Methods and compositions for TUSC2 immunotherapy

Abstract

This invention provides a method for treating subjects with cancer. [Solution] A method comprising the step of administering TUSC2 therapy, which is one of the tumor suppressor therapies, wherein the subject has been treated with or is currently being treated with at least one immune checkpoint inhibitor. Preferably, the method wherein at least one immune checkpoint inhibitor comprises an anti-PD1 agent.

Description

[Technical Field]

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

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

[0003] 2. Explanation of related technologies As the molecular and genetic mechanisms of tumorigenesis become clearer, the focus of cancer treatment is shifting from the tissue level to the gene 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 deletions or mutations 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 led to the identification of several 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 for enhancing the effectiveness of such therapies remain unclear. [Overview of the Initiative]

[0004] In a first embodiment, a method is provided for treating a subject with cancer, comprising the step of administering a tumor suppressor therapy (e.g., TUSC2 therapy) in combination with an immune checkpoint inhibitor. Thus, a method is provided for treating a subject with cancer who is currently being treated with (or has previously been treated with) at least one immune checkpoint inhibitor, comprising the step of administering a tumor suppressor therapy such as TUSC2 therapy to the subject. For example, a subject to be treated with TUSC2 therapy may be a subject that has 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. The TUSC2 therapy as used herein may be any type of therapy that provides or induces the expression of TUSC2 polypeptide in cancer cells (see, for example, U.S. Patent No. 7,902,441 incorporated herein by reference). For example, TUSC2 therapy may include the delivery of TUSC2 polypeptide or a TUSC2 expression vector to cancer cells. The therapy may be delivered, for example, via nanoparticles or, in the case of nucleic acid expression vectors, through the use of a viral vector.

[0005] In one embodiment, a method is provided for treating a subject with cancer, comprising the step of 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 concurrently 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 an amount therapeutically effective for treating cancer.

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

[0007] In one embodiment, the administration of TUSC2 therapy includes the administration of a TUSC2 expression vector, such as a DNA plasmid encoding TUSC2. Expression vectors for use in the embodiments provided herein generally include regulatory elements for the expression of the TUSC2 coding sequence. For example, a vector may include promoter and enhancer elements effective for expression in cancer cells of interest. In one aspect, for example, TUSC2 expression is brought about by a CMV promoter or a recombinant version thereof, such as the CMV promoter construct described in U.S. Patent Application Publication 20070092968, incorporated herein by reference. In one embodiment, a vector provided herein includes a modified CMV promoter. In one embodiment, a vector provided herein includes a mini-CMV promoter. Further expression regulatory elements may also be included, for example, introns, drug response elements, RNA stabilization or destabilization sequences, cell localization signals, polyadenylation signal sequences, and / or optimized translation initiation codons. Plasmid DNA vectors may also include sequences that help promote DNA production, bacterial replication origins, and / or drug resistance markers. In certain contexts, the TUSC2 expression vector is the pLJ143 / KGB2 / FUS1 plasmid.

[0008] Methods for delivering expression vectors to cells (e.g., in vivo delivery) are well known in the art, and these methods include, but are not limited to, nanoparticles (e.g., liposome 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. Those skilled in the art recognize that vector delivery can be optimized by tuning various properties of the liposomes. For example, the liposomes may be tuned to have a certain size range and / or a specific ratio of DNA to lipids; DNA to cholesterol; or lipids 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 having an average particle size of about 50 to about 500 nm (e.g., 200 to 500 nm). In an even 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 [specific characteristics].

[0009] In a further embodiment, TUSC2 therapy may include the administration of a TUSC2 polypeptide. Methods for the administration of a TUSC2 polypeptide are described, for example, in U.S. Patent Applications Publications 20060251726 and 20090023207, which are incorporated herein by reference. The TUSC2 polypeptide may be modified to enhance its activity and / or ability to enter cancer cells. For example, the polypeptide can be modified with a lipid component (e.g., myristoylation). 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] TUSC2 therapies and / or immune checkpoint inhibitors according to the embodiments provided herein are typically formulated in a pharmaceutically acceptable carrier. Therapies according to these embodiments may be delivered, for example, intravenously, intradermally, intraarterially, intraperitoneally, intrafocally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreously, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, intramucosally, intrapericardially, intraocularly, orally, locally, by 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 conjugated with DOTAP:cholesterol liposomes can be administered by intravenous infusion. In certain situations, TUSC2 therapy is administered intravenously in doses ranging from approximately 0.01 mg / kg to approximately 0.10 mg / kg, for example, approximately 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10 mg / kg. In further situations, TUSC2 therapy may 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 may vary, but is not limited to these, and may include approximately 1, 2, or 3 days, approximately 1, 2, or 3 weeks, or more than 1 month between doses.

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

[0012] In a further aspect, the methods provided herein further comprise administering a further anti-cancer treatment. The further anti-cancer treatment can include, but is not limited to, surgical therapy, chemotherapy (e.g., administration of a protein kinase inhibitor or an EGFR-targeted therapy), radiation therapy, cryotherapy, hyperthermia, phototherapy, radiation ablation therapy, hormone therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenesis therapy, cytokine therapy, or biological therapy such as, for example, monoclonal antibodies, siRNA, antisense oligonucleotides, ribozymes or gene therapy. Biological therapy can include, but is not limited to, gene therapy such as, for example, 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-angiogenesis factor gene therapy.

[0013] Thus, in yet a further aspect, there are provided compositions, therapies, and methods for treating a subject having cancer, comprising administering to the subject a TUSC2 therapy (e.g., a TUSC2 polypeptide or a TUSC2 expression vector) in combination with an immune checkpoint inhibitor and a further anti-cancer agent, such as, for example, a chemotherapeutic agent. For example, the chemotherapeutic agent can be a protein kinase inhibitor such as a Src or Akt kinase inhibitor. In some aspects, the chemotherapeutic agent is an epidermal growth factor receptor (EGFR) inhibitor.

[0014] Accordingly, in one embodiment, a method is provided for treating a subject with cancer, comprising the step of 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 may be administered before, after, or essentially concurrently with the protein kinase inhibitor. Accordingly, 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, mbritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, salakatinib, sorafenib, sunitinib, and tras This may include tuzumab, vandetanib, AP23451, vemurafenib, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridafololimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof. In some contexts, protein kinase inhibitors are AKT inhibitors (e.g., MK-2206, GSK690693, A-443654, VQD-002, miltefosine, or perifosine).

[0015] EGFR-targeted therapies for use according to this embodiment include, but are not limited to, inhibitors of EGFR / ErbB1 / HER, ErbB2 / Neu / HER2, ErbB3 / HER3, and / or ErbB4 / HER4. A wide range of such inhibitors are known, and they include, but are not limited to, tyrosine kinase inhibitors and EGFR-binding antibodies or aptamers that are active against the receptor. For example, EGFR inhibitors 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 topically. 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 concurrently with TUSC2 therapy. For example, the therapies may be administered simultaneously, such as by co-administration via intravenous infusion. In one embodiment, the TUSC2 inhibitor and the EGFR inhibitor can be administered in any amount effective to treat the cancer. In one embodiment, the compositions, therapies, and methods provided herein include the step of administering the TUSC2 inhibitor and the EGFR inhibitor at a lower dose than either composition administered alone. In one embodiment, the compositions, therapies, and methods include the step of administering the TUSC2 inhibitor and the EGFR inhibitor at a lower dose that reduces side effects. In one embodiment, the compositions, therapies, and methods include the step of administering the TUSC2 therapy, the immune checkpoint inhibitor, and the EGFR inhibitor at a dose effective to provide an additive, cooperative, or synergistic effect than that provided by either composition administered alone. In one aspect, the cancer for treatment with such therapy may be any of the cancers described herein, such as lung cancer (e.g., non-small cell lung cancer). In one preferred aspect, the cancer for treatment with combination therapy is an EGFR-expressing cancer.In certain embodiments, cancers that express EGFR comprise 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% tumor cells that express EGFR.

[0016] In yet a further aspect, provided herein is a method for treating a subject having a cancer previously determined to express EGFR, the method comprising administering to the subject, in combination with an immune checkpoint inhibitor and an EGFR inhibitor, a TUSC2 therapy. In certain embodiments, provided herein is a method for treating a subject having cancer, the method comprising determining whether the cancer expresses EGFR, and administering to the subject TUSC2, an immune checkpoint inhibitor, and an EGFR inhibitor. Methods for assessing the EGFR expression status of cancer are described, for example, in U.S. Patent Application Publication No. 20110052570, which is incorporated herein by reference. In certain aspects, cancers that express EGFR can be cancers that express mutant EGFR, such as cancers that express EGFR having L858R and / or T790M mutations. In certain embodiments, the compositions and therapies provided herein are administered to a patient having a cancer that expresses EGFR and comprises 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% tumor cells that express EGFR. In a further aspect, the subject for treatment has a cancer previously determined to express EGFR and at least 10% of the cancer cells are apoptotic. In certain embodiments, the methods provided herein further comprise 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 the cells of the cancer that expresses EGFR are apoptotic.

[0017] In some aspects, cancer for treatment or evaluation may exist as a tumor, such as a primary tumor or a metastatic tumor. Cancer may be early-stage cancer, or metastatic or late-stage cancer. In some aspects, cancer may be oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine cancer, hematological cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, prostate cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, These include parathyroid cancer, pituitary tumors, adrenal tumors, osteogenic sarcomas, multiple neuroendocrine type I and II tumors, 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, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colorectal cancer, rectal cancer, or skin cancer. In a further context, cancer may be defined as cancer that is resistant to one or more anticancer treatments, such as chemotherapy-resistant cancer. For example, cancer may be cancer that is resistant to platinum-based chemotherapeutic agents such as cisplatin.

[0018] In some aspects of the above embodiment, the step of administering TUSC2 therapy and at least one immune checkpoint inhibitor is performed in the tumor to include NK cells and / or CD8 + This leads to an increase in T cell density. In certain situations, CD8 + T cell density increases by at least three times, e.g., four, five, six, seven, eight, nine, or ten times. In some cases, the step of administering TUSC2 therapy and at least one immune checkpoint inhibitor results in serum levels of CcL3, CcL4, CcL21a, and / or CcL19.

[0019] In a further embodiment, a kit comprising a TUSC2 therapeutic agent and at least one immune checkpoint inhibitor is provided herein. For example, in some aspects, the kit provided herein comprises a TUSC2 therapeutic agent, at least one immune checkpoint inhibitor, and reagents for testing a subject to determine its response to the TUSC2 therapeutic agent and / or immune checkpoint inhibitor. For example, the reagents for testing a subject to determine its response to the TUSC2 therapeutic agent could be reagents for determining the level of apoptosis in the cancer cells of the subject. In a further aspect, the kit further comprises one or more anti-inflammatory agents or kinase inhibitors. In a further aspect, the kit may comprise one or more additional components, including, but not limited to, pharmaceutically acceptable diluents, syringes, infusion bags, infusion lines, and / or a set of instructions for using the kit.

[0020] Any method or composition described herein is intended to be applicable in relation to any other method or composition described herein. Similarly, aspects of this embodiment discussed in the context of methods for treating a subject are similarly applicable to methods for predicting a reaction in a subject, and vice versa.

[0021] When used in conjunction with the term “including” in the claims and / or specification, the use of the words “a” or “an” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.”

[0022] In this specification, the term "essentially absent" with respect to a particular component means that not even a small amount of the component is intentionally included in the composition and / or is present only as an impurity or in trace amounts. Therefore, the total amount of the component resulting from any unintentional inclusion of the component in the composition is far less than 0.01%. Most preferably, the composition is one in which no amount of the component can be detected by standard analytical methods.

[0023] As used herein and in the claims, “a” or “an” may mean one or more. As used herein and in the claims, “a” or “an” may mean one or more when used in conjunction with the word “including.” As used herein and in the claims, “another” or “further” may mean at least a second or subsequent one.

[0024] As used herein and in the claims, the term “about” is used to indicate that the value includes inherent variations in the error of the device or method used to determine the value, or variations present among the subjects of the test.

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

[0026] The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the particular embodiments presented herein. [Figure 1A]Combined treatment with TUSC2 and anti-PD1 enhanced antitumor activity in a CMT167 subcutaneous model. (A) Sequential treatment strategy showing tumor inoculation, treatment schedule and dose, blood and spleen collection for immunocytochemistry analysis, tumor recovery for immunohistochemical examination, and RNA isolation. (B) Surface expression levels of PD-L1 on CMT167-luc cells were determined by flow cytometry. (C), (D) Tumor growth curves in 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-free) vector. TUSC2+PD1 therapy resulted in the greatest inhibition of tumor growth, followed by TUSC2, PD1, and the control. (E) Representative images of tumor-bearing mice from each treatment group with bioluminescent signals, imaged by IVIS 200 imaging. Data are representative of four independent studies. Image intensity between treatment groups was compared using the CONTRAST statement in the PROC MIXED method of SAS. All analyses were performed using SAS version 9.4 and S-Plus version 8.04. Unless otherwise specified, statistical values ​​are shown at a significance level of p<0.05. *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 1B] See the explanation in Figure 1A. [Figure 1C] See the explanation in Figure 1A. [Figure 1D] See the explanation in Figure 1A. [Figure 1E] See the explanation in Figure 1A. [Figure 2A]The combination of TUSC2 and anti-PD1 upregulates natural killer cells and cytotoxic T cells, and downregulates regulatory cells. TUSC2 + anti-PD1 treatment altered immune cell populations in peripheral blood and spleen. The control group was treated with nanovesicles loaded with empty (TUSC2 gene-free) vectors. (A) Effects 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 different immune populations (T cells, B cells, NK cells & Lin-negative cells) were sorted from the spleen, and TUSC2 expression was determined by RT-PCR. (B) Effects of TUSC2 treatment and TUSC2 + anti-PD1 treatment on natural killer (NK) cells, T cells, and B cells at 2 weeks after tumor transplantation. (C) TUSC2 treatment altered the MDSC status. Mononuclear and granulocytic MDSCs were determined using the following gating strategy: CD45+>CD3->MHCII low>CD11b+>Gr-1+. CD11b+ Gr-1 high was considered granulocytic MDSC, and CD11b+ Gr-1 low was considered mononuclear MDSC. Data are presented as mean percentage ± SD, n=5. *, P<0.05; **, P<0.01; ***, P<0.001. (D) Effects 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 percentage ± 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 percentage ± SD, n=5, *, P<0.05;**, P<0.01;*** P<0.001. (F) Effects of TUSC2 and anti-PD1 treatment are shown as the ratio of NK / MDSC cells and CD8 T effector / Treg cells in peripheral blood leukocytes. Data are presented as mean ± SD, n=5.Statistical analysis of flow data was performed using the PROC GENMOD method in SAS with a general linear regression model and the CONTRAST statement. *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 2B] See the explanation in Figure 2A. [Figure 2C] See the explanation in Figure 2A. [Figure 2D] See the explanation in Figure 2A. [Figure 2E] See the explanation in Figure 2A. [Figure 2F] See the explanation in Figure 2A. [Figure 3A]The combination of TUSC2 with anti-PD1 increased the infiltration of NK cells and CD8 T cells, while inhibiting the infiltration of MDSCs and Tregs. (A) Subcutaneous tumors were treated with nanovesicles loaded with empty (TUSC2 gene-free) vectors (control), TUSC2 nanovesicles, anti-PD1, and TUSC2 + anti-PD1. Formalin-fixed excised 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 (20×) were acquired by imaging 25% of the tumor area using the Vectra automated imaging system. N=5 tumor sections were imaged per group. Approximately 100 images per treatment group were analyzed using InForm software for H-scoring. A generalized linear regression model was used for statistical analysis of H-scores between treatment groups. A composite symmetric covariance structure was used to explain the inter-mouse variability and repeated measures of the data. H scores were compared between pairs of treatment groups using the ESTIMATE statement in the PROC MIXED method of SAS. *, P<0.05; **, P<0.01; ***, P<0.001. (B) RNA was extracted from freshly excised tumors from the treatment groups (n=3 tumors per treatment group), and chemokine gene expression was determined by NanoString technology. Data were normalized, and the fold change in expression was analyzed using nCounter analysis software. The fold change was compared with the control sample. The bar shows the mean fold change (n=3). (C) Levels of CCL4 and CCL5 chemokines in serum induced by TUSC2 treatment are shown. Subcutaneous tumor-bearing mice were treated with TUSC2 according to the protocol described in the method. Serum was collected 10 days after treatment, and Luminex multiplex ELISA was performed. Chemokines between treatment groups were compared using a linear regression model. Chemokines were compared between treatment group pairs using the ESTIMATE statement in the PROC MIXED method 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 the explanation in Figure 3A. [Figure 3C] See the explanation in Figure 3A. [Figure 4A] The antitumor activity of TUSC2 is dependent on natural killer cells that produce a Th1-mediated immune response. Cytokines IL-15 and IL-18 were associated with natural killer cell regulation. (A) NK cell depletion suppressed treatment efficacy and antitumor immune response. Graphs of tumor bioluminescence intensity show signals from tumors treated with TUSC2 and combinations in NK-depleted and non-depleted mice. NK1.1 antibody was injected every 3 days for 5 times to deplete NK cells. The control group was treated with nanovesicles loaded with empty (TUSC2 gene-free) vectors. *, 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 the Luminex assay in NK-depleted and non-depleted mice. Bars are shown as the ratio of IFN-γ (Th1) to 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 changed after treatment in NK-depleted and non-depleted mice. Serum cytokines were measured using the 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) Expression ratios of IL-15Ra and IL-18R1 in NK cells selected from mice treated with TUSC2 compared to the control. Data are presented as mean ± SD, n=3, determined by multiple t-tests; *, P<0.05, **, P<0.01. (F) NanoString analysis of tumors treated with TUSC2 and TUSC2+PD1 to compare the fold changes in mRNA expression of IL-15Ra and IL-18R1. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 4E] See the explanation in Figure 4A. [Figure 4F] See the explanation in Figure 4A. [Figure 5A]Combination treatment with TUSC2 and anti-PD1 significantly improved survival and recruited natural killer cells to tumor-bearing lungs in a KRAS-mutated lung metastasis mouse model. (A) 344SQ-luc cells were used for this experimental metastasis model with knock-ins of the KrasG12D allele and the Trp53R172HΔG allele. PD-L1 expression levels were determined by flow cytometry and compared to those of CMT167-luc cells. (B) Sequential treatment with checkpoint blockade (anti-PD1 and anti-CTLA4) and TUSC2 is illustrated. (C) Survival after treatment is shown by Kaplan-Meier curves. 344SQ cells were intravenously injected, and mice were treated with TUSC2 and / or checkpoint blockade alone or in combination (shown as groups). The control group was treated with nanovesicles loaded with an empty (TUSC2 gene-free) vector, and survival time was recorded (n=10 mice per group). For statistical analysis, the Univariate Cox model was used to compare overall survival among treatment groups. Maximum survival was observed in the TUSC2+PD1 group (left), followed by TUSC2, PD1, and the control. Similarly, maximum survival was observed in the TUSC2+PD1+CTL4 group (right), followed by TUSC2, PD1+CTL4, and the control. (D) Bioluminescence images of tumor-bearing mice acquired by IVIS 200 showed lung-specific engraftment of tumor cells. Levels of signal intensity are shown between treatment groups. Representative figures from three independent experiments are shown. (E) Images of dissected lungs showing tumor nodule status 2 weeks after tumor transplantation. (F)(G)(H) and (I) Single cells were prepared from metastatic lungs from different groups according to the protocols described in Methods, and CD49b+NK cells, CD4+CD25+Treg, Gr-1+MDSC, and PD-L-1 and PD-L2(+)ve leukocyte infiltration were determined by flow cytometry. Data were normalized based on 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 presented as gram ± SD of cells / tissue, n=5. *, P<0.05, **, P<0.01, ***, P<0.001.The CONTRAST statement in the SAS PROC GENMOD method was used to compare flow data for statistical analysis. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 5E] See the explanation in Figure 5A. [Figure 5F] See the explanation in Figure 5A. [Figure 5G] See the explanation in Figure 5A. [Figure 5H] See the explanation in Figure 5A. [Figure 5I] See the explanation in Figure 5A. [Figure 6-1]Combination treatment with TUSC2 and anti-PD1 altered the immunogene expression profile in the tumor microenvironment. Gene expression analysis of a 776-gene NanoString pan-cancer immunotherapy 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, and gene profiles and statistical analyses were quantified. Positive controls, housekeeping genes, and negative controls were used to adjust for sample preparation variability, background noise, and RNA quantity variability. A linear model was used to evaluate the overall treatment effect, and pairwise comparisons of interests were performed using contrast. A beta-uniform mixture (BUM) model was used to model the resulting p-values, determine the false detection rate (FDR) cutoff, and identify significantly differentially expressed genes. (A) The heatmap shows the 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. The Volcano plot shows the segregation of genes upregulated and downregulated by the treatment. Statistically significant genes are shown in color. (C) Selected genes known in the antitumor immune response were upregulated at least twice as strongly in the combination treatment group compared to monotherapy. (D), (E), and (F) show the fold changes in CD8, INF-γ, and transcription factor (Tbx21, Gata3) expression by TUSC2, anti-PD1, and combination treatment, respectively, compared to the control. All gene expression data shown here were generated by NanoString technology. [Figure 6-2] See the explanation in Figure 6-1. [Figure 6-3] See the explanation in Figure 6-1. [Figure 7] The gating strategies of peripheral blood leukocytes and spleen cells have been shown to determine immune subpopulations using multicolor flow cytometry assays. [Figure 8A]The effect of NK depletion antibody (NK1.1) on other immune cells. NK1.1 was administered intraperitoneally five times according to the protocol described in the method. The effect of NK depletion was evaluated three days after the last injection. Splenocytes were analyzed by flow cytometry for T cells, B cells, and NK cells. (A) Gating strategy used in the analysis. (B) Percentage of each population, pooled together for CD45, CD3, CD19, and CD49b antibody staining of N=3 mouse samples, shown in bar charts. Representative scatter plots show the effectiveness of NK depletion. [Figure 8B] See the explanation in Figure 8A. [Figure 9] Effects of CD8 T depletion on other immune cells. Intraperitoneal injection of CD8 T cell depletion antibody was administered five times according to the protocol described in the methods. The effect of NK cell depletion was evaluated three days after the last injection. Splenocytes were analyzed by flow cytometry for T cells, B cells, and NK cells. Representative scatter plots show the efficacy 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 digit changes for all 13 genes are listed. A linear model was used to evaluate the overall treatment effect, and contrast was used for pairwise comparisons of the target of interest. A beta-uniform mixture (BUM) model was used to model the resulting p-values, determine the false detection rate (FDR) cutoff, and identify significantly differentially expressed genes. [Modes for carrying out the invention]

[0027] Description of exemplary embodiments Cancer development involves the disruption of several cellular pathways that control normal cell growth. Healthy cells express several tumor suppressor genes, which act as molecular gatekeepers and prevent uncontrolled cell division. Therefore, a crucial step in cancer cell development is the disruption of tumor suppressor signaling pathways. Given this, one promising approach to 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 of tumor suppressor genes function to enhance the effectiveness of tumor suppressor therapies such as TUSC2 therapy.

[0028] The research presented in this patent application is the first to demonstrate 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 the body's own immune cells to inhibit tumor growth. In contrast, therapies using tumor suppressors, such as TUSC2 therapy, aim to reverse the transformed phenotype of cancer cells. Since the latter therapies tend to make tumor cells "less transformed" and as immunogenic as possible, it has previously been counterintuitive to attempt to use TUSC2 therapy in combination with immune checkpoint inhibitors. Nevertheless, the research presented herein demonstrates that the antitumor effect of immune checkpoint inhibitors (e.g., anti-PD1 and / or CTLA4) is actually significantly enhanced when the therapy is combined with TUSC2 therapy (see, for example, Figure 1).

[0029] Specifically, in this study, anti-PD1 showed limited effects in suppressing tumor growth and extending survival in syngeneic mouse models of lung adenocarcinoma with varying levels of PDL-1 expression, specifically in two Kras mutations, G12V and G12D. However, when combined with TUSC2 gene restoration, the effects on tumor reduction and survival were far superior. TUSC2 altered both the innate and adaptive immune cell populations. This was demonstrated by a significant increase in circulating NK cells and CD8+ T cells, and a decrease in myeloid-derived suppressor cells (MDCS), regulatory T cells (Treg), B cells, T cell checkpoint receptor PD1 and T lymphocyte-associated protein 4 (CTLA-4), and mucin domain-3 (TIM-3). The proliferation of tumor-infiltrating NK cells and CD8+ T cells was induced by the TUSC2-anti-PD1 combination. In vivo depletion of NK cells or CD8+ T cells, respectively, completely and partially reduced the effect of this combination, suggesting that CD8+ T cells may contribute to TUSC2-enhancing sensitivity to anti-PD1, and that NK cells are required for this synergy. Cytokine levels of interferon-gamma (IFNγ), interleukin 15 and 18 (IL15 and IL18) significantly increased after TUSC2 recovery, which also enhanced survival with dual checkpoint blockers, anti-PD1 + anti-CTLA-4. Gene expression profile analysis showed changes in the tumor microenvironment due to TUSC2-anti-PD1 combination treatment. These data suggest that this novel combination therapy may be a potential strategy for treating Kras-mutated lung adenocarcinoma.

[0030] Therefore, the method detailed herein provides a novel method that is effective in treating cancer by 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 components of the immune system that deliver inhibitory signals to their constituents in order to modulate 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. Pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR are recognized in the art as constituting immune checkpoint pathways, similar to CTLA-4 and PD-1-dependent pathways (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 between a PD-1 axis-binding partner and one or more of its binding partners, resulting in the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing) and eliminating T cell dysfunction caused by 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 interferes with signaling 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 also be a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In certain contexts, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling 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, PD-1 binding antagonists include MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pizilizumab), or AMP-224.

[0034] The term "PD-L1-binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with 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 to its binding partners. In certain contexts, a PD-L1-binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. PD-L1-binding antagonists may include anti-PD-L1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with 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, PD-L1-binding antagonists reduce negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediate PD-L1 signaling, thereby reducing dysfunction in dysfunctional T cells (e.g., enhancing effector responses 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 interferes with 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 may also be a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. For example, a PD-L2-binding antagonist inhibits the binding of PD-L2 to PD-1. Such PD-L2 antagonists include anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1.

[0036] An "immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockage. Specifically, immune checkpoint proteins are human immune checkpoint proteins. Therefore, immune checkpoint protein inhibitors are specifically inhibitors of human immune checkpoint proteins.

[0037] Accordingly, this disclosure provides a method for enhancing the effects of immune checkpoint blockade by administering tumor suppressors such as TUSC2 therapy. As described above, immune checkpoints either increase (e.g., co-stimulatory molecules) or decrease signaling. 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 activator gene-3 (LAG3), programmed cell death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitor 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, for example, International Patent Publication 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 their analogues may be used, in particular chimeric, humanized, or human forms of antibodies. Those skilled in the art will understand that alternative names and / or equivalent names may be used for certain antibodies mentioned herein. Such alternative names and / or equivalent names are interchangeable in the context of the present invention. For example, lambrolizumab is known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0039] It is intended that any of the immune checkpoint inhibitors known in the art to stimulate an immune response may be used. These include inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. These immune checkpoint inhibitors include, but are not limited to, agents that target immune checkpoint proteins and pathways including PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, LAG3 inhibitors known in the art include soluble LAG3 (IMP321 disclosed in WO2009044273 incorporated herein by reference, or LAG3-Ig), as well as mouse antibodies or humanized antibodies that block human LAG3 (e.g., 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, though not limited to these, is provided by the use of BTLA-blocking agents, including antibodies that block the interaction of human BTLA with its ligand (such as 4C7 disclosed in WO2011014438 incorporated herein by reference). Yet another example, though not limited to these, is provided by the use of B7H4-neutralizing agents, including antibodies against human B7H4 (disclosed in WO 2013025779 and WO2013067492, respectively, incorporated herein by reference), or soluble recombinant forms of B7H4 (such as those disclosed in US20120177645, incorporated herein by reference). Yet another example, though not limited to these, is provided by B7-H3-neutralizing agents, including antibodies that neutralize human B7-H3 (e.g., MGA271 disclosed as BRCA84D and its derivative in US 20120294796, incorporated herein by reference).Further examples are provided by TIM3-targeting agents, including, but not limited to, antibodies that target human TIM3 (e.g., antibodies such as those disclosed in WO 2013006490 A2, and 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 antagonist T-cell dysfunction or anergy occurs simultaneously with the inducible and sustained 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 of treating cancer by inhibiting the PD-L1 / PD-1 interaction in combination with the administration of tumor suppressors such as TUSC2 therapy is provided.

[0041] For example, PD-1 axially coupled antagonists include PD-1-binding antagonists, PDL1-binding antagonists, and PDL2-binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0042] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain contexts, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain contexts, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain contexts, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the manner provided herein are known in the art, including those described in U.S. Patent Applications No. US20140294898, No. US2014022021, and No. US20110008369, all of which are incorporated herein by reference.

[0043] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing the extracellular portion or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP-224. Nivolumab is the anti-PD-1 antibody described in WO2006 / 121168, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Further PD-1 binding antagonists include pizilizumab, 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 IMP321 and BMS-986016, but is not limited to these. The immune checkpoint inhibitor may also be an adenosine A2a receptor (A2aR) antagonist, such as PBF-509.

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

[0046] Therefore, antibodies used herein may be nonglycosylated. Antibody glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate component to the side chain of an asparagine residue. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid other than proline), are recognition sequences for the enzymatic attachment of carbohydrate components to the asparagine side chain. Therefore, 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 following sugars—N-acetylgalactosamine, galactose, or xylose—to a hydroxylated amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of a glycosylation site from an antibody is easily achieved by modifying the amino acid sequence to remove one of the above tripeptide sequences (of the N-linked glycosylation site). The modification may be made by substituting an asparagine, serine, or threonine residue within the glycosylation site for another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0047] The antibody or its antigen-binding fragment may be produced using methods known in the art, for example, by culturing host cells containing nucleic acids encoding any of the 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 then recovering the antibody or fragment.

[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 it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is analogous to the T cell costimulatory protein CD28, both of which bind to CD80 and CD86, also known as B7-1 and B7-2, on antigen-presenting cells, respectively. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0049] In some embodiments, immune checkpoint inhibitors are anti-CTLA-4 antibodies (e.g., human antibodies, humanized antibodies, or chimeric antibodies), their antigen-binding fragments, immunoadhesins, fusion proteins, or oligopeptides.

[0050] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art may 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 tisilimunab), US Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the method disclosed herein. The teachings of each of the aforementioned 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 Application No. WO2001014424, International Patent Application No. WO2000037504, and U.S. Patent No. US8017114, all of which are incorporated herein by reference.

[0051] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and their variants (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the CDR or VR of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on CTLA-4 as the antibody described above and / or binds to that same epitope. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., 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. Patent Nos. US5844905, US5885796, and International Patent Applications WO1995001994 and WO1998042752, all of which are incorporated herein by reference, as well as immunoadhesins, such as those described in U.S. Patent No. US8329867, which are incorporated herein by reference.

[0053] C. Killer immunoglobulin-like receptor (KIR) Another immune checkpoint inhibitor for use in this disclosure is an anti-KIR antibody. Anti-human KIR antibodies (or VH / VL domains derived therefrom) suitable for use in this method can be prepared using methods well known in the art.

[0054] Alternatively, anti-KIR antibodies recognized in the art can be used. Anti-KIR antibodies may cross-react with multiple inhibitory KIR receptors, enhancing the cytotoxicity of NK cells possessing one or more of these receptors. For example, anti-KIR antibodies may bind to KIR2D2DL1, KIR2DL2, and KIR2DL3, 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, anti-KIR antibodies do not bind to KIR2DS4 and / or KIR2DS3. For example, monoclonal antibodies 1-7F9 (also known as IPH2101), 14F1, 1-6F1, and 1-6F5 can be used, as described in WO 2006 / 003179, the teaching of which is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to KIR can also be used. Further anti-KIR antibodies recognized in the art that may be used include, for example, the antibodies 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 lirirumab (also known as BMS-986015 or IPH2102). In other embodiments, the anti-KIR antibody comprises the complementarity-determining regions (CDRs) or variable regions (VRs) of the heavy and light chains of lirirumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable (VH) region of lirirumab, as well as the CDR1, CDR2, and CDR3 domains of the light chain variable (VL) region of lirirumab. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with lirirumab.

[0056] II. Tumor Suppressor Factor Therapy In a certain respect, this relates to compositions and methods for delivering nucleic acids or polypeptides to cells. Specifically, this specification provides nanoparticle-nucleic acid complexes or nanoparticle-polypeptide complexes, and methods for administering such complexes to a target. The complex comprises a TUSC2 polypeptide and / or nucleic acid associated with nanoparticles. As used herein, “association” means physical association, chemical association, or both. For example, association may include covalent bonding, hydrophobic interaction, encapsulation, or surface adsorption.

[0057] Polypeptides and nucleic acids typically have difficulty crossing the cell membrane. Both types of molecules contain charged residues, which hinder membrane binding and intracellular membrane transport. This embodiment overcomes this difficulty by providing a nanoparticle complex that promotes cellular uptake.

[0058] According to this embodiment, polypeptides and / or nucleic acids can associate with nanoparticles to form nanoparticle complexes. In some embodiments, the nanoparticles are liposomes or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP: cholesterol vesicles). Liposomes used in cancer therapy enhance liposome concentration at tumor sites by taking advantage of increased fenestration in neovascular structures of cancer.

[0059] In other embodiments, the nanoparticles are non-lipid nanoparticles, such as iron oxide-based superparamagnetic nanoparticles. Superparamagnetic nanoparticles with a diameter in the range of approximately 10–100 nm are small enough to avoid capture by the spleen and large enough to avoid clearance by the liver. Particles of this size can penetrate very small capillaries and be efficiently distributed within body tissues. Superparamagnetic nanoparticle complexes are used as MRI contrast agents to identify and track cells that take up the therapeutic complex. 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 may be nanoshells containing a gold layer covering 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 may be fullerenes or nanotubes (Gupta et al., 2005).

[0060] According to this embodiment, nanoparticle complexes can target specific tissues and cells. This can be achieved by conjugating cell-targeting components to nanoparticles. Targeting components may be, but are not limited to, proteins, peptides, lipids, steroids, sugars, carbohydrates, or synthetic compounds. Cell-targeting components, such as ligands, recognize and bind to their alloreceptors on the cell surface. Similarly, antibodies can function as cell-targeting components by recognizing their alloreantigens on the cell surface. In some embodiments, the targeted nanoparticle complexes provided herein can enhance the specificity of disease treatment and increase the amount of therapeutic agent entering target cells.

[0061] A. Nanoparticles As used herein, the term “nanoparticles” refers to any material having dimensions in the range of 1 to 1,000 nm. In some embodiments, nanoparticles have dimensions in the range of 50 to 500 nm. Nanoparticles used in this embodiment include nanoscale materials 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 into nanoparticles provides structures with applicability for targeted delivery, controlled release, enhanced cellular uptake and intracellular transport, 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; Michaelet 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 may be positively charged, negatively charged, or neutral. In one embodiment, lipid-based nanoparticles are charged to neutral (e.g., DOPC liposomes).

[0063] "Liposome" is a general term encompassing a variety of single-layer and multi-layer lipid vesicles formed by the creation of a surrounding lipid bilayer or aggregate. Liposomes can be characterized as having a vesicle structure with a bilayer generally containing phospholipids and an internal medium generally containing an aqueous composition. Liposomes provided herein include monolayer liposomes, multilayer liposomes, and multi-vesicle liposomes. Liposomes provided herein may be positively charged, negatively charged, or neutrally charged. In some embodiments, liposomes are neutrally charged.

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

[0065] In certain contexts, polypeptides or nucleic acids may, for example, be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that associate with both liposomes and polypeptides / nucleic acids, encapsulated within liposomes, or form complexes with liposomes.

[0066] The 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 make up about 5% of the weight of the composition. An excess of tert-butanol is added to the mixture so that the volume of tert-butanol is at least 95%. The mixture is vortexed and frozen in a dry ice / acetone bath and freeze-dried overnight. The freeze-dried preparation can be stored at -20°C and used for up to 3 months. If necessary, the freeze-dried 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 at least 10 times the expected volume of the 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 volume of liposomes. The composition can be further dried in a desiccator under vacuum. Due to the tendency of dried lipids to degrade over time, they are generally discarded after about one week.

[0068] The dried lipids can be hydrated to approximately 25-50 mM phospholipids with pyrogenically decontaminated sterile water by shaking until all lipid films are resuspended. The aqueous liposomes can then be divided into aliquots, each placed in a vial, freeze-dried, 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 in a suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in a vortex mixer. The liposome pellet is washed by removing any further unencapsulated substances, such as hormones, drugs, and nucleic acid constructs, by centrifugation at 29,000 × g, but not limited to these. The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50–200 mM. The amount of further encapsulated substances or activators can be determined by standard methods. After determining the amount of further encapsulated substances or activators in the liposome preparation, the liposomes may be diluted to an appropriate concentration and stored at 4°C until use. Pharmaceutical compositions containing liposomes typically include a sterile, pharmaceutically acceptable carrier or diluent, e.g., water or saline solution.

[0070] In other alternative methods, liposomes can be prepared according to other known experimental techniques (see, for example, Bangham et al., 1965; Gregoriadis, 1979; Deamer and Uster, 1983; Szoka and Papahadjopoulos, 1978, each incorporated herein by reference in the relevant parts). Further liposomes that may 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 exception. The process for producing 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 may be used in certain embodiments are disclosed in U.S. Patent No. 5,855,911, which is incorporated herein by reference. These methods differ in their respective ability to capture aqueous substances and in their respective aqueous space-to-lipid ratios.

[0071] The size of liposomes varies depending on the synthesis method. Liposomes in this embodiment can be of a variety of sizes. In one embodiment, liposomes are small, for example, with an outer diameter of about 100 nm, about 90 nm, about 80 nm, about 70 nm, less than about 60 nm, or less than about 50 nm. For example, generally, DOTAP:cholesterol liposomes for use in this embodiment include a size of about 50–500 nm before nucleic acid incorporation. 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 before nucleic acid incorporation. 400This includes [the following]. Similarly, the overall charge of such particles in solution can be defined by a zeta potential of approximately 50–80 mV.

[0072] Any protocol described herein or a protocol known to those skilled in the art may be used in the preparation of such liposomes. Further non-limiting examples of liposome preparation are described in U.S. Patents Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706, each incorporated herein by reference; 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.

[0073] In one embodiment, the lipid-based nanoparticles are neutral liposomes (e.g., DOPC liposomes). 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 within a given population (e.g., a population of liposomes), only a very small number of lipid components, if any, contain a charge that is not canceled out by the opposite charge of another component (i.e., less than 10%, more preferably less than 5%, and most preferably less than 1%, of components contain an uncharged charge). In one embodiment, the neutral liposome may primarily consist of lipids and / or phospholipids that are neutral themselves under physiological conditions (i.e., at about pH 7).

[0074] The liposomes and / or lipid-based nanoparticles of this embodiment may contain phospholipids. In one embodiment, a single type of phospholipid may be used in the preparation of liposomes (for example, a neutral phospholipid such as DOPC may be used to prepare neutral liposomes). In another embodiment, two or more types of phospholipids may be used to prepare liposomes.

[0075] Phosphatidylethanolamine and phosphatidylcholine are uncharged under physiological conditions (i.e., at approximately pH 7), and these compounds may be particularly useful for producing neutral liposomes; therefore, phospholipids include, for example, phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine. In one embodiment, phospholipid DOPCs are used to produce uncharged liposomes. In another embodiment, non-phospholipid lipids (e.g., cholesterol) may be used.

[0076] Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylcholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilaurilloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), and 1-palmi Toyl-2-myristoylphosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoylphosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"), dilaurioylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol DSPG ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoyl phosphatidylethanolamine ("DSPE"), dioleoyl phosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl Phosphatidylserine ("DMPS"), dipalmitoylphosphatidylserine ("DPPS"), cerebral phosphatidylserine ("BPS"), cerebral sphingomyelin ("BSP"), dipalmitoylsphingomyelin ("DPSP"), dimyristilphosphatidylcholine ("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1,It contains 2-diecocenoyl-sn-glycero-3-phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyleoylphosphatidylcholine ("POPC"), palmitoyleoylphosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.

[0077] Phospholipids may be derived from natural or synthetic sources. However, phospholipids from natural sources, such as phosphatidylcholine from eggs or soybeans, phosphatidic acid from brains, phosphatidylinositol from brains or plants, cardiolipin from hearts, and phosphatidylethanolamine from plants or bacteria, may not be used as the main phosphatide (i.e., constituting 50% or more of the total phosphatide composition) in some embodiments, because they may result in liposome instability and leakage.

[0078] 2. DOTAP: Cholesterol nanoparticles In one embodiment, the lipid-based vesicles are DOTAP:cholesterol nanoparticles. DOTAP:cholesterol nanoparticles are prepared by mixing the cationic lipid DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane) with cholesterol. Vesicles prepared with DNA can form a structure (called a "sandwich") in which the DNA appears to condense between two lipid bilayers (U.S. Patents 6,770,291 and 6,413,544).

[0079] DOTAP:cholesterol-nucleic acid complexes can be prepared as in the following non-limiting example. DOTAP:cholesterol (DC) nanoparticles (size 50-500 nm) 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 in a rotary evaporator for 30 minutes and freeze-dried 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 successively filtered 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 It should be uniformly dispersed at a zeta potential of 50-80 mV. The residual CHCl3 level should be less than 60 ppm.

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

[0081] B. Target-directed nanoparticles Targeted delivery is achieved by attaching ligands without impairing the nanoparticles' ability to deliver their 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. Target ligands may associate with nanoparticles noncovalently or covalently and can be conjugated to nanoparticles in a variety of ways as described herein.

[0082] Examples of proteins or peptides that can be used to direct nanoparticles to a target 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 in which the nucleic acid sequence can be replicated. The nucleic acid sequence may 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 position 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 well capable of constructing vectors by standard recombination 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 gene construct containing a nucleic acid that codes for RNA capable of transcription. 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 “regulatory sequences,” which refer to nucleic acid sequences required for the transcription and possibly translation of functionally linked coding sequences in a particular host cell. In addition to regulatory sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions and are described below.

[0085] In one embodiment, the use of a nucleic acid TUSC2 coding sequence is provided herein. For example, such a vector can be used for recombinant production of a TUSC2 polypeptide and / or in vivo expression of TUSC2 in a subject. The sequence may be modified to take into account that one amino acid is encoded by multiple different codons, while still coding the same protein or polypeptide. Codon selection optimization may be done in light of the specific organism used in recombinant expression, or it may be optimized for maximal expression in human cells (e.g., cancer cells). Vectors for use according to this embodiment further include elements that control gene expression and / or assist in vector production and purification.

[0086] 1. Promoter and Enhancer A “promoter” is a regulatory sequence, a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain a genetic element to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of the nucleic acid sequence. The statements “functionally positioned,” “functionally linked,” “under control,” and “transcriptionally regulated” mean that the promoter is in the correct functional position and / or orientation in relation to the nucleic acid sequence and controls the transcription initiation and / or expression of that sequence.

[0087] Promoters generally contain sequences that function to position the start site of RNA synthesis. The best-known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the SV40 late gene, a separate element covering the start site itself helps to fix the start location. Further promoter elements regulate the frequency of transcription initiation. Typically, these are located in a region 30–110 bp upstream of the start site, but some promoters have been shown to also contain functional elements downstream of the start site. To guide the coding sequence "under the control" of the promoter, the 5' end of the transcription start site in the transcription reading frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of DNA and promotes the expression of the encoding RNA.

[0088] The spacing between promoter elements is often mobile to ensure promoter function is maintained when elements invert or move in relation to each other. In tk promoters, the spacing between promoter elements can be increased up to 50 bp before activity begins to decrease. 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 elements 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 one that can be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be called “endogenous” or “homologous.” Similarly, an enhancer may be an enhancer located downstream or upstream of the nucleic acid sequence, naturally associated with that nucleic acid sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant exogenous or heterologous promoter, which refers to a promoter not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers not normally associated with the nucleic acid sequence in its natural environment. Such promoters or enhancers may include viral promoters and enhancers, such as the CMV promoter.

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

[0091] In addition, any promoter / enhancer combination (e.g., from 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 assist cytoplasmic transcription from a particular bacterial promoter if a suitable bacterial polymerase is provided as part of a delivery complex or as an additional gene expression construct.

[0092] 2. Translation start signal Specific start signals may also be required for efficient translation of coding sequences. These signals include ATG start codons or adjacent sequences. In some cases, it may be necessary to provide exogenous translational regulatory signals, including ATG start codons. Those skilled in the art can readily determine this and provide the necessary signals. It is well known that the start codon must be "in-frame" with the reading frame of the coding sequence desired to ensure translation of the entire insertion fragment. Exogenous translational regulatory signals and start codons may be native or synthetic. Expression efficiency may be enhanced by the inclusion of appropriate transcriptional enhancer elements.

[0093] 3. Multiple cloning sites A vector may include multiple cloning sites (MCS), which are nucleic acid regions containing multiple restriction enzyme sites that can be digested in conjunction with standard recombination techniques (see, for example, Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, 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 within 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, a vector is linearized or fragmented using restriction enzymes that cleave within the MCS, allowing for ligation of an exogenous sequence into the vector. “Ligation” refers to the process of forming a phosphate diester bond between two nucleic acid fragments that may or may not be in close proximity to each other. Restriction enzymes and the techniques involved in ligation reactions are well known to those skilled in the art in the field of recombination techniques.

[0094] 4. Splicing site Many transcribed eukaryotic RNA molecules undergo RNA splicing, in which introns are removed from the primary transcript. Vectors containing eukaryotic genome sequences may require donor and / or acceptor splicing sites to ensure appropriate transcript processing for protein expression (see, for example, Chandler et al., 1997, incorporated herein by reference). The inclusion of such splicing sites can also enhance expression by preventing the resulting nonsense mutation-dependent degradation mechanism of the RNA transcript.

[0095] 5. Termination Signals The vectors or constructs of this embodiment generally include at least one termination signal. The “termination signal” or “terminator” consists of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in one embodiment, a termination signal is intended to end the production of an RNA transcript. The terminator may be required in vivo to achieve a desired message level.

[0096] Terminators intended for use in this embodiment include, but are not limited to, any known transcriptional terminators described herein or known to those skilled in the art, including, for example, gene termination sequences, e.g., bovine growth hormone terminators, or viral termination sequences, e.g., SV40 terminators. In some embodiments, termination signals may lack a transcribed or translatable sequence due to sequence breaks or the like.

[0097] 6. Polyadenylation signal In expression, particularly in eukaryotic expression, typically a polyadenylation signal is included that results in appropriate polyadenylation of the transcript. The nature of the polyadenylation signal is not considered to be decisive to the success of the implementation of this embodiment, and any such sequence may be used. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, which are known to be simple and function well in a variety of target cells. Polyadenylation may increase the stability of the transcript or facilitate cytoplasmic transport.

[0098] 7. Origin of replication To amplify the vector in the host cell, it may contain one or more origin sites (often called "ori"), which are specific nucleic acid sequences from which replication is initiated. Alternatively, if the host cell is yeast, an ARS (arrow-response sequence) can be used.

[0099] 8. Selectable and screenable markers In one embodiment, cells containing nucleic acid constructs provided herein can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker would impart a identifiable change to the cell, enabling easy identification of the cell containing the expression vector. Generally, a selectable marker is a marker that provides a property that enables selection. A selectable positive marker is a marker whose presence enables selection, and a selectable negative marker is a marker whose presence hinders selection. An example of a selectable positive marker is a drug resistance marker.

[0100] Typically, the inclusion of drug selection markers aids in the cloning and identification of transformants; for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeosin, and histidinol are useful selectable markers. In addition to phenotypic markers that enable the identification of transformants based on the implementation of conditions, other types of markers are also considered, 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 likely know how to utilize immunomarkers in conjunction with FACS analysis. The markers used are not considered critical as long as they have the ability to be 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 one embodiment, plasmid vectors are intended for use in transforming host cells. Generally, plasmid vectors containing replicons and regulatory sequences derived from species compatible with host cells are used in association with these hosts. Vectors typically contain replication sites, as well as marking sequences that can provide 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 the E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance, thus providing a convenient means for identifying transformed cells. The pBR plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, a promoter that can be used by the microorganism for the expression of its own proteins.

[0102] In addition, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can also be used as transformation vectors in relation to these hosts. For example, phage λGEM(trademark)-11 can be used in the preparation of recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

[0103] Further useful plasmid vectors include the pIN vector (Inouye et al., 1985) and the pGEX vector, which are used to construct glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage. Other suitable fusion proteins include those involving β-galactosidase and ubiquitin.

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

[0105] 10. Viral vectors The ability of certain viruses to infect or enter cells via receptor-dependent endocytosis, integrate into the host cell genome, and stably and efficiently express viral genes makes them attractive candidates for the delivery of exogenous nucleic acids into cells (e.g., mammalian cells). Therefore, viruses encoding and expressing TUSC2 may be utilized. A non-limiting list of viral vectors that may be used to deliver TUSC2 nucleic acids is provided below.

[0106] Adenovirus vectors. Certain methods for nucleic acid delivery involve the use of adenovirus expression vectors. Adenovirus vectors are known to have a low ability to integrate into genomic DNA, but this characteristic is offset by the high efficiency of gene transfer provided by these vectors. An “adenovirus expression vector” means that it contains a construct that (a) supports the packaging of the construct and (b) contains enough adenovirus sequences to ultimately express the tissue-specific or cell-specific construct cloned therein. Knowledge of the genetic makeup, or adenovirus, which is a 36kb linear double-stranded DNA virus, allows for the replacement of large fragments of adenovirus DNA with exogenous sequences of up to 7 kb (Grunhaus and Horwitz, 1992).

[0107] AAV vectors. Nucleic acids may be introduced into cells using adenovirus-mediated transfection. Increased transfection efficiency has been reported in cell systems using adenovirus-coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated viruses (AAVs) have high integration rates and can infect non-dividing cells, making them useful, for example, for gene delivery into mammalian cells in tissue culture (Muzyczka, 1992) or in vivo. AAVs have a broad host range in terms of infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details relating to the preparation and use of rAAV vectors are described in U.S. Patents No. 5,139,941 and No. 4,797,368, which are incorporated herein by reference, respectively.

[0108] Retroviral vectors. Retroviruses have the ability to integrate their genes into the host genome, introduce large amounts of exogenous genetic material, infect a broad spectrum of species and cell types, and package within specific cell lines (Miller, 1992). To construct a retroviral vector, nucleic acid (e.g., nucleic acid encoding a protein of interest) is inserted into the viral genome, replacing a specific viral sequence, to produce a virus with replication defects. To generate virions, a packaging cell line is constructed that contains the gag, pol, and env genes but lacks LTR and packaging components (Mann et al., 1983). When a recombinant plasmid containing cDNA along with retroviral LTRs and packaging sequences is introduced into a specific cell line (e.g., by calcium phosphate precipitation), the packaging sequences allow the recombinant plasmid's RNA transcript 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 culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

[0109] Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain 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. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are constructed by multiple attenuation of HIV pathogenic genes, for example, by deleting genes env, vif, vpr, vpu, and nef, to make 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 several attractive features to various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0111] Modified viruses. The nucleic acids to be delivered may be contained within infectious viruses that have been engineered to express a specific binding ligand. Thus, the viral particles specifically bind to allogeneic receptors on target cells and deliver their contents to the cells. A novel approach designed to enable the specific targeting of retroviral vectors has been developed based on the chemical modification of retroviruses by chemically adding lactose residues to the viral envelope. This modification enables specific infection of hepatocytes via sialycoprotein receptors.

[0112] Another approach to targeting recombinant retroviruses was designed, utilizing biotinylated antibodies against retroviral envelope proteins and specific cell receptors. The antibodies were coupled via the biotin component using streptavidin (Roux et al., 1989). In vitro infection of diverse human cells possessing the surface antigens of ecotropic viruses was demonstrated using antigens against major histocompatibility complex class I and class II antigens (Roux et al., 1989).

[0113] III. Pharmaceutical preparations The pharmaceutical compositions provided herein comprise effective amounts 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 acceptable" means, where applicable, molecular entities and compositions that do not cause adverse drug reactions, allergic reactions, or other adverse reactions when administered to animals, such as humans. The preparation of pharmaceutical compositions containing at least TUSC2 nucleic acids, peptides, or nanoparticle complexes, or further active ingredients, is known to those skilled in the art in light of this 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 the preparations should meet the standards of sterility, pyrogenicity, general safety, and purity required by the FDA Office of Biological Standards.

[0114] As used herein, “pharmaceutically acceptable carriers” include any and all solvents, dispersions, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobials, antifungals), isotonic agents, absorption retarders, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, such substances, and any combination thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289–1329, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is intended.

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

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

[0117] In one preferred embodiment, the pharmaceutical composition is administered orally to treat cancer (e.g., gastrointestinal cancer).

[0118] In one embodiment, the actual dose of a composition administered to a patient can be determined by physical and physiological factors, such as body weight, severity of condition, type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and route of administration. In any case, the administerer will determine the concentration and appropriate dose of the active ingredient in the composition for each individual subject.

[0119] In one embodiment, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound. In another embodiment, the active compound may contain about 2% to about 75% of the weight of one unit, or, for example, about 25% to about 60%, or any range derived therefrom. In other non-limiting examples, doses may also include approximately 1 microgram / kg / body weight, approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 15 micrograms / kg / body weight, approximately 20 micrograms / kg / body weight, approximately 25 micrograms / kg / body weight, approximately 30 micrograms / kg / body weight, approximately 35 micrograms / kg / body weight, approximately 0.04 milligrams / kg / body weight, approximately 0.05 milligrams / kg / body weight, approximately 0.06 milligrams / kg / body weight, approximately 0.07 milligrams / kg / body weight, approximately 0.08 milligrams / kg / body weight, approximately 0.09 milligrams / kg / body weight, approximately 0.1 milligrams / kg / body weight, approximately 0.2 milligrams / kg / body weight to approximately 0.5 mg / kg / body weight or more, and any range derived therefrom. In non-limiting examples of the ranges derived from the numbers listed herein, doses such as approximately 0.01 mg / kg / body weight to approximately 0.1 mg / kg / body weight, and approximately 0.04 micrograms / kg / body weight to approximately 0.08 milligrams / kg / body weight can be administered based on the above values.

[0120] In any case, the composition may contain various antioxidants to slow the oxidation of one or more components. In addition, inhibition of microbial activity can be achieved by preservatives such as various antimicrobial 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 into compositions in free base form, neutral form, or salt form. Pharmacovigilant salts include acid addition salts, such as those formed by free amino groups of the proteinaceous composition, 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. Salts formed by free carboxyl groups can be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0122] In embodiments where the composition is in liquid form, the carrier may be a solvent or dispersion medium, but is not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin; by maintaining the required particle size by dispersion on a carrier such as a liquid polyol or lipid; by the use of 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 solutions or sprays, aerosols, or inhalants may be used in this embodiment. Such compositions are generally designed to suit the type of target tissue. In non-limiting examples, nasal solutions are typically aqueous solutions designed to be administered into the nasal cavity as drops or sprays. Nasal solutions are prepared to resemble nasal secretions in many respects so as to maintain normal ciliary function. 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, ophthalmic preparations, antimicrobial preservatives similar to those used in pharmaceuticals, or appropriate drug stabilizers may be included in the formulation if necessary. For example, various commercially available nasal preparations are known and contain drugs such as antibiotics or antihistamines.

[0124] In one embodiment, one or more polypeptides, nucleic acids, or nanoparticle complexes are prepared for administration via a route such as oral ingestion. In these embodiments, the solid composition may include, for example, a solution, suspension, emulsion, tablet, pill, capsule (e.g., hard or soft-shell gelatin capsule), sustained-release formulation, buccal composition, lozenge, elixir, suspension, syrup, cachet, or a combination thereof. The oral composition may be taken directly with food in a meal. Preferred carriers for oral administration include inert diluents, absorbable food carriers, or a combination thereof. In other embodiments, the oral composition may be prepared as a syrup or elixir. The syrup or elixir may include, for example, at least one activator, sweetener, preservative, flavoring agent, colorant, or a combination thereof.

[0125] In one preferred embodiment, the oral composition may comprise one or more binders, excipients, disintegrants, lubricants, flavorings, and combinations thereof. In one embodiment, the composition may comprise one or more of the following: binders, e.g., tragacanth gum, acacia, corn starch, gelatin, or combinations thereof; excipients, e.g., dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; disintegrants, e.g., corn starch, potato starch, alginic acid, or combinations thereof; lubricants, e.g., magnesium stearate; sweeteners, e.g., sucrose, lactose, saccharin, or combinations thereof; flavorings, e.g., peppermint, wintergreen oil, cherry flavor, orange flavor; or combinations thereof. If the dosage unit form is a capsule, in addition to the above types of materials, a carrier such as a liquid carrier may be included. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both.

[0126] Further formulations suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, typically administered for insertion into the rectum, vagina, or urethra. After insertion, the suppository softens, melts, or dissolves in the lumen. Generally, conventional carriers in suppositories may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In one embodiment, a suppository 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] Injectable sterile solutions are prepared by combining the required amount of the active compound with the various other components listed above in a suitable solvent, and then, if necessary, by sterilizing by filtration. Generally, dispersions are prepared by combining various sterilized active ingredients with a sterile vehicle containing a basic dispersion medium and / or other components. For sterile powders for the preparation of injectable sterile solutions, suspensions, or emulsions, the preferred preparation method is a vacuum drying or freeze-drying technique, which yields a powder of the active ingredient from the liquid medium that has been pre-sterilized and filtered, with any additional desired components added. The liquid medium should be appropriately buffered if necessary, and the liquid diluent should first be isotonic with sufficient saline or glucose before injection. Preparation of high-concentration compositions for direct injection is also intended, where the use of DMSO as a solvent is assumed to provide extremely rapid penetration and delivery of high concentrations of the active ingredient to a narrow area.

[0128] The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Endotoxin contamination should be kept to a minimum, at a safe level, for example, less than 0.5 ng / mg of protein.

[0129] In certain embodiments, prolonged absorption of an injectable composition can be achieved by using an absorption-delaying agent in the composition, such as aluminum monostearate, gelatin, or a combination thereof.

[0130] IV. Combination Therapy To enhance the efficacy of the nucleic acid, polypeptide, or nanoparticle complexes of this embodiment, 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 carried out with the TUSC2 therapeutic agents and / or immune checkpoint inhibitors of this embodiment in conjunction with other anticancer agents. The “anticancer” agents can negatively affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, suppressing or inhibiting cancer progression, or increasing the lifespan of the subject having cancer. More generally, these other compositions will be provided in an aggregate amount effective in killing or inhibiting the proliferation of cells. This process may involve a step of simultaneously contacting cells with anticancer peptides or nanoparticle complexes and agents or multiple factors. This may be achieved by simultaneously contacting cells with a single composition or pharmaceutical formulation containing both agents, or by simultaneously contacting cells with two different compositions or formulations, one of which contains an anticancer peptide or nanoparticle complex and the other contains a second agent. In a particular embodiment, the anti-cancer peptide can be one agent, and the anti-cancer nanoparticle complex can be the other agent.

[0132] Treatment with anti-cancer peptides or nanoparticle complexes may be performed prior to or after treatment with other agents, at intervals ranging from minutes to weeks. In embodiments where the other agents and anti-cancer peptides or nanoparticle complexes are applied to cells separately, the goal is generally to avoid a significant period of time passing between the delivery of each, so that the agents and anti-cancer peptides or nanoparticle complexes can still exert a favorable combined effect on the cells. In such cases, it is intended that both modalities and cells be brought into contact within approximately 12–24 hours of each other, more preferably within approximately 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) pass between each administration.

[0133] Similarly, in certain situations, 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". TIFF2026086498000002.tif17128

[0134] In one embodiment, the administration of the TUSC2 therapy and / or immune checkpoint inhibitors of this embodiment to the patient shall follow the general protocol for the administration of chemotherapeutic agents, taking into consideration the toxicity of the vector, if any. If necessary, the treatment cycle is expected to be repeated. Various standard therapies and surgical interventions may also be applied in combination with the hyperproliferative cell therapy described.

[0135] a. Chemotherapy Cancer treatment also includes a variety of combination therapies. In some aspects, the TUSC2 therapeutic agents and / or immune checkpoint inhibitors of this embodiment are administered (or formulated) in combination with chemotherapeutic agents. For example, in some aspects, the chemotherapeutic agents are protein kinase inhibitors such as 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. Non-exclusive examples of protein kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, and dasatinib. Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mbritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Salakatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, V This includes QD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridafololimus, arbocidib, genistein, selumetinib, AZD-6244, batalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0136] Further combination chemotherapy includes, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquan, metsuredopa, and uredopa; ethyleneimines and methylamelamellamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); briostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duoc Lumycin (including synthetic analogs, KW-2189 and CB1-TM1); Eloyterobin; Pancratistatin; Sarcodictiin; Spongistatin; Nitrogen mustards such as chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, uracil mustard; Nitrosourea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; Antibiotics such as engine antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1); Dinemycin including Dinemycin A; Bisphosphonates such as clodronate; Esperamycin;Furthermore, neocardinostatin chromofoa and related pigment proteins enediin antibiotics chromofoa, acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidoxorubicin, and deoxydoxorubicinone), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin Icin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; antiadrenal agents such as mitotane and trilostane; frolinic acid Folic acid supplements such as acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diazicone; erflorumitin; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidynin; meitansinoids such as meitansin and ansamitosin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lyzoxin;Schizophyllan; spirogermanium; tenuazonic acid; triadiquan; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, bergarin A, loridine A, and anguidin); urethanes; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinosides ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxelge This includes mucitabine; 6-thioguanine; mercaptopurine; platinum-coordinate complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycinone; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, precomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, trans platinum, and any pharmaceutically acceptable salts, acids, or derivatives of the above. In some embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, this embodiment may be carried out in combination with Gleevec (for example, the patient may be administered about 400 to about 800 mg / day of Gleevec). In some embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.

[0137] b. Radiation therapy Other widely used factors that cause DNA damage include gamma rays, X-rays, and / or direct delivery of radioisotopes to tumor cells, as are commonly known. Other forms of DNA damage factors, such as microwave and UV irradiation, are also considered. All of these factors are likely to cause extensive damage to DNA, DNA precursors, DNA replication and repair, as well as chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens over long periods (3–4 weeks) to a single dose of 2000–6000 roentgens. The dose range for radioisotopes is highly variable and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by newly formed cells.

[0138] The terms “contact” and “exposure” are used herein to describe a process by which, when applied to cells, a therapeutic composition and a chemotherapeutic agent or radiotherapeutic agent are delivered to or directly juxtaposed with target cells. To achieve cell toxicity or quiescence, both agents are delivered to the cells in an aggregate amount effective in killing the cells or preventing cell division.

[0139] c. Immunotherapy Immunotherapy generally relies on the use of immune effector cells and molecules that target and destroy cancer cells. Immune effectors may be, for example, antibodies specific to certain markers on the surface of tumor cells. Antibodies may function alone as effectors of therapy, or they may mobilize other cells to actually cause cell killing. Antibodies may also be conjugated with drugs or toxins (such as chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and function solely as targeting agents. Alternatively, effectors may be lymphocytes possessing surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0140] Therefore, immunotherapy can be used as part of a combination therapy in combination with the TUSC2 therapy of this embodiment. A general approach to combination therapy is described below. Generally, tumor cells must have several markers that are applicable to targeting, i.e., markers that are not present in the majority of other cells. Numerous tumor markers exist, and any of these may be suitable for targeting in the context of this embodiment. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tract 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 a gene therapy in which a therapeutic polynucleotide is administered before, after, or concurrently with the therapeutic composition. Viral vectors for the expression of gene products are well known in the art and include eukaryotic expression systems such as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia virus, and papillomaviruses including SV40. Alternatively, the administration of the expression construct can be achieved by liposomes or lipid-based vectors such as 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 would have a combined anti-overgrowth effect on target tissues. A variety of proteins are included within the scope of this embodiment, some of which are described below.

[0143] i. Inhibitors of cell proliferation As mentioned above, tumor suppressor oncogenes have the function of inhibiting excessive cell proliferation. Inactivation of these genes destroys their inhibitory activity, resulting in uncontrolled proliferation.

[0144] Genes that can be used 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 fusion, p21 / p27 fusion, 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 significant role in regulating apoptosis and enhancing cell viability in response to diverse 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). Evolutionarily conserved Bcl-2 proteins are now recognized as members of a family of related proteins and can be classified as either death agonists or death antagonists.

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

[0147] e. Surgery Approximately 60% of humans with cancer undergo some type of surgical procedure, including prophylactic, diagnostic or staging, curative, and palliative surgery. Curative surgical procedures are cancer treatments that can be used in combination with other therapies such as the treatments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies provided herein.

[0148] Curative surgical procedures include resections in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumorectomy refers to the physical removal of at least part of a tumor. In addition to tumorectomy, surgical procedures include laser surgery, cryosurgery, electro-surgery, and microsurgery (Mohs surgery). This aspect is further contemplated to be used in combination with the removal of superficial cancer, pre-cancer, or an incidental amount of normal tissue.

[0149] A cavity may be formed in the body by the excision of cancerous cells, tissue, or part or all of a tumor. Treatment may be achieved by perfusion, direct injection, or local application of further anticancer treatment to the area. Such treatment may 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 may also be administered in various doses.

[0150] f. Anti-inflammatory drugs In certain contexts, TUSC2 therapy and / or immune checkpoint inhibitors are administered in combination with anti-inflammatory agents. Anti-inflammatory agents are defined herein as agents known or suspected to be useful in treating or preventing inflammation in a subject. Corticosteroids are a major class of anti-inflammatory agents. Corticosteroids can act for short, intermediate, or long periods and can be delivered in a variety of ways. A non-exclusive list of corticosteroids contemplated in this embodiment includes oral corticosteroids such as cortisone, hydrocortisone, prednisone, and dexamethasone.

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

[0152] Other anti-inflammatory agents include antirheumatic agents such as gold salts (e.g., sodium aurothiomalate, 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, mechloretamine, cyclophosphamide, chlorambucil, cyclosporine, and azathioprine). Other immunosuppressants targeted by this embodiment are tacrolimus and everolimus. Tacrolimus inhibits 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, as well as other members of this class, and their mechanisms of action and applications.

[0153] g. Other agents Other agents may be used in combination with the compositions provided herein to improve the therapeutic efficiency of the treatment. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and gap junctions, cell division inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferating 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. Upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL, is further intended to enhance the apoptosis-inducing ability of the compositions provided herein by establishing autocrine or paracrine action on hyperproliferating cells. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on nearby hyperproliferating cell populations. In other embodiments, cell division inhibitors and differentiation agents can be used in combination with the compositions provided herein to enhance the anti-hypertrophic effect of the treatment. Cell adhesion inhibitors are intended to enhance the effects of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further intended that other agents that increase the sensitivity of hyperproliferating cells to apoptosis, such as antibody c225, can be used in combination with the compositions provided herein to enhance the treatment effect.

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

[0155] V. Examples The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in carrying out the invention and thus constitute a preferred mode for its implementation. However, those skilled in the art should understand that, in light of this disclosure, many modifications are made in certain embodiments that still achieve similar or identical results without departing from the spirit and scope of the invention.

[0156] Example 1 - TUSC2 therapy in combination with an immune checkpoint inhibitor Combination treatment with TUSC2 and anti-PD1 effectively inhibits tumor growth in a G12V Kras mutant syngeneic subcutaneous lung model: Mouse lung cancer cell line CMT / 167-luciferase, carrying the Kras G12V mutation and low levels of TUSC2 expression, was subcutaneously transplanted into 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. 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). Anti-PD1 showed limited effect in suppressing tumor growth, while TUSC2 significantly inhibited tumor growth (Figure 1C). The combination further enhanced tumor reduction with TUSC2. The mean volumes for isotype control, anti-PD1, TUSC2, and TUSC2 + anti-PD1 were 800 mm³. 3 , 600mm 3 , 300mm 3 , and 180mm 3 ( * p<0.05; ** p<0.01; and *** (p<0.001). IVS imaging, which measures bioluminescence intensity in tumors as total flux per second, also shows a combined effect (Figure 1D-E). The posterior probability of synergistic action between TUSC2 and anti-PD1 was over 99%. These results suggest that in this model, TUSC2 exhibits a synergistic effect with anti-PD1 in reducing tumor growth.

[0157] Combination treatment with TUSC2 and anti-PD1 is effective for NK cells and CD8 +To illustrate the immunological effects of TUSC2 in combination with anti-PD1, which increases T cell density and suppresses regulatory cells, the main immune populations in peripheral blood leukocytes (PBLs) and spleen cells were characterized using 10-color panel flow cytometry. Figure 2A shows the effects of intravenous delivery of TUSC2 nanovesicles on peripheral NK cells, B cells, and T cells in tumor-free mice. There was no difference between TUSC2 and TUSC2 + anti-PD1 in tumor-free mice. The gating strategy for flow cytometry analysis is shown in Supplementary Figure 1. After tumor cell inoculation, TUSC2 affected NK cells and CD8 cells. + It was found that the combination treatment significantly and moderately induced T cell density (p<0.001 and p<0.05), while simultaneously significantly reducing B cells, MDSCs, Tregs, and T cells expressing PD1, CTLA4, and Tim3 (Figures 2B-E). Anti-PD1 did not have a 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 biggest difference in the combination treatment was the ratio of NK cells to MDSCs and the ratio of CD8 to Tregs. + The result was an enhancement of the T cell ratio (p<0.001) (Figure 2F). In summary, these results suggest that the TUSC2-anti-PD1 synergy is likely due to the enhancement of NK cells and CD8 cells. + This indicates a correlation with increased T cell proliferation.

[0158] Combination treatment with TUSC2 and anti-PD1 is effective against tumor-infiltrating NK cells and CD. +To enhance 8T cells: To determine whether TUSC2 + anti-PD1 treatment is associated with higher density tumor immune cell infiltration, immunoinfiltration was analyzed using the Vectra high-throughput pathology system covering 25% of the area of ​​each tumor (N=5 per treatment group). The entire subcutaneous tumor was uniformly excised for treatment, and multiple sections from each tumor were analyzed to account for varying tumor sizes between different treatment groups and eliminate any potential sampling bias. H-score values ​​were used in conjunction with the percentage of positive cells, taking staining intensity into consideration. Compared to control or anti-PD1, the combination was associated with higher CD8 in the tumor. + T cell density was increased tenfold and threefold, respectively (p<0.0001; Figure 3A). CD8 in the TUSC2 group + T cell infiltration was lower in TUSC2-treated tumors than in the combination, but not statistically significant. 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 to TUSC2 or the combination. Conversely, TUSC2 and TUSC2+anti-PD1 significantly suppressed MDSCs with tumor-infiltrating Foxp3-positive T cells (p<0.0001) and tumor-suppressive granulocytic marker 1 (Gr-1) (p<0.0001), markers expressed by Tregs. Anti-PD1 slightly reduced Foxp3 density (p=0.18) but had no effect on GR1. These results suggest that TUSC2 and the combination altered the tumor immunomicroenvironment.

[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. Upregulation of the set of chemokine genes associated with T lymphocyte and NK cell migration was observed after exposure to TUSC2 and the combination (Figure 3B). Expression of CcL3 and CcL4, which are involved in NK cell migration via CCR5 recognition, more than doubled, while CcL21a and CcL19 (Viola et al., 2012; Griffith et al., 2014), which interact with the CCR7 receptor and recruit T cells and dendritic cells to tumors, increased more than four-fold compared to untreated controls. Serum chemokine levels of CcL4 and CcL5 also increased with TUSC2 and TUSC2 + anti-PD1 treatment compared to the control group (Figure 3C).

[0160] NK cells or CD8 + T cell depletion completely or partially eliminates the combined antitumor effects of NK cells and CD cells, respectively. + The finding that both densities were strongly upregulated after combination therapy is related to CD8 + This suggests that T cells and NK cells regulate TUSC2+ anti-PD1-induced tumor reduction. To confirm this hypothesis, we used anti-NK1.1 antibodies or anti-CD8 + Intraperitoneal injection of T cell antibodies to NK cells or CD8 + T cells were depleted in CMT167 tumor-bearing mice (Figures 8 and 9). As shown in Figure 4A, treatment with anti-NK1.1 antibody completely eliminated the combination of tumor reduction, whereas treatment with anti-CD8 + Treatment with T antibodies partially reduced it (Figure 4B). Furthermore, NK cell depletion rendered TUSC2-induced tumor growth inhibition ineffective, whereas CD8 + T cell depletion had no effect. Neither type of depletion had any effect on the anti-PD1 response. These findings are related to CD8 +While T cells may contribute to the enhancement of anti-PD1 sensitivity by TUSC2, NK cells are suggested to be essential for this synergistic effect.

[0161] Next, analysis of serum cytokines using the Luminex assay revealed that both TUSC2 and the combination induced a potent Th1-mediated immune response (control vs. TUSC2: p<0.0001; control vs. combination: p=0.007 (Figure 4C)), which was negated by NK depletion (TUSC2 vs. TUSC2 / NK1.1: p=0.008; combination vs. NK1.1: p=0.0009). This suggests that NK cells play a crucial role in inducing the Th1-mediated immune response against TUSC2 and the combination. However, there was no significant difference in the Th1 / Th2 ratio between these two treatment groups, regardless of the presence or absence of NK depletion. TUSC2 + anti-PD1 therapy promoted higher levels of IL-15 (p=0.0001) and IL-18 (p<0.0001) cytokines compared to its untreated or anti-PD1-treated counterparts (Figure 4D). IL-15 was induced at the same level in both the TUSC2 and combination groups, while IL-18 levels were significantly higher in TUSC2 than in the combination group. Depletion of NK cells significantly reduced IL-15 (control vs. TUSC2: p=0.03) and IL-18 (control vs. TUSC2: p=0.0005). A significant difference in IL-18 levels existed between TUSC2 and the combination group regardless of NK depletion, but not for IL-15. Finally, expression profiles of selected NK cells and tumor tissue using qPCR and nanostring techniques showed significantly higher IL-15R and IL-18R expression in the TUSC2-treated group compared to the untreated and anti-PD1-treated counterpart groups (p=0.01 and p=0.001, respectively; Figures 4E, F).

[0162] Combination treatment with TUSC2 and anti-PD1 improved survival in a syngeneic G12D Kras mutation lung metastasis model: The efficacy of TUSC2 + anti-PD1 was evaluated in a second Kras metastatic model using 129Sv mice intravenously inoculated with 344SQ-luciferase lung cancer cells carrying the K-rasG12D mutation. PD-L1 expression levels in 344SQ cells were only 4.5% (Figure 5A). The sequential treatment strategy is shown in Figure 5B. The treatment groups were similar to those in the aforementioned 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 because of reports of enhanced clinical efficacy compared to each drug group alone (Larkin et al., 2015). TUSC2 significantly improved survival compared to the untreated group, the anti-PD1 group, and the anti-PD1 + anti-CTLA4 treated group (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 extended survival (combination vs. control: p<0.0001; combination vs. anti-PD1: p<0.001; combination vs. TUSC2: p=0.024). The combination of TUSC2 with anti-PD1 and anti-CTLA4 extended survival by several days compared to TUSC2 + anti-PD1 treatment. Bioluminescence imaging of tumors supported these findings (Figure 5D). Figure 5E shows impressive clearance of tumor nodules in lungs treated with TUSC2 + anti-PD1 at week 2. These results demonstrate the effectiveness of the TUSC2 + anti-PD1 combination and suggest that the combination of dual checkpoint blockade, anti-PD1 and anti-CTLA4, with TUSC2 has 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 combination treatment group of anti-PD1 with anti-CTLA4 (p<0.001) (Figure 5F). The effects of TUSC2 + anti-PD1 or TUSC2 + anti-PD1 + anti-CTLA4 were slightly higher than those of TUSC2 alone. In contrast, Treg and MDSC cell infiltration was significantly suppressed by anti-PD1 (p=0.004; p=0.0003), and the effect was further enhanced by TUSC2 and combinations (Figures 5G and 5H). These results were consistent with those observed in the evaluation of the CMT167 subcutaneous tumor model (Figure 2).

[0164] The combination of TUSC2 with anti-PD1 altered the immunogene expression profile in the tumor microenvironment: To identify specific immunogenes 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 immunogenes covering both adaptive and innate responses with 40 housekeeping controls. Welch's t-test, corrected for false discovery rate (q<0.05), was applied to derive statistically significant differences in gene expression between treatment groups. A p-value of <0.05 was considered significant. Results were visualized using Volcano plots and heatmaps (Figure 6A, B). Since 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 that were significantly upregulated in the combination groups were identified. This includes Cd1d2, Ltf, Klra21, H60a, Tnfsf18, and Bcl6. Another cluster that is significantly downregulated was found, consisting of Egr3, Cd46, Ncr1, Klra5, Ccl1, Il12rb2, and Cd59b (Figure 6B). All of these genes are found in NK cells and CD8 +It is 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 are important for NK and CD8 + This supports T immunoprofiling and tumor invasion data (Figures 2-3).

[0165] Example 2 - Materials and Methods Cell culture and reagents: KRasG12 / CMT167-luc cells 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 medium supplemented with 10% fetal bovine serum (Atlanta Biological, GA) and 1% penicillin and streptomycin (life science technologies). Isotypes, anti-PD1, anti-CTLA4, InVivoPlus anti-NK1.1 (clone PK136), and anti-CD8 + The 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 (Albaster, AL). The synthesis and preparation of DC-TUSC2 were previously described (Ito et al., 2004).

[0166] Animal Testing: All animal methods were reviewed and approved by the Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center. In the CMT167-luc syngeneic model, 6-8 week old female C57BL / 6-Elite mice (Charles River Laboratories, Houston, TX) were subjected to 1 × 10⁶ doses. 6CMT167-luc cells were subcutaneously injected into the right flank, and 10 mice each were randomized to one of the following treatment groups: 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 3 cycles, and 0.25 mg of anti-PD1 antibody was injected intraperitoneally (ip) every 4 days for 3 cycles. Tumor volume was calculated using the formula: 1 / 2 (length × width). 2 Calculations were performed using ). Mice were euthanized 3–4 weeks after tumor cell injection, and tumors and spleens were collected. In the 344SQ metastasis model, 100,000 344SQ-luc cells were intravenously injected into 6–8 week old female 129 / Sv mice. The treatment groups / 10 mice were as follows: control (empty vector nanovesicles, isotype antibodies), 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 double-blind. For immunophenotypic analysis, animals were killed 2 weeks after tumor cell injection, lungs were collected, and peripheral blood was collected via cardiac puncture.

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

[0168] Multicolor flow cytometry: PBLs were isolated and stained according to standard flow cytometry protocols. Multicolor panels were developed and optimized using the 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 and staining buffer, incubated with a mouse Fc receptor binding inhibitor for 10 minutes, and stained with the indicated antibodies. Data were analyzed using FlowJo software version 10 (FlowJo, Ashland, OR).

[0169] Immunohistochemical Analysis: Tumors recovered using CMT167 were fixed with 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 the Imaging Core Facility of MD Anderson (Houston, 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). H-scores ranging from 0 to +3 were 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 the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Relative expression levels were normalized and measured using the 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 were used per treatment group, and the assay was repeated twice. Briefly, seven standards were prepared according to the manufacturer's protocol, 25 μL of serum sample was mixed with the antibody-coated beads indicated, and incubated at room temperature for 2 hours with agitation at 500 rpm. The ProcartaPlex multiplex immunoassay was performed using 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: For quality control and expression profile analysis using NanoString Technology, total tumor RNA extracted from three replicas from each treatment group using the Qiagen RNeasy Mini Kit was submitted to the Genomic Core Facility at Baylor College of Medicine (Houston, TX). The NanoString PanCancer mouse immunoprofiling panel used profiles 776 genes associated with specific immune cell types and immune cell functions. Data were analyzed at the Bioinformatics Core Facility at MD Anderson.

[0173] Statistical Analysis: Tumor growth was analyzed for the CMT167 model using a generalized linear regression model. All data were expressed as mean ± SD. Statistical significance of differences between treatments was tested using two-way ANOVA and side-by-side t-tests, 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 examine the difference in survival time between groups. Regression analysis of survival time data based on the Cox proportional hazards model was performed for OS, defined as the time from treatment initiation to death.

[0174] Statistical analysis of flow cytometry and Luminex data was performed using generalized linear regression models to compare different treatment groups. For immunohistochemical data, generalized linear regression models were used for statistical analysis of H-scores between treatment groups. The ESTIMATE statement in the SAS PROC MIXED method was used between each pair. For NanoString analysis, data were normalized before use in gene profile quantification and statistical analysis. Positive controls, housekeeping genes, and negative controls were used to adjust for sample preparation variability, background noise, and RNA quantity variability. The overall treatment effect was evaluated using linear models, and pairwise comparisons of interests were performed using contrast. The resulting p-values ​​were modeled using a beta-uniform mixture (BUM) model to determine the false detection rate (FDR) cutoff and identify significantly differentially expressed genes. All statistical analyses were performed using the statistical analysis software R.

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

[0176] References The following references are incorporated herein by reference to the extent that they provide exemplary techniques or other details that supplement what is described herein. TIFF2026086498000003.tif189150TIFF2026086498000004.tif231150TIFF2026086498000005.tif231141TIFF2026086498000006.tif231150TIFF2026086498000007.tif230150TIFF2026086498000008.tif86128

Claims

[Claim 1] The invention described in the specification of this application.

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