embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein;

[0137] b. Radiation therapy Other widely used agents that cause DNA damage include what are commonly known as gamma rays, X-rays, and / or the direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwave and UV radiation, are also contemplated. All of these agents likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0138] The terms "contacting" and "exposing," when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell killing or stasis, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0139] c. Immunotherapy Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody may function alone as an effector of therapy or may recruit other cells to actually cause cell killing. The antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function solely as a targeting agent. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0140] Thus, immunotherapy can be used as part of a combination therapy in conjunction with the TUSC2 therapy of this embodiment. A general approach to combination therapy is described below. Generally, tumor cells must have some markers that are amenable to targeting, i.e., that are not present on the majority of other cells. Numerous tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

[0141] d. Gene therapy In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the therapeutic composition. Viral vectors for the expression of gene products are well known in the art, including eukaryotic expression systems such as adenovirus, adeno-associated virus, retrovirus, herpesvirus, lentivirus, poxvirus including vaccinia virus, and papillomavirus including SV40. Alternatively, administration of the expression construct can be achieved by lipid-based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, for example, Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).

[0142] Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues: A variety of proteins are encompassed within the scope of this embodiment, some of which are described below.

[0143] i. Cell proliferation inhibitors As mentioned above, tumor suppressor oncogenes function to inhibit excessive cell proliferation. Inactivation of these genes destroys their inhibitory activity, resulting in uncontrolled proliferation.

[0144] Genes that may be utilized as secondary treatments according to this embodiment include p53, p16, Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27 / p16 fusions, p21 / p27 fusions, antithrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors), MCC, and other genes listed in Table IV.

[0145] ii. Regulators of programmed cell death Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing cancer development (Kerr et al., 1972). Bcl-2 family proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. Bcl-2 proteins, discovered in association with follicular lymphoma, play a prominent role in regulating apoptosis and enhancing cell survival in response to various apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, Proc. Nat'l. Acad. Sci. USA, 82(21):7439-43, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein is now recognized as a member of a family of related proteins, which can be classified as death agonists or death antagonists.

[0146] Following its discovery, Bcl-2 was shown to act to suppress cell death induced by a variety of stimuli. It is now clear that there is a family of Bcl-2 cell death-regulating proteins that share common structural and sequence homology. These various family members have similar functions to Bcl-2 (e.g., Bcl XL , Bcl W , Bcl S , Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0147] e. Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventative, diagnostic or pathologic, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatments provided herein, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0148] Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). It is further contemplated that this embodiment may be used in conjunction with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue.

[0149] A cavity can be formed in the body by removing part or all of cancerous cells, tissues, or tumors. Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer treatment to the area. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also be treatments with various dosages.

[0150] f. Anti-inflammatory agents In certain aspects, TUSC2 therapy and / or immune checkpoint inhibitors are administered in conjunction with anti-inflammatory agents.Anti-inflammatory agents are defined herein as agents that are known or suspected to be useful in treating or preventing inflammation in subjects.Corticosteroids are a major class of anti-inflammatory agents.Corticosteroids can be short-, medium-, or long-acting, and can be delivered in various ways.A non-limiting list of corticosteroids contemplated in this embodiment includes oral corticosteroids such as cortisone, hydrocortisone, prednisone, and dexamethasone.

[0151] Another major class of anti-inflammatory drugs is nonsteroidal anti-inflammatory drugs. Nonsteroidal anti-inflammatory drugs include a class of drugs used to treat inflammation and pain. The exact mechanism of action of this class of drugs is unknown. Examples of members of this class of drugs include, but are not limited to, ibuprofen, ketoprofen, flurbiprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, flufenamic acid, diflunisal, oxaprozin, rofecoxib, and celecoxib. Those skilled in the art are familiar with these drugs. This category includes salicylates and salicylate derivatives, such as acetylsalicylic acid, sodium salicylate, choline salicylate, choline magnesium salicylate, and diflunisal.

[0152] Other anti-inflammatory agents include antirheumatic agents such as gold salts (e.g., gold sodium thiomalate, aurothioglucose, and auranofin), antirheumatic agents (e.g., chloroquine, hydroxychloroquine, and penicillamine), antihistamines (e.g., diphenhydramine, chlorpheniramine, clemastine, hydroxyzine, and triprolidine), and immunosuppressants (e.g., methotrexate, mechlorethamine, cyclophosphamide, chlorambucil, cyclosporine, and azathioprine). Other immunosuppressants contemplated by this embodiment are tacrolimus and everolimus. Tacrolimus suppresses interleukin-2 production associated with T cell activation and inhibits the differentiation and proliferation of cytotoxic T cells. Today, tacrolimus is recognized worldwide as a cornerstone of immunosuppressive therapy. Those skilled in the art are familiar with these agents and other members of this class of agents, as well as their mechanisms of action and applications.

[0153] g. Other agents It is contemplated that other agents may be used in combination with the compositions provided herein to improve the therapeutic efficacy of treatment. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing factors. Immunomodulators include tumor necrosis factor; interferon α, β, and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL, enhances the apoptosis-inducing ability of the compositions provided herein by establishing autocrine or paracrine effects on hyperproliferative cells. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatics and differentiation agents can be used in combination with the compositions provided herein to improve the anti-hyperproliferative effect of treatment. It is contemplated that cell adhesion inhibitors can improve the effect of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with the compositions provided herein to improve the effect of treatment.

[0154] In some embodiments, hormone therapy can also be used in conjunction with this embodiment, or in combination with any other cancer treatment previously described.The use of hormones can be used to reduce or block the action of certain hormones, such as testosterone or estrogen, in the treatment of certain cancers, such as breast cancer, prostate cancer, ovarian cancer or cervical cancer.This treatment is often used in combination with at least one other cancer treatment, as a treatment option or to reduce the risk of metastasis. [Example]

[0155] V. Working Example The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention and, as such, are considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made in the specific embodiments which are disclosed and which will still obtain a like or similar result, without departing from the spirit and scope of the invention.

[0156] Example 1 - TUSC2 therapy in combination with immune checkpoint inhibitors Combination treatment with TUSC2 and anti-PD1 effectively inhibits tumor growth in a syngeneic lung subcutaneous model with a G12V Kras mutation: The murine lung carcinoma cell line CMT / 167-luciferase, which harbors the Kras G12V mutation and low levels of TUSC2 expression, was implanted subcutaneously in C57BL / 6 mice. Ten mice were assigned to each group: DOTAP:cholesterol (DC)-empty vector / isotype; anti-PD1 antibody; DC-TUSC2 nanoparticles; and DC-TUSC2 nanoparticles + anti-PD1 antibody. The randomized sequential treatment with TUSC2 (iv) and anti-PD1 (ip) is shown in Figure 1A. No toxicity was associated with the combination treatment. Tumor volume and bioluminescence intensity were measured by caliper and IVIS imaging, respectively. PD-L1 expression in CMT / 167 cells was 23.7% (Figure 1B). While anti-PD1 showed limited efficacy in suppressing tumor growth, TUSC2 significantly inhibited tumor growth (Figure 1C). The combination further enhanced tumor shrinkage induced by TUSC2. The mean volumes of the isotype control, anti-PD1, TUSC2, and TUSC2 + anti-PD1 were 800 mm, respectively. 3 , 600mm 3 , 300mm 3 , and 180mm 3 It was ( * p<0.05; ** p<0.01; and *** p<0.001). IVS imaging, measuring bioluminescence intensity in the tumor as total flux per second, also demonstrates a combination effect (Figure 1D-E). The posterior probability of cooperation between TUSC2 and anti-PD1 was greater than 99%. These results suggest that in this model, TUSC2 synergizes with anti-PD1 in reducing tumor growth.

[0157] Combination treatment with TUSC2 and anti-PD1 increased NK cell and CD8 +Increased T cell density and suppression of regulatory cells: To demonstrate the immune impact of the combination of TUSC2 with anti-PD1, the main immune populations in peripheral blood leukocytes (PBLs) and splenocytes were characterized using 10-color panel flow cytometry. The effect of intravenous delivery of TUSC2 nanovesicles on peripheral NK cells, B cells, and T cells in tumor-free mice is shown in Figure 2A. There was no difference between TUSC2 and TUSC2 + anti-PD1 in tumor-free mice. The gating strategy for flow cytometry analysis is shown in Figure 1. After tumor cell inoculation, TUSC2 increased the density of NK cells and CD8 + It was found that TUSC2 significantly and moderately induced T cell density (p<0.001 and p<0.05), respectively, while significantly reducing B cells, MDSCs, Tregs, and T cells expressing PD1, CTLA4, and Tim3 (Figures 2B-E). Anti-PD1 had no clear effect on NK cells, T cells, and B cells, but reduced MDSCs, Tregs, and T cells expressing PD1, CTLA4, and Tim3. The combination treatment had the same effect as TUSC2 alone. The greatest difference in the combination treatment was in the ratio of NK cells to MDSCs and CD8 to Tregs. + The proportion of CD8 T cells was enhanced (p<0.001) (Figure 2F). Collectively, these results suggest that TUSC2-anti-PD1 synergy likely enhances the proportion of NK cells and CD8 T cells. + Shown to be associated with increased T cell proliferation.

[0158] Combination treatment with TUSC2 and anti-PD1 significantly increased tumor-infiltrating NK cells and CD4+ +Enhanced 8T Cells: To determine whether TUSC2 + anti-PD1 treatment was associated with denser tumor immune cell infiltration, immune infiltration was analyzed using a Vectra high-throughput pathology system covering 25% of each tumor's area (N=5 per treatment group). Entire subcutaneous tumors were uniformly excised for treatment, and multiple sections from each tumor were analyzed to account for the variable tumor size between different treatment groups and eliminate any potential sampling bias. H-score values ​​were used in conjunction with the percentage of positive cells, taking into account staining intensity. Compared to control or anti-PD1, the combination significantly increased intratumoral CD8 + The CD8 T cell density in the TUSC2 group increased 10-fold and 3-fold, respectively (p<0.0001; Figure 3A). + T cell infiltration was lower than that of the combination, but not significantly. Activated NK cell infiltration was highest in tumors treated with TUSC2 (p<0.0001), followed by the combination (p<0.0001) (Figure 3A). Anti-PD1 slightly increased NK cell infiltration compared with TUSC2 or the combination. Conversely, TUSC2 and TUSC2 + anti-PD1 significantly suppressed tumor-infiltrating Foxp3-positive T cells (p<0.0001), a marker expressed by Tregs, and MDSCs bearing the tumor-suppressive granulocytic marker 1 (Gr-1) (p<0.0001). Anti-PD1 slightly reduced Foxp3 density (p=0.18), while having no effect on GR-1. These results suggest that TUSC2 and the combination altered the tumor immune microenvironment.

[0159] Combination treatment with TUSC2 and anti-PD1 enhanced the expression of chemokines associated with NK cells and T lymphocytes. Serum chemokine expression profiles were analyzed using Nanostring technology. A set of chemokine genes associated with T lymphocyte and NK cell migration was upregulated after exposure to TUSC2 and the combination (Figure 3B). Expression of CcL3 and CcL4, which are involved in NK cell migration via CCR5 recognition, increased more than twofold, whereas expression of CcL21a and CcL19, which interact with the CCR7 receptor and recruit T cells and dendritic cells to tumors (Viola et al., 2012; Griffith et al., 2014), increased more than fourfold compared to untreated controls. CcL4 and CcL5 serum chemokine levels were also increased by TUSC2 and TUSC2 + anti-PD1 treatment compared to controls (Figure 3C).

[0160] NK cells or CD8 + Depletion of T cells completely and partially abolishes the antitumor activity of the combination: NK and CD4+, respectively + 8 The finding that both densities were strongly upregulated after combination therapy suggests that CD8 + These results suggest that T cells and NK cells mediate TUSC2+anti-PD1-induced tumor regression. To confirm this hypothesis, we investigated the effects of anti-NK1.1 or anti-CD8 + via intraperitoneal injection of T cell antibodies, NK cells or CD8 + T cells were depleted in CMT167 tumor-bearing mice (Figs. 8 and 9). As shown in Fig. 4A, treatment with anti-NK1.1 antibody completely abolished the combination tumor regression, whereas anti-CD8 + Treatment with T antibody partially attenuated it (Fig. 4B). Furthermore, NK cell depletion abolished TUSC2-induced tumor growth inhibition, whereas CD8 + T cell depletion had no effect. Neither depletion had any effect on anti-PD1 responses. These findings support the conclusion that CD8 +These results suggest that T cells may contribute to the TUSC2-mediated enhancement of sensitivity to anti-PD1, whereas NK cells are essential for this synergy.

[0161] Next, analysis of serum cytokines using a Luminex assay revealed that both TUSC2 and the combination induced potent Th1-mediated immune responses (control vs. TUSC2: p<0.0001; control vs. combination: p=0.007 (Figure 4C)), an effect that was abolished by NK depletion (TUSC2 vs. TUSC2 / NK1.1: p=0.008; combination vs. NK1.1: p=0.0009). This suggests that NK cells are important in inducing Th1-mediated immune responses to TUSC2 and the combination. However, there was no significant difference in the Th1 / Th2 ratio between these two treatment groups, regardless of whether NK depletion was performed. TUSC2 + anti-PD1 therapy promoted higher levels of IL-15 (p=0.0001) and IL-18 (p<0.0001) cytokines compared with its untreated or anti-PD1-treated counterparts (Figure 4D). IL-15 was induced at similar levels in the TUSC2 and combination groups, whereas IL-18 levels were significantly higher in TUSC2 than in the combination. Depletion of NK cells significantly reduced IL-15 (control vs. TUSC2: p = 0.03) and IL-18 (control vs. TUSC2: p = 0.0005) levels. There was a significant difference in IL-18 levels, but not IL-15, between TUSC2 and the combination, regardless of NK depletion. Finally, expression profiles of sorted NK cells and tumor tissues using qPCR and Nanostring technology showed significantly higher expression of IL-15R and IL-18R in TUSC2-treated than in their untreated and anti-PD1-treated counterparts (p = 0.01 and p = 0.001, respectively; Figure 4E, F).

[0162] Combination treatment with TUSC2 and anti-PD1 improved survival in a syngeneic G12D Kras mutant lung metastasis model: The efficacy of TUSC2 plus anti-PD1 was evaluated in a second Kras metastatic model using 129Sv mice intravenously inoculated with 344SQ-luciferase lung cancer cells harboring the K-ras G12D mutation. The level of PD-L1 expression in 344SQ cells was only 4.5% (Figure 5A). The sequential treatment strategy is shown in Figure 5B. Treatment groups were similar to those in the previous model, with the addition of two groups: anti-PD1 combined with anti-CTLA4, and TUSC2 combined with anti-PD1 and anti-CTLA4. The former combination was used in this experiment due to its reported enhanced clinical efficacy compared with each drug group alone (Larkin et al., 2015). TUSC2 significantly improved survival compared with the untreated, anti-PD1, and anti-PD1 + anti-CTLA4-treated groups (TUSC2 vs. control: p<0.0001; TUSC2 vs. anti-PD1: p<0.001; TUSC2 vs. anti-PD1 + anti-CTLA4: p<0.001) (Figure 5C). Combining TUSC2 with anti-PD1 significantly prolonged survival (combination vs. control: p<0.0001; combination vs. anti-PD1: p<0.001; combination vs. TUSC2: p=0.024). Combining TUSC2 with anti-PD1 and anti-CTLA4 extended survival by several days compared with TUSC2 + anti-PD1 treatment. Bioluminescence imaging of tumors confirmed these findings (Figure 5D). Figure 5E shows impressive clearance of tumor nodules in the lungs of mice receiving TUSC2 + anti-PD1 at week 2. These results demonstrate the efficacy of the TUSC2 + anti-PD1 combination and suggest that the combination of dual checkpoint blockade, anti-PD1 and anti-CTLA4, with TUSC2 may have translational utility.

[0163] Analysis of immune cell infiltration using single-cell analysis showed higher NK cell infiltration with TUSC2 compared to the control group or the anti-PD1 / anti-CTLA4 combination treatment group (p<0.001) (Figure 5F). The effects of TUSC2 + anti-PD1 or TUSC2 + anti-PD1 + anti-CTLA4 were slightly higher than those of TUSC2. In contrast, Treg and MDSC cell infiltration were significantly suppressed by anti-PD1 (p=0.004; p=0.0003), an effect further enhanced by TUSC2 and the combination (Figures 5G and 5H). These results were consistent with those observed in the evaluation of the CMT167 subcutaneous tumor model (Figure 2).

[0164] Combining TUSC2 with anti-PD1 altered the immune gene expression profile in the tumor microenvironment: To identify specific immune genes differentially expressed in the TUSC2 + anti-PD1 combination, RNA from tumor samples was subjected to digital multiplex profiling (NanoString Technologies Inc.) using a mouse pan-cancer panel consisting of 770 mouse immune genes covering both adaptive and innate responses, with 40 housekeeping controls. Welch's t-test with false discovery rate (q < 0.05) correction was applied to derive statistically significant gene expression differences between treatment groups. A p value of < 0.05 was considered significant. Results were visualized using volcano plots and heat maps (Figure 6A, B). Because TUSC2 addition enhanced the response to anti-PD1 treatment, pairwise comparisons were performed between the anti-PD1 group and the TUSC2 + anti-PD1 group. Pairwise comparisons were also performed between all other groups. Initially, six gene clusters were found to be significantly upregulated in the combination group. These include Cd1d2, Ltf, Klra21, H60a, Tnfsf18, and Bcl6. Another cluster of significantly downregulated genes was found, consisting of Egr3, Cd46, Ncr1, Klra5, Ccl1, Il12rb2, and Cd59b (Figure 6B). All of these genes are involved in NK cell and CD8 +These results are important for the regulation of T cells (Deng et al., 2013; Shevach and Stephens, 2006; Orr et al., 2009). The combination treatment also upregulated the expression of genes related to T cell-mediated antitumor function in the tumor microenvironment (Figure 6D-F). These results support the upregulation of NK and CD8 + Corroborating T immune profiling and tumor infiltration data (Figures 2-3).

[0165] Example 2 - Materials and Methods Cell culture and reagents: KRasG12 / CMT167-luc and K-RasG12D / 344SQ-luc cells were kindly provided by Dr. Alan Fields (Mayo Clinic) and Dr. Frank R. Jirik (University of Calgary). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Atlanta Biological, GA) and 1% penicillin and streptomycin (Life Science Technologies). Antibodies against isotype, anti-PD1, anti-CTLA4, InVivoPlus anti-NK1.1 (clone PK136), and anti-CD8 were used. + T (clone 2.43) anti-mouse monoclonal antibody was purchased from Bio X Cell (West Lebanon, NH). DOTAP and cholesterol were purchased from Avanti Polar Lipids (Alberta, AL). The synthesis and preparation of DC-TUSC2 was previously described (Ito et al., 2004).

[0166] Animal studies: All animal procedures were reviewed and approved by the Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. For the CMT167-luc syngeneic model, 6- to 8-week-old female C57BL / 6-Elite mice (Charles River Laboratories, Houston, TX) were inoculated with 1 × 10 6CMT167-luc cells were injected subcutaneously into the right flank and 10 mice each were randomized into treatment groups as follows: control (empty vector nanovesicles, isotype antibody), anti-PD1, TUSC2 nanovesicles, and TUSC2 + anti-PD1. Briefly, 25 μg of TUSC2 was injected intravenously every 48 hours for three cycles, and 0.25 mg of anti-PD1 antibody was injected intraperitoneally (ip) every four days for three cycles. Tumor volume was calculated using the formula: 1 / 2(length × width) 2 ) were calculated. Mice were euthanized 3–4 weeks after tumor cell injection, and tumors and spleens were harvested. For the 344SQ metastasis model, 6–8-week-old female 129 / Sv mice were intravenously injected with 100,000 344SQ-luc cells. Treatment groups (10 mice) were as follows: control (empty vector nanovesicles, isotype antibody), anti-PD1, TUSC2, anti-CTLA4, TUSC2 + anti-PD1, anti-PD1 + anti-CTLA4, and TUSC2 + anti-PD1 + anti-CTLA4. In both models, animals were routinely monitored, and tumors were imaged using IVIS. All treatments and measurements were performed double-blind. For immunophenotyping analysis, animals were sacrificed 2 weeks after tumor cell injection, lungs were harvested, and peripheral blood was collected via cardiac puncture.

[0167] NK cells or CD8 + Depletion of T cells: NK cells or CD8 in tumor-bearing CMT167 mice + To deplete T cells, mice were injected with neutralizing monoclonal antibodies anti-NK1.1 (clone PK136) or anti-CD8+ T (clone 2.43) anti-mouse monoclonal antibodies (100 μg, i.p.) every 3 days for 4 cycles starting on day 0 after subcutaneous inoculation of tumor cells. + T cell depletion was monitored via flow cytometry analysis of splenocytes. Tumor volume was measured and bioluminescence intensity was quantified by IVIS.

[0168] Multicolor flow cytometry: PBLs were isolated and stained according to standard protocols for flow cytometry. Multicolor panels were developed and optimized using a Gallios Flow Cytometer Research System (Beckman Coulter, Brea, CA). Mouse antibodies were purchased from BioLegend (San Diego, CA). Single-cell suspensions were washed with fluorescence-activated cell sorting staining buffer, incubated with mouse Fc receptor binding inhibitors for 10 minutes, and stained with the indicated antibodies. Data were analyzed using FlowJo software version 10 (FlowJo, Ashland, OR).

[0169] Immunohistochemistry: Tumors harvested from CMT167 were fixed in 10% paraformaldehyde, and 8-μm sections of formalin-fixed, paraffin-embedded tissue were stained with anti-CD8, anti-Foxp3, anti-Gr-1, and anti-NKp46 mouse antibodies. All immunohistochemical analyses were performed at the MD Anderson Histology Core Laboratory (Smithville, TX). At least five tumor samples from each group were stained with each antibody and imaged and analyzed using a 200-slide Vectra 3.0 automated quantitative pathology imaging system (PerkinElmer, Waltham, MA) at the Imaging Core Facility at MD Anderson (Houston, TX). An H-score ranging from 0 to +3 was generated for each cell.

[0170] Quantitative PCR: Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using the SuperScript III Kit (Invitrogen, Carlsbad, CA). Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Relative expression levels were normalized and measured using an ABI Viia7 Real-Time PCR System (Applied Biosystems). Relative quantification was performed using the comparative CT method described by the manufacturer.

[0171] Luminex Assay: To identify serum cytokines and chemokines, the Affymetrix (eBioscience) ProcartaPlex 36-plex immunoassay (Affymetrix, Santa Clara, CA) was used according to the manufacturer's instructions. Three samples per treatment group were run in duplicate. Briefly, seven standards were prepared according to the manufacturer's protocol, and 25 μL of serum sample was mixed with beads coated with the indicated antibodies and incubated for 2 hours at room temperature with agitation at 500 rpm. The ProcartaPlex multiplex immunoassay used Luminex xMAP (multi-analyte profiling) technology. Plates were read using a Luminex 200 system (Luminex, Austin, TX), and standard curves were plotted. Data were analyzed using ProcartaPlex Analyst software version 1.0.

[0172] Gene Expression Analysis: Total tumor RNA extracted from three replicates per treatment group using the Qiagen RNeasy Mini Kit was submitted to the Genomic Core Facility at Baylor College of Medicine (Houston, TX) for quality control and expression profile analysis by NanoString Technology. The NanoString PanCancer Mouse Immune Profiling Panel used profiles 776 genes associated with specific immune cell types and immune cell functions. Data were analyzed at MD Anderson's Bioinformatics Core Facility.

[0173] Statistical Analysis: For the CMT167 model, tumor growth was analyzed using a generalized linear regression model. All data are expressed as mean ± SD, and the statistical significance of differences between treatments was tested by two-way ANOVA and two-tailed t-test, with P<0.05 considered significant. For the 344SQ model, the distribution of overall survival (OS) was estimated using the Kaplan-Meier method. A log-rank test was performed to test for differences in survival time between groups. Regression analysis of survival data based on the Cox proportional hazards model was performed with OS defined as the time from treatment initiation to death.

[0174] Statistical analysis of flow cytometry and Luminex data was performed using a generalized linear regression model to compare different treatment groups. For immunohistochemistry data, a generalized linear regression model was used for statistical analysis of H scores between treatment groups. The ESTIMATE statement in the PROC MIXED procedure of SAS was used for each pairwise comparison. For NanoString analysis, data were normalized before gene profile quantification and statistical analysis. Positive controls, housekeeping genes, and negative controls were used to adjust for sample preparation variation, background noise, and RNA abundance variation. Linear models were used to assess overall treatment effects, and contrasts were used for pairwise comparisons of interest. A beta uniform mixture (BUM) model was used to model resulting p-values, determine false discovery rate (FDR) cutoffs, and identify significantly differentially expressed genes. All statistical analyses were performed using the statistical analysis software R.

[0175] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of this disclosure.The compositions and methods of the present invention have been described in terms of preferred embodiments, and it will be apparent to those skilled in the art that modifications may be made to the methods described herein and the steps or order of steps of the methods without departing from the concept, spirit and scope of the present invention.More specifically, it will be apparent that certain chemically and physically related agents may be substituted for the agents described herein, as long as they achieve the same or similar results.All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0176] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007825465000002.tif189150TIFF0007825465000003.tif231150TIFF0007825465000004.tif231141TIFF0007825465000005.tif231150TIFF0007825465000006.tif230150TIFF0007825465000007.tif86128

Claims

1. A pharmaceutical composition for treating cancer in a subject, comprising a TUSC2 polypeptide, a TUSC2 nucleic acid, or a TUSC2 expression vector, used in combination with at least one immune checkpoint inhibitor, wherein the subject has been treated or is currently being treated with at least one immune checkpoint inhibitor, and the at least one immune checkpoint inhibitor is an anti-PDL1 antibody.

2. 10. The pharmaceutical composition of claim 1, wherein at least one immune checkpoint inhibitor is administered to the subject before, after, or simultaneously with administration of the pharmaceutical composition.

3. 10. The pharmaceutical composition of claim 1, wherein the subject has been administered at least one immune checkpoint inhibitor within two weeks prior to administration of the pharmaceutical composition.

4. The pharmaceutical composition of claim 1, comprising a TUSC2 expression vector.

5. The TUSC2 expression vector (a) It is a plasmid DNA; (b) pLJ143 / KGB2 / FUS1; and / or (c) provided in liposomes; 5. The pharmaceutical composition of claim 4.

6. 5. The pharmaceutical composition of claim 4, wherein the TUSC2 expression vector is provided in a liposome that is a DOTAP:cholesterol liposome.

7. 7. The pharmaceutical composition of claim 6, wherein the DOTAP:cholesterol ratio is about 1.5:1 to 1:1.5 or about 10:

9.

8. 7. The pharmaceutical composition of claim 6, wherein the TUSC2 expression vector and DOTAP:cholesterol liposome are administered at a dose of about 0.01 mg / kg to about 0.10 mg / kg.

9. 10. The pharmaceutical composition of claim 1, which is administered more than once.

10. 10. The pharmaceutical composition of claim 1, used in combination with an anti-inflammatory agent or an additional anti-cancer treatment.

11. The pharmaceutical composition of claim 1, comprising a TUSC2 polypeptide.

12. The pharmaceutical composition of claim 11, wherein the TUSC2 polypeptide is myristoylated or contained in a nanoparticle.

13. The pharmaceutical composition of claim 11, wherein the TUSC2 polypeptide is contained in a nanoparticle that is a lipid-based nanoparticle, a superparamagnetic nanoparticle, a nanoshell, a semiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle, a metal-based nanoparticle, a fullerene, or a nanotube.

14. 11. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is used in combination with an additional anti-cancer treatment, wherein the additional anti-cancer treatment is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, cytokine therapy, or an EGFR inhibitor.

15. 2. The pharmaceutical composition of claim 1, wherein the cancer is oral cavity cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer, or blood cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteogenic sarcoma tumor, multiple neuroendocrine tumors type I and type II, breast cancer, lung cancer, non-small cell lung cancer, metastatic lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

16. 10. The pharmaceutical composition of claim 1, wherein the cancer is resistant to at least a first chemotherapy or is resistant to a platinum-based chemotherapy.

17. Administration of the pharmaceutical composition and at least one immune checkpoint inhibitor (a) NK cells and / or CD8 in tumors ＋ resulting in increased T cell density, (b) CD8 in tumors ＋ At least a three-fold increase in T cell density, or (c) resulting in increased serum levels of CcL3, CcL4, CcL21a, and / or CcL19; 10. The pharmaceutical composition of claim 1.

18. 15. The pharmaceutical composition of claim 14, wherein the EGFR inhibitor is a tyrosine kinase inhibitor, an EGFR-binding antibody or aptamer, gefitinib, erlotinib, cetuximab, matuzumab, panitumumab, AEE788, CI-1033, HKI-272, HKI-357, or EKB-569.

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