Combination of IL-12 gene therapy and immune checkpoint inhibitors for treatment of cancer

Abstract

The present disclosure relates to combination therapies comprising an immune checkpoint inhibitor, a nanoparticle formulated plasmid comprising an IL-12 encoding nucleic acid, and optionally at least one adjuvant chemotherapeutic drug, as well as methods of treatment using such combination therapies and / or compositions.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application 63 / 375,529, filed on September 13, 2022, which is hereby incorporated by reference in its entirety.

[0003] Regarding Electronically Submitted Sequence Listing

[0004] The content of the electronically submitted sequence listing in the.XML file submitted with the application (Name: 2437_082PC01_Sequencelisting_ST26; Size: 102,830 bytes; and Creation Date: August 31, 2023) is hereby incorporated by reference in its entirety herein. Technical Field

[0005] This disclosure relates to the fields of cancer therapy, gene therapy, and immunology. Background Art

[0006] IL - 12 is one of the most active cytokines used to stimulate an immune response against cancer. However, when administered as a recombinant protein, the pharmacokinetics of IL - 12 require its administration by frequent, large boluses, resulting in severe toxicities that limit its use. GEN - 1 is an IL - 12 DNA plasmid vector formulated using a lipid - polymer delivery system. GEN - 1 can be delivered locally (e.g., intraperitoneally), offering the potential for the cytokine to be specifically expressed in the tumor microenvironment, with the goal of achieving increased efficacy while minimizing potential systemic toxicity.

[0007] Traditional chemotherapy regimens are designed to inhibit tumor growth through cytotoxic mechanisms, while immunocytokine therapies are designed to elicit tumor killing by enhancing the immune system against cancer cells. GEN - 1 reduces the toxicity issues associated with IL - 12. Its nanoparticle characteristics allow for cell transfection, followed by continuous local secretion of IL - 12 at therapeutic levels while avoiding the toxicities associated with recombinant IL - 12. Summary of the Invention

[0009] Certain aspects of this disclosure relate to combination therapies that include: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin - 12 (IL - 12) formulated with a lipid - polymer (e.g., a nanoparticle); and (ii) an immune checkpoint inhibitor.

[0010] Certain aspects of the present disclosure relate to methods of treating a subject having cancer, the method comprising administering to the subject a combination therapy comprising: (i) a nucleic acid carrier (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer (e.g., a nanoparticle); and (ii) an immune checkpoint inhibitor.

[0011] In some aspects, the polynucleotide encodes human IL-12.

[0012] In some aspects, the nucleic acid carrier (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL12.

[0013] In some aspects, the promoter is a CMV promoter.

[0014] In some aspects, the nucleic acid carrier (e.g., a plasmid) comprises an intron, a 3’UTR (e.g., hGH 3’UTR), an antibiotic resistance gene, or any combination thereof (e.g., Figure 1 elements).

[0015] In some aspects, the lipid polymer comprises polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups (e.g., Figure 2 lipid polymer).

[0016] In some aspects, the combination further comprises an anti-cancer agent.

[0017] In some aspects, the anti-cancer agent is a chemotherapeutic agent.

[0018] In some aspects, the chemotherapeutic agent is selected from the group consisting of: topoisomerase inhibitors (e.g., irinotecan, topotecan, doxorubicin, epirubicin, idarubicin), anti-microtubule agents (e.g., paclitaxel, docetaxel), alkylating agents (e.g., cyclophosphamide, dacarbizine), platinum drugs (cisplatin, carboplatin, oxaliplatin), anti-metabolites (e.g., gemcitabine, methotrexate, 5-fluorouracil), or combinations thereof.

[0019] In some aspects, the chemotherapeutic agent is selected from the group consisting of: doxorubicin, paclitaxel, carboplatin, docetaxel, albumin-bound paclitaxel, olaparib, and any combination thereof.

[0020] In some aspects, the anti-cancer agent is doxorubicin.

[0021] In some aspects, the anti-cancer agent is paclitaxel.

[0022] In some aspects, the anti-cancer agent is carboplatin.

[0023] In some aspects, the anti-cancer agent is docetaxel.

[0024] In some aspects, the anti-cancer agent is albumin-bound paclitaxel.

[0025] In some aspects, the anti-cancer agent is olaparib.

[0026] In some aspects, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2) and / or LAG-3.

[0027] In some aspects, the immune checkpoint inhibitor is an antibody.

[0028] In some aspects, the immune checkpoint inhibitor is a small molecule inhibitor.

[0029] In some aspects, the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and cemiplimab.

[0030] In some aspects, the immune checkpoint inhibitor is a PD-1 antagonist, wherein the PD-1 antagonist is nivolumab.

[0031] In some aspects, the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0032] In some aspects, the immune checkpoint inhibitor is a CTLA-4 antagonist, which is ipilimumab.

[0033] In some aspects, the immune checkpoint inhibitor is a LAG-3 antagonist, wherein the LAG-3 antagonist is relatlimab.

[0034] In some aspects, the combination therapy further comprises (c) a second immune checkpoint inhibitor.

[0035] In some aspects, the second immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2) and / or LAG-3.

[0036] In some aspects, the second immune checkpoint inhibitor is an antibody.

[0037] In some aspects, the second immune checkpoint inhibitor is a small molecule inhibitor.

[0038] In some aspects, the second immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and cemiplimab.

[0039] In some aspects, the second immune checkpoint inhibitor is nivolumab.

[0040] In some aspects, the second immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0041] In some aspects, the second immune checkpoint inhibitor is a CTLA-4 antagonist, which is ipilimumab.

[0042] In some aspects, the second immune checkpoint inhibitor is a LAG-3 antagonist, wherein the LAG-3 antagonist is relatlimab.

[0043] In some aspects, the method further includes performing surgery (e.g., intermediate cytoreductive surgery) in a subject to remove all or part of the tissue or tumor.

[0044] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered intratumorally or intraperitoneally.

[0045] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered intravenously.

[0046] In some aspects, the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, intravenously, intracystically, or any combination thereof.

[0047] In some aspects, the immune checkpoint inhibitor is administered intratumorally or intraperitoneally.

[0048] In some aspects, the immune checkpoint inhibitor is administered intravenously.

[0049] In some aspects, the immune checkpoint inhibitor is administered intracystically.

[0050] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the immune checkpoint inhibitor.

[0051] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the anti-cancer agent.

[0052] In some aspects, an anti-cancer agent (e.g., the first) is administered, followed by the administration of the nucleic acid carrier formulated with a lipid polymer (e.g., the second), and then followed by the administration of the immune checkpoint inhibitor (e.g., the third).

[0053] In some aspects, an anti-cancer agent (e.g., the first) is administered, followed by the administration of the nucleic acid carrier formulated with a lipid polymer (e.g., the second), followed by the immune checkpoint inhibitor (e.g., the third), and then followed by surgery to remove all or part of the tissue or tumor (e.g., intermediate cytoreductive surgery) (e.g., the fourth).

[0054] In some aspects, surgery is administered to remove all or part of a tissue or tumor (e.g., intermediate debulking surgery) (e.g., first), followed by administration of a nucleic acid carrier formulated with a lipid polymer (e.g., second), and then followed by administration of an immune checkpoint inhibitor (e.g., third, fourth, etc., depending on how many immune checkpoint inhibitors are administered).

[0055] In some aspects, an anti-cancer agent is administered, followed by administration of a nucleic acid carrier (e.g., a DNA plasmid) comprising a polynucleotide encoding IL-12, followed by an immune checkpoint inhibitor, followed by intermediate debulking surgery.

[0056] In some aspects, administration of the anti-cancer agent comprises administering paclitaxel at a dose of about 25 - 250 mg / m 2 and optionally, subsequently administering carboplatin IV at a dose of about AUC 4 - 6.

[0057] In some aspects, administration of the anti-cancer agent comprises administering docetaxel at a dose of 25 - 250 mg / m 2 and optionally, subsequently administering carboplatin IV at a dose of about AUC 4 - 6.

[0058] In some aspects, administration of the anti-cancer agent comprises administering albumin-bound paclitaxel at a dose of 25 - 350 mg / m 2 and optionally, subsequently administering carboplatin IV at a dose of about AUC 4 - 6.

[0059] In some aspects, interleukin-12 (IL-12) formulated with a lipid polymer (e.g., nanoparticles) is administered at a dose of about 35 mg / m 2 to about 80 mg / m 2 .

[0060] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer (e.g., glioblastoma), lung cancer, any combination thereof, and metastases of any of the foregoing cancers.

[0061] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0062] In some aspects, the subject is a human.

[0063] In some aspects, the lipid polymer (e.g., nanoparticles) is administered at a dose of about 60 mg / m 2 .

[0064] In some aspects, the immune checkpoint inhibitor is nivolumab, and wherein nivolumab is administered at about 240 mg.

[0065] In some aspects, nivolumab and a lipid polymer (e.g., a nanoparticle) are administered every 1 - 4 weeks (e.g., every 2 weeks) during treatment.

[0066] In some aspects, the second inhibitor is ipilimumab, and wherein ipilimumab is administered at about 1 mg / kg.

[0067] In some aspects, ipilimumab is administered once every 2 - 8 weeks (e.g., every 6 weeks) during treatment.

[0068] In some aspects, the nucleic acid carrier is a plasmid.

[0069] In some aspects, the lipid polymer is a nanoparticle. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 An exemplary human IL-12 (hIL-12) expression plasmid is shown.

[0071] Figure 2 A PEG-PEI-cholesterol structure is shown. DETAILED DESCRIPTION OF THE INVENTION

[0073] I. DEFINITIONS

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the present application, including definitions, will control. Unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0075] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and the claims.

[0076] Unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include plural referents. The terms “a” (or “an”) and the terms “one or more” and “at least one” are used interchangeably herein. In certain aspects, the term “a” or “an” means “single.” In other aspects, the term “a” or “an” includes “two or more” or “plural.”

[0077] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in connection with a numerical range, it modifies the range by extending the boundaries above and below the numerical values. Generally, the term "about" is used herein to modify a numerical value that varies by up to 10% above and below the stated value, up or down (higher or lower).

[0078] Throughout this disclosure, various aspects are presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of this disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. The recited numerical ranges include the numbers defining the range and include every integer within the defined range.

[0079] Units, prefixes, and symbols are expressed in their internationally accepted SI form. Numerical ranges include the numbers defining the range. In the case of a recited numerical range, it should be understood that every intermediate integer value and every fraction thereof between the upper and lower limits of the range are also specifically disclosed, as well as every sub-range between these values. The upper and lower limits of any range may be independently included in or excluded from the range, and every range that includes either limit, excludes a limit, or includes both limits is also covered in this disclosure. Accordingly, the ranges recited herein are understood to be a shorthand for all values within that range, including the recited endpoints. For example, a range of 1 to 10 should be understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0080] In the case of expressly recited values, it should be understood that values that are approximately the same quantity or amount as the recited values are also within the scope of this disclosure. In the case of a disclosed combination, every sub-combination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, in the case of separately disclosed different elements or groups of elements, their combinations are also disclosed. In the case where any disclosed element is disclosed as having multiple alternatives, examples of this disclosure are also hereby disclosed in which each alternative is excluded, either individually or in any combination with other alternatives; more than one element of this disclosure may have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0081] As used herein, the term "and / or" shall be taken to specifically disclose each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0082] It should be understood that, whatever aspects are described in this text using the language "comprising", other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0083] As used herein, the term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" means the amount or quantity of a drug or pharmaceutically active substance sufficient to elicit the desired or expected therapeutic response, or in other words, an amount sufficient to elicit a significant biological response when administered to a patient.

[0084] "Transfecting" or "transfection" shall mean the transport of nucleic acid from the extracellular environment of a cell to the intracellular environment, particularly to the cytoplasm and / or the nucleus. Without being bound by any particular theory, it is understood that the nucleic acid can be delivered to the cell after being encapsulated in or adhered to or entrained by one or more cationic polymer / nucleic acid complexes. Specific examples of transfection deliver nucleic acid to the nucleus. Nucleic acids include DNA and RNA and their synthetic homologs. Such nucleic acids include missense, antisense, nonsense, and protein-producing nucleotides, nucleotides that control the on and off and rate regulation of protein, peptide, and nucleic acid production. In particular, but not limited to, they can be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences, or synthetic or semi-synthetic sequences, and are of natural or artificial origin. In addition, the size of the nucleic acid can be variable, ranging from oligonucleotides to chromosomes. These nucleic acids can be of human, animal, plant, bacterial, viral, or synthetic origin. They can be obtained by any technique known to those skilled in the art.

[0085] As used herein, the term "agent" or "drug" or any other similar term refers to any chemical or biological material or compound suitable for administration by methods previously known in the art and / or by methods taught by the present disclosure, which induces a desired biological or pharmacological effect, the desired biological or pharmacological effect may include, but is not limited to, (1) having a prophylactic effect on an organism and preventing undesired biological effects, such as preventing infection, (2) alleviating the symptoms caused by a disease, such as alleviating pain or inflammation caused by a disease, and / or (3) alleviating, reducing or completely eliminating a disease from an organism. The effect can be local, such as providing a local anesthetic effect, or it can be systemic.

[0086] As used herein, the terms "biocompatible" or "biodegradable" are defined as the conversion of a material into less complex intermediates or end products by solubilization hydrolysis or by the action of bioformed entities, which entities can be enzymes and other products of an organism.

[0087] As used herein, "effective amount" means an amount of a nucleic acid or bioactive agent sufficient to provide the desired local or systemic effects and performance attendant to any medical treatment at a reasonable risk / benefit ratio.

[0088] As used herein, "peptide" means a peptide of any length and includes proteins. The terms "polypeptide" and "oligopeptide" are used herein without any specific expected size limitation, unless a specific size is otherwise indicated.

[0089] As used herein, "derivatives" of carbohydrates include, for example, the acid forms of sugars, such as glucuronic acid; amines of sugars, such as galactosamine; phosphates of sugars, such as mannose-6-phosphate, etc.

[0090] As used herein, "administer" and similar terms mean delivering a composition to an individual to be treated such that the composition can circulate systemically, wherein the composition binds to target cells and is taken up by endocytosis. Thus, the composition is preferably administered systemically to an individual, typically by subcutaneous, intramuscular, transdermal, intravenous or intraperitoneal routes. Injectables for such uses can be prepared in conventional forms, as liquid solutions or suspensions, or in solid forms suitable for preparation as solutions or suspensions in a liquid prior to injection, or as emulsions. Suitable excipients for administration include, for example, water, saline, dextrose, glycerol, ethanol, etc.; and, if desired, small amounts of auxiliary substances such as wetting agents or emulsifying agents, buffering agents, etc.

[0091] As used herein, "efficacy" and similar terms mean disappearance of a tumor or reduction in tumor size or reduction in tumor density or increase in lymphocyte count or increase in neutrophil count or improvement in survival, or all of the above.

[0092] As used herein, "toxicity" is defined as any treatment-related adverse effect on clinical observations, including but not limited to abnormal hematological or serum chemical results or organ toxicity.

[0093] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / regulatory sequence. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell. The term "inducible" promoter means a nucleotide sequence that, when operably linked to a polynucleotide encoding a specified gene product, substantially causes the production of the gene product in a cell only when an inducer corresponding to the promoter is present in the cell.

[0094] As used herein, the term "expression" refers to the process by which a gene produces a biochemical substance such as a polypeptide. The process includes any manifestation of the functional presence of a gene within a cell, including but not limited to gene knockdown as well as transient and stable expression. It includes but is not limited to the transcription of a gene into messenger RNA (mRNA) and the translation of such mRNA into a polypeptide. The expression of a gene produces a "gene product".

[0095] As used herein, a gene product can be a nucleic acid, such as messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. The gene products described herein also include nucleic acids with post-transcriptional modifications (such as polyadenylation), or polypeptides with post-translational modifications (such as methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, etc.).

[0096] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses incorporating recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0097] As used herein, the term "operably linked" or "transcriptionally controlled" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence and a second nucleic acid sequence are operably linked when they are arranged in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be adjacent to each other, for example, in cases where two protein-coding regions need to be joined, the DNA sequences are in the same reading frame.

[0098] As used herein, the term "transfer vector" refers to a composition comprising a nucleic acid of interest and a substance that can be used to deliver the nucleic acid of interest into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. The term transfer vector should also be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.

[0099] As used herein, the term "host cell" can be any type of cell, such as primary cells, cells in culture, or cells from a cell line. In a particular aspect, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parental cell that was transfected with the nucleic acid molecule, for example, due to mutations that may occur in subsequent generations or environmental influences or integration of the nucleic acid molecule into the host cell genome.

[0100] "Percent (%) amino acid sequence identity" with respect to a polypeptide sequence as described herein is defined as the percentage of amino acid residues in a candidate sequence of interest to be compared that are identical to the amino acid residues in a specific polypeptide sequence as described herein (e.g., a specific polypeptide sequence characterized by a sequence identifier in a sequence listing), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The sequence alignment for determining the percent amino acid sequence identity can be performed according to procedures known in the art, such as those described in, for example, EP 1 241 179B1, which is incorporated herein by reference, particularly including lines 35 on page 9 to line 40 on page 10, which has the definitions used therein and Table 1 regarding possible conservative substitutions. For example, one of ordinary skill in the art can use publicly available computer software. Computer program methods for determining sequence identity include, but are not limited to, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. According to one embodiment, the software alignment program used can be BLAST. One of ordinary skill in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. According to one embodiment, the WU-BLAST-2 computer program (Altschul et al., 1996, Methods in Enzymology 266:460-480, which is incorporated herein by reference) can be used to generate % identity values. According to one embodiment, when the WU-BLAST-2 computer program is executed, the following parameters are used: Most WU-BLAST-2 search parameters are set to default values. The adjustable parameters are set to the following values: Overlap span = 1, Overlap fraction = 0.125, Word threshold (T) = 11, Scoring matrix = BLOSUM62. The HSP S and HSP S2 parameters are dynamic values used by BLAST-2 and are established by the program itself based on the composition of the sequence of interest and the composition of the database searched for that sequence. However, these values can be adjusted to increase sensitivity. The % sequence identity value can be determined by dividing by the number of: (a) identical amino acid residues that match between a specific amino acid sequence as described herein (e.g., a specific polypeptide sequence characterized by a sequence identifier in a sequence listing) being compared and a candidate amino acid sequence of interest to be compared, e.g., the number of identical amino acid residues that match as determined by WU-BLAST-2, (b) the total number of amino acid residues in the polypeptide sequence as described herein (e.g., a specific polypeptide sequence characterized by SEQ ID.NO. in a sequence listing) being compared.

[0101] "Percent (%) nucleic acid sequence identity" with respect to a nucleic acid sequence as described herein is defined as the percentage of nucleotides in a candidate sequence of interest to be compared that are identical to the nucleotides in a specific nucleic acid sequence as described herein (e.g., a specific polypeptide sequence characterized by a sequence identifier in a sequence listing), after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity.

[0102] As used herein, the terms "homology" or "identity" refer to the identity of subunit sequences between two polymeric molecules, e.g., between two nucleic acid molecules, such as between two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in the two molecules are occupied by the same monomeric subunit; e.g., if the position in each of two DNA molecules is occupied by adenine, they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half of the positions in two sequences (e.g., 5 positions in a polymer of 10 subunits) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

[0103] In the context of two or more nucleic acid or polypeptide sequences, percent identity refers to two or more identical sequences. When comparing and aligning for maximum correspondence in a comparison window or specified region, as measured by using one of the following sequence comparison algorithms or by manual alignment and visual inspection, two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (e.g., 60% identity over a specified region, or if not specified, over the entire sequence, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity). Optionally, the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. For sequence comparison, a reference sequence is typically used to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. Subsequently, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. As noted above, methods for sequence alignment for comparison are well known in the art.

[0104] A "coding sequence" or a sequence that "encodes" a particular molecule (e.g., a therapeutic molecule) is a nucleic acid that, when operably linked to appropriate regulatory sequences (e.g., a promoter), is transcribed (in the case of DNA) or translated (in the case of mRNA) in vitro or in vivo into a polypeptide. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxyl) terminus. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it can be considered part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region.

[0105] Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. Transcription termination sequences are typically located at the 3' of the coding sequence.

[0106] As used herein, the term "recombinant DNA / RNA technology" refers to the manipulation of nucleic acid sequences outside of a living organism. The technology includes, but is not limited to: combining nucleic acid sequences derived from multiple sources (e.g., coding sequences, regulatory elements (e.g., promoters, enhancers, silencers, termination sequences), linkers (e.g., spacers, internal ribosome entry sites, cleavage sites)); inserting nucleic acid sequences from multiple sources into a suitable vector (e.g., a delivery vector, an expression vector, an integration vector); modifying or altering nucleotide sequences (e.g., by mutagenesis, inserting modified nucleotides, 5'-capping, polyadenylation); synthesizing artificial nucleotide sequences. A variety of techniques well known in the art (e.g., molecular cloning, polymerase chain reaction (PCR), digestion with restriction enzymes, in vitro ligation, mutagenesis, site-directed mutagenesis, transformation or transduction of prokaryotic and eukaryotic cells, in vitro DNA / RNA synthesis, in vitro RNA-5'-capping, in vitro RNA-polyadenylation, complementary DNA (cDNA) synthesis, nucleic acid isolation, etc.) can be used to manipulate nucleic acid sequences outside of a living organism (see, e.g., Green & Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-3, 4th edition).

[0107] As used herein, the term "recombinant" refers to any nucleic acid (e.g., DNA or RNA), peptide (e.g., oligopeptide, polypeptide, or protein), cell, or organism prepared by combining genetic material from two or more different sources. In some aspects, the recombinant nucleic acid, peptide, cell, or organism contains a portion of the genetic material from at least one source. In some aspects, a "recombinant DNA" molecule can include a DNA molecule derived from one organism and inserted into a host organism to create a new genetic combination. In some aspects, a "recombinant RNA" molecule (e.g., a recombinant mRNA molecule) can include an RNA molecule derived from one organism and inserted into a host organism to produce the expression of a desired genetic product in the host organism. In some aspects, a "recombinant peptide" molecule can include amino acid molecules derived from an organism or cell and expressed by a recombinant nucleic acid molecule.

[0108] As used herein, the term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the substances with which it coexists in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment such as a host cell.

[0109] As used herein, the term "tumor" refers to any mass of tissue caused by excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0110] As used herein, the term "primary tumor" refers to the original or first tumor that forms in a subject.

[0111] As used herein, the terms "metastasis," "metastatic," "secondary tumor," or "metastatic tumor" refer to a cancer (e.g., a tumor) formed by cancer cells derived from a primary cancer (e.g., a tumor) that has spread to other locations or regions of the body.

[0112] As used herein, the term "specifically binds" refers to an antigen-binding molecule of a protein that recognizes and binds a binding partner (such as a tumor antigen) present in a sample, but the antigen-binding molecule substantially does not recognize or bind other molecules in the sample.

[0113] As used herein, the term "tumor heterogeneity" refers to the molecular biological or genetic changes exhibited by the daughter cells of a tumor after multiple rounds of division and proliferation during tumor growth, resulting in differences in aspects such as the growth rate, invasive ability, drug sensitivity, and prognosis of the tumor. This is one of the characteristics of malignant tumors.

[0114] As used herein, the term "cancer" refers to a large group of diverse diseases characterized by the uncontrolled growth of abnormal cells (such as malignant cells) in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues by local spread and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. In some aspects, the methods of the present disclosure can be used to reduce the size of a primary or metastatic tumor, or to treat a primary or metastatic tumor. Conditions that can be treated or prevented by the methods of the present disclosure include, for example, various tumors, including benign or malignant tumors, various hyperplasias, and the like. The methods of the present disclosure can achieve inhibition and / or reversal of the undesired overproliferative cell growth involved in such conditions. In some aspects, the cancer can be ovarian cancer.

[0115] As used herein, "ovarian cancer" refers to cancer that originates in or involves the ovary (such as ovarian epithelium). As used herein, the terms "cancer" or "tumor" refer to uncontrolled cell growth that interferes with the normal functioning of body organs and systems. A subject having cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can ultimately lead to the death of the subject through deterioration of the function of the affected organs. Ovarian cancer is typically treated by cytoreductive surgery (also referred to herein as "debulking") followed by administration of chemotherapy. As used herein, "cytoreductive surgery" refers to surgically removing at least a portion of the ovarian cancer tissue from a subject. Cytoreductive surgery can remove different amounts of tumor tissue from the subject, depending on the location and characteristics of the tumor tissue, the health of the subject, and complex factors that can be evaluated by those skilled in the art. In some embodiments, cytoreductive surgery can remove at least 10% of the tumor tissue, such as 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the tumor tissue present in the subject.

[0116] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include primary cells of a subject and their progeny.

[0117] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In one embodiment, a refractory cancer can be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer can become drug-resistant during treatment. A refractory cancer is also referred to as a drug-resistant cancer. In some aspects, refractory or recurrent malignancies can be treated using the methods disclosed herein.

[0118] As used herein, "recurrence" refers to the reappearance of the signs and symptoms of a disease (such as cancer) or the reappearance of a disease (such as cancer during a period of improvement), for example, after treatment, such as after a previous treatment for cancer treatment.

[0119] As used herein, the term "combination therapy" means a therapy that includes more than one treatment (e.g., an active agent or surgery). In some aspects, the combination therapy includes at least gene therapy, an anti-cancer agent, and at least one immune checkpoint inhibitor, which can be administered together or separately. In some aspects, the compositions of the combination therapy are formulated together in a single composition or as separate compositions.

[0120] As used herein, the terms "treat", "treated", and "treating" refer to therapeutic and prophylactic treatment or preventive measures, wherein the aim is to reverse, mitigate, improve, reduce, inhibit, slow down the progression, development, severity, or recurrence of an undesired symptom, complication, condition, disorder, or disease, or a biochemical marker thereof, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to: alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; a stable (i.e., not worsening) state of a condition, disorder, or disease; delayed onset or slowed progression of a condition, disorder, or disease; improvement or alleviation (whether partial or complete), whether detectable or not, of a condition, disorder, or disease state; improvement of at least one measurable physical parameter, which need not be discernible by the patient; or enhancement or improvement of a condition, disorder, or disease. In some aspects, treatment includes eliciting a clinically significant response without an excessive level of side effects. In some aspects, treatment includes extended survival compared to expected survival in the absence of treatment. As used herein, the term "amelioration" or "ameliorating" refers to a reduction in the severity of at least one indicator of a condition or disease. As used herein, the terms "preventing" or "prevention" refer to delaying or precluding the onset, development, or progression of a condition or disease for a period of time, including weeks, months, or years. As used herein, the term "prevention" (e.g., "preventive agent", "preventive treatment", "preventively effective amount") refers to any complete or partial prevention of a disease or its symptoms and / or can be therapeutic in terms of partial or complete cure of the disease and / or adverse reactions and / or symptoms caused by the disease.

[0121] As used herein, the terms "individual" and "subject" have the same meaning herein and can be a human and animals from other species. As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some aspects, the subject is a mammal, such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some aspects, the subject is a human. In some aspects, a patient is a subject who has a disease, disorder, or condition, or is at risk of having a disease, disorder, or condition, or otherwise requires the compositions and methods provided herein.

[0122] As used herein, the terms "therapeutically effective amount", "therapeutically effective", "effective amount", or "in an effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, substance, or composition that is effective in achieving a particular biological result as described herein (such as, but not limited to, treating or reducing the growth of cancer or a tumor). When indicating an "immunologically effective amount", "antitumor effective amount", "tumor-suppressive effective amount", or "therapeutically effective amount", the precise amount of the immune effector cells and therapeutic agents of the present disclosure to be administered can be determined by a physician considering the age, body weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). An effective amount of immune effector cells refers to, but is not limited to, the number of immune effector cells capable of increasing, enhancing, or prolonging the antitumor activity of immune effector cells; increasing the number of antitumor immune effector cells or activated immune effector cells; and promoting tumor regression, tumor shrinkage, and / or tumor necrosis.

[0123] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, formulations, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0124] The term "excipient" refers to any substance that is not itself a therapeutic agent and that can be used in a composition to deliver an active therapeutic agent to a subject or in combination with an active therapeutic agent (e.g., to produce a pharmaceutical composition) to improve its handling or storage characteristics or to allow or facilitate the formation of dosage units of the composition. Excipients include, but are not limited to: solvents, permeation enhancers, wetting agents, antioxidants, lubricants, emollients, substances added to improve the appearance or texture of the composition, and substances used to form hydrogels. Any such excipient can be used in any dosage form according to the present disclosure. The foregoing classes of excipients are not meant to be exhaustive but are merely illustrative, as one of ordinary skill in the art will recognize that additional types and combinations of excipients can be used to achieve the desired goal of delivering a drug. Excipients can be inert substances, inactive substances, and / or non-pharmaceutically active substances. Excipients can be used for a variety of purposes.

[0125] One or more excipients can be selected by those skilled in the art through routine experiments and without undue burden according to specific desired characteristics. The amount of each excipient used can vary within the range conventional in the art. The techniques and excipients available for formulating dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0126] As used herein, the term "immune response" refers to a biological response in an organism against foreign agents or abnormal cells (e.g., tumor cells), wherein the response protects the organism from such agents / cells and the diseases caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by these cells or the liver, including antibodies, cytokines, and complement, so as to selectively target, bind, damage, destroy, and / or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells, or other abnormal cells in the organism, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. In some aspects, the immune response includes, for example, activating or inhibiting T cells, such as effector T cells or Th cells, such as CD4+ or CD8+ T cells, or inhibiting regulatory T cells (Treg cells).

[0127] As used herein, the term "autologous" refers to any material derived from an individual that will subsequently be re-introduced into the same individual.

[0128] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0129] Papain digestion of a complete antibody generates two identical antigen-binding fragments, called "Fab" fragments, each of which contains the variable domains of the heavy and light chains (VH and VL, respectively), as well as, in addition, the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Thus, the term "Fab fragment" refers to an antibody fragment that comprises a light chain containing the VL domain and the CL domain and a heavy chain fragment containing the VH domain and the CH1 domain.

[0130] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0131] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds an antigen that is bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0132] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs), including complementarity-determining regions (CDRs) (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).

[0133] As used interchangeably herein, "paratope" or "antigen-binding site" refers to the part of an antibody that recognizes and binds an antigen. The antigen-binding site is formed by the juxtaposition of several individual amino acid residues from the variable regions of the heavy and light chains of the antibody, which are spatially close in the tertiary structure of the Fv region. In one embodiment, the antigen-binding site is defined as a set of six CDRs contained within a homologous VH / VL pair.

[0134] As used herein, the term "complementary determining region" or "CDR" refers to each region of an antibody variable domain that is highly variable in sequence and contains antigen - contacting residues. Typically, an antibody contains six CDRs: three in the VH domain (CDR - H1, CDR - H2, CDR - H3), and three in the VL domain (CDR - L1, CDR - L2, CDR - L3). Unless otherwise specified, CDR residues and other residues (such as FR residues) in the variable domain are numbered herein according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0135] "Recipient human framework" for purposes herein is a framework containing an amino acid sequence of a light - chain variable domain (VL) framework or a heavy - chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.

[0136] As used herein, "framework" or "FR" refers to the variable - domain amino acid residues other than the CDR residues. The framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Thus, CDR and FR amino acid sequences typically occur in the following sequences in (a) the VH domain: FR1 - CDR - H1 - FR2 - CDR - H2 - FR3 - CDR - H3 - FR4; and (b) in the VL domain: FR1 - CDR - L1 - FR2 - CDR - L2 - FR3 - CDR - L3 - FR4.

[0137] As used herein, "affinity" refers to the strength of the sum of non - covalent interactions between a single binding site of a molecule (such as an antibody) and its binding partner (such as an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (such as an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.

[0138] As used herein, the term "epitope" refers to a site on an antigen (protein antigen or non-protein antigen) to which an antibody binds. An epitope can be formed by a continuous segment of amino acids (linear epitope) or contain non-contiguous amino acids (conformational epitope), for example being spatially proximate due to folding of the antigen (i.e., through the tertiary folding of a protein antigen). After a protein antigen is exposed to a denaturing agent, linear epitopes are generally still bound by antibodies, while conformational epitopes are generally disrupted upon treatment with a denaturing agent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.

[0139] Screening of antibodies that bind to a specific epitope (i.e., antibodies that bind the same epitope) can be carried out using conventional methods in the art, such as but not limited to alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0140] Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-assisted Profiling (MAP), allows for the binning of monoclonal antibodies that specifically bind to an antigen based on the binding profile of each of a plurality of antibodies to a chemically or enzymatically modified antigen surface (see, e.g., US2004 / 0101920). Antibodies within each bin bind to the same epitope, which can be a unique epitope that is distinct or partially overlapping with the epitope represented by another bin. Competitive binding can also be used to readily determine whether an antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding. For example, an "antibody that binds to the same epitope as a reference antibody" means that in a competition assay, the antibody blocks the binding of the reference antibody to its antigen by 50% or more, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Similarly, for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to its antigen under saturating conditions. After removing the excess reference antibody, the ability of the antibody in question to bind to its antigen is evaluated. If the antibody is able to bind to its antigen after the reference antibody has bound saturatingly, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question is unable to bind to its antigen after the reference antibody has bound saturatingly, the antibody in question may bind to the same epitope as the epitope to which the reference antibody binds. To confirm whether the antibody in question binds to the same epitope or is simply sterically hindering binding, conventional experiments (e.g., peptide mutagenesis and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. The assay should be performed in two settings, i.e., both antibodies are saturating antibodies. If in both settings only the first (saturating) antibody is able to bind to the antigen of interest, it can be concluded that the antibody in question and the reference antibody compete for binding to the same antigen.

[0141] Sometimes, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations that reduce or eliminate the binding of one antibody in the antigen also reduce or eliminate the binding of the other antibody. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of the other antibody, the two antibodies are considered to have "overlapping epitopes".

[0142] As used herein, the term "anti-tumor effect" refers to a biological effect that can manifest in various ways, including but not limited to: for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in tumor cell proliferation, and a decrease in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. The "anti-tumor effect" can also be expressed by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent or reduce the frequency of tumorigenesis.

[0143] As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers to the various chemotherapeutic agents that can be used in accordance with the embodiments of the present invention. The term "chemotherapy" refers to the treatment of cancer with drugs. "Chemotherapeutic agent" is used to denote a compound or composition administered in cancer treatment.

[0144] The term "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredient is effective and which does not contain additional components that are unacceptably toxic to the subject to which the composition will be administered. The composition can be sterile.

[0145] Those skilled in the art will readily recognize that the vectors, polynucleotides, and pharmaceutical compositions of the present disclosure, or combinations thereof, can be readily incorporated into one of the established kit forms well known in the art.

[0146] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenics, microbiology, recombinant DNA, and immunology within the skill of the art. These techniques are well explained in the literature.

[0147] II. GEN-1 (nanoparticle-formulated IL-12 DNA)

[0148] In animal models, recombinant IL-12 has been shown to induce strong T cell-mediated anti-tumor effects, resulting in the regression of established tumors, followed by systemic immune memory. See The Oncologist, 1996, vol. 1, 88. However, in several experimental trials and the initial human trials, systemic administration of recombinant IL-12 resulted in dose-limiting toxicity. See Lab Invest., 1994, vol. 71, 862; Science, 1995, vol. 270, 908; J. Interferon Cytokine Res., 1995, vol. 14, 335. In recent human clinical trials, dose-limiting toxicity was also observed with intraperitoneal administration of recombinant IL-12. Clin. Cancer Res., 2002, vol. 8, 3686. Gene delivery methods that can locally provide therapeutic levels of IL-12 at the tumor site would have the advantage of generating an anti-cancer response without causing systemic toxicity.

[0149] Both viral and non-viral gene delivery systems have been used for IL-12 gene delivery in cancer animal models. Due to toxicity issues, viral methods have severe practical limitations, mainly because of the increased cancer incidence and the strong immune response of the host system to viral antigens. Because of the lower toxicity of non-viral gene delivery systems, there has been considerable interest in their development. Delivery of IL-12 using polyvinylpyrrolidone (PVP), a non-viral gene delivery system, has been demonstrated for the treatment of renal carcinoma (Renca) and colon carcinoma (CT26). See Gene Ther., 1999, Vol.6, 833. When tumors received this gene therapy, they showed all the characteristics of IL-12 protein therapy, such as increased infiltration of NK cells, CD4 and CD8 T cells, and increased expression of major histocompatibility complex (MHC) class I molecules. IL-12 gene delivery was well tolerated and highly efficient in Renca- and CT26-bearing animals. Tumor-rejected mice were also protected from subsequent re-challenge, indicating the presence of a durable systemic immunity. Functionalized and less toxic water-soluble lipid polymers (WSLP) have been tested for delivery of the IL-12 gene to CT26 colon carcinoma tumors. See Mahato et al, Mol.Ther., 2001, vol.4, 130. Treatment with IL-12 plasmid (pIL-12) and WSLP (pIL-12 / WSLP) resulted in higher levels of intratumoral gene expression than naked DNA.

[0150] Interleukin-12 (IL-12) is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16:495-521 (1998). IL-12 functions primarily as a 70 kDa heterodimeric protein composed of two disulfide-linked p35 and p40 subunits. IL-12p40 homodimers do exist, but they do not appear to mediate biological responses except as antagonists that bind the IL-12 receptor. Ibid. The precursor form of the IL-12p40 subunit (e.g., NM_002187; NP_002178; also known as IL-12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2) is 328 amino acids in length, while its mature form is 306 amino acids in length. The precursor form of the IL-12p35 subunit (e.g., NM_000882; NP_000873; also known as IL-12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids in length, and its mature form is 197 amino acids in length. Ibid. The genes for the IL-12p35 and p40 subunits are located on different chromosomes and are regulated independently of each other. Gately, MK et al., Annu Rev Immunol. 16:495-521 (1998). Many different immune cells (e.g., dendritic cells, macrophages, monocytes, neutrophils, and B cells) produce IL-12 upon antigen stimulation. The active IL-12 heterodimer forms after protein synthesis. Ibid.

[0151] Since the IL-12 protein is able to activate NK cells and cytotoxic T cells, it has been investigated as a promising anti-cancer therapeutic agent since 1994. See Nastala, C.L. et al., J Immunol 153:1697-1706 (1994). However, despite high expectations, early clinical studies did not produce satisfactory results. Lasek W. et al., Cancer Immunol Immunother 63:419-435, 424 (2014). In most patients, repeated administration of IL-12 results in an adaptive response and a progressive decline in IL-12-induced interferon gamma (IFNγ) levels in the blood. Ibid. In addition, although it has been recognized that the anti-cancer activity induced by IL-12 is primarily mediated by the secondary secretion of IFNγ, IL-12 simultaneously induces IFNγ as well as other cytokines (e.g., TNF-a) or chemokines (IP-10 or MIG) causing severe toxicity. Ibid.

[0152] In addition to negative feedback and toxicity, the marginal efficacy of IL-12 therapy in a clinical setting may be caused by a strong immunosuppressive environment in the human body.

[0153] Furthermore, when compared to naked DNA, secondary effects of cytokine IL-12 production, namely, higher levels of IFN-γ and nitric oxide (NO) were also observed in tumors treated with WSLP. Single injection of the pIL-12 / WSLP complex had a suboptimal effect on tumor growth and animal survival, while repeated delivery produced better efficacy, indicating insufficient systemic delivery. J. Control Release 2003, Vol. 87, 177. Similarly, intratumoral injection of IL-12 plasmid in another polymeric carrier, PAGA, only produced partial inhibition of CT26 tumors. See Gene Ther., 2002, Vol. 9, 1075. These results demonstrate the need for more efficient delivery systems. Despite their deficiencies in early preclinical trials, the excellent molecular flexibility of polymeric gene carriers allows for complex modifications and new functionalizations, which are necessary for the development of more effective gene delivery systems.

[0154] To obtain the desired results from combinatorial approaches involving gene therapeutics, it is important to select an appropriate gene delivery system. The gene delivery system used in the above combinatorial experiment (Molecular Therapy, 2004, Vol. 9, 829) was the water-soluble lipid polymer PEI-cholesterol (WSLP).

[0155] IL-12 is mainly secreted by antigen-presenting cells (phagocytes, dendritic cells) in response to pathogens, promoting the differentiation of CD4+ cells into Th1 cells. It has a positive co-proliferative effect on pre-activated NK and T cells, and independently and / or synergistically enhances the cytolytic capacity of both NK and CD8+ T cells by upregulating genes encoding cytotoxic cell granule-associated proteins. It also increases ADCC in antibody-coated tumors at concentrations much lower than IL-2. Effector cells stimulated by IL-12 produce several cytokines, such as GMCSF and TNF-α, but mainly IFN-γ. IL-12 is composed of p35 and p40 subunits, the latter of which is also shared by IL-23. The IL-12 receptor (IL-12R) consists of two chains (IL-12Rβ1 and IL-12Rβ213) and mainly signals through STAT4. IL-12R is mainly expressed by activated NK and T cells; it is barely detectable in resting T cells but is expressed at low levels in NK, which may explain their rapid response to IL-12. However, TCR activation and co-stimulatory signals such as B7, IFN-α, IFN-γ, and IL-12 upregulate IL-12R expression (especially IL-12Rβ2).

[0156] The anti-tumor activity of systemic or local administration of IL-12 has been determined in preclinical studies against various tumor cell lines. Dose- and model-dependent responses and memory anti-tumor effects have been observed. Other studies have shown that IL-12 is more effective against early or microscopic tumors than advanced disease. T cell responses are responsible for its anti-tumor activity, and NK / NKT cell activation appears to prevent metastasis. In addition to effector T cell (Teff)-mediated tumor control, IL-12 also increases the Teff / Treg ratio in vitro and in vivo by inhibiting T cell IL-2 production, inducing apoptosis, or through IFN-γ-mediated cell arrest to inhibit tumor-derived regulatory T cells (Tregs). IL-12-mediated reprogramming of intratumoral myeloid-derived suppressor cells (MDSCs) has also been reported to enhance CTL activity in the B16 melanoma model and reverse its in vivo inhibitory effects. IL-12Rβ2 also emerges as a potential tumor suppressor gene, especially in hematological malignancies, where epigenetic IL-12Rβ2 gene silencing by promoter hypermethylation or gene downregulation has been observed in multiple B cell myeloma samples, and IL-12 significantly reduces tumor burden upon receptor restoration. Consistently, IL-12Rβ2- / - mice develop spontaneous malignancies and lymphoproliferative diseases. IL-12 also has "direct" anti-tumor activity in tumors expressing IL-12Rβ1 through IFN-γ-mediated upregulation of MHC class I molecules.

[0157] In a phase II trial of patients with metastatic RCC and melanoma, the initial clinical trial was temporarily halted after an unexpected toxicity-related death was observed, despite a safe MTD having been established in a previous phase I trial. This was attributed to a minor but biologically significant change in the treatment schedule, including elimination of the test dose prior to the 5-day course. Evaluation showed that the test dose attenuated the subsequent effect of IL-12 on IFN-γ production by an unknown mechanism. Subsequently, various IL-12 regimens were tested, but had modest clinical outcomes in patients with solid tumors and more promising results in hematological malignancies. Since IL-12 is a key mediator of the Th1 response, efforts have focused on using IL-12 as an adjuvant to vaccine therapy against tumor-associated antigens for both resectable and metastatic disease. In general, IL-12 has improved immune responses, but this has not translated into clinically significant anti-tumor effects.

[0158] Preclinical models have demonstrated that local delivery of IL-12 is effective without the detriment of systemic administration, which has facilitated the development of ways to express IL-12 intratumorally (IT). Studies injecting IL-12 into the primary tumors of patients with head and neck cancer have shown that induced B cell activation is associated with increased survival. Enabling tumor cells to produce IL-12 has been of interest, and many small studies using viral vectors or plasmid DNA have tested this approach, but with limited results. Antibody formation against viral vectors and unrecorded gene delivery efficiency are limiting factors. A new method of local IL-12 administration is by injecting plasmid DNA encoding IL-12 and electroporating superficial tumors to facilitate cell entry. The results of phase I studies have demonstrated antitumor activity at both the local injection site and non-injected sites systemically. Two trials in patients with gynecologic malignancies evaluated IL-12 plasmid formulated with PEG-PEI-cholesterol lipid polymers delivered by intraperitoneal administration, but clinical benefit was modest. Current studies are also testing IL-12 as a gene therapy, including gene electrotransfer-mediated plasmid transfer, adenoviral vectors combined with activating ligands administered orally, or mesenchymal engineered cells for IL-12 expression. NHS-IL-12 is an immunocytokine consisting of two IL-12 molecules fused to a tumor necrosis-targeted human IgG1 with a longer half-life. It has now entered phase II clinical trials for testing.

[0159] Carson et al. conducted an NCI-sponsored Phase I trial to determine the safety and optimal biological dose of IL-12 when administered in combination with trastuzumab. Patients with metastatic HER2-positive malignancies received trastuzumab (initial 4 mg / kg and then 2 mg / kg thereafter) on Day 1 of each weekly cycle. Starting from Week 3, patients also received intravenous (i.v.) injections of IL-12 on Days 2 and 5 of the weekly cycle. The IL-12 component was dose-escalated (30, 100, 300, or 500 ng / kg) in a cohort of three patients. Fifteen patients were treated. The regimen was well tolerated, and no drug-related grade 3 or 4 toxicities were recorded. Assessment of dose-limiting toxicity and biological endpoints indicated that the 300 ng / kg dose was both the maximum tolerated dose and the optimal biological dose of IL-12 when used in combination with trastuzumab. Correlative assays showed that NK cells produced IFN-γ continuously only in three patients who showed a good response to the regimen. Elevated circulating levels of NK cell-derived chemokines (MIP-1α, IL-8, and RANTES) were also detected in these three patients. These factors exert a strong chemotactic effect on naive and activated T cells, and their presence is associated with infiltration of CD8+ T cells into tumor tissue. Importantly, in vitro NK cell-mediated ADCC against tumor targets appeared to be independent of clinical response or the dose of IL-12. Thus, as predicted, the clinical response to IL-12 / trastuzumab therapy was associated with the production of IFN-γ and chemokines by immune cells.

[0160] Carson et al. conducted a follow-up trial using IL-12 in combination with trastuzumab and paclitaxel. Paclitaxel was administered at 175 mg / m 2Administered intravenously (i.v.) every 3 weeks. Trastuzumab (initially 4 mg / kg and thereafter 2 mg / kg) was administered on day 1 of each week, and starting from cycle 2, IL-12 was co-injected on days 2 and 5. This trial enrolled 21 patients with metastatic 2+ and 3+ HER2-positive tumors, 16 of whom had received prior chemotherapy. The IL-12 component was dose-escalated (100 ng / kg and then 300 ng / kg) in a cohort of three patients, but due to dose-limiting grade 3 fatigue at the 300 ng / kg dose level, the IL-12 component was reduced to 200 ng / kg subcutaneously. The overall clinical benefit rate was 52%. Activation of ERK in peripheral blood mononuclear cells increased significantly, and plasma levels of IFN-γ increased, with clinical benefit (complete response, partial response, or disease stabilization), but not in patients with progressive disease. None of the patients with progressive disease had measurable levels of IFN-γ in their circulation. In any given cycle, IFN-γ levels typically peaked after injection of IL-12 (range 124 - 1612 pg / ml) and then declined to baseline. Analysis of patient time-to-disease-progression data using the non-parametric Mann-Whitney U test showed that induction of IFN-γ was associated with a statistically significant increase in progression-free survival (p = 0.004). Intracellular flow cytometry analysis of cryopreserved PBMCs from day 5 of the treatment cycle showed high levels of IFN-γ within CD56+ NK cells only in those patients who demonstrated clinical response or disease stabilization. Circulating levels of MIP-1α were also associated with clinical response, as were levels of IP-10 and MIG (two anti-angiogenic factors induced by IFN-γ).

[0161] The GOG trial evaluated GEN-1 in 16 patients with persistent or recurrent platinum-resistant EOC. In this study, higher doses of GEN-1 were evaluated in combination with: intravenous pegylated liposomal doxorubicin (PLD) 40 mg / m 2 (dose levels 1 and 2) or 50 mg / m 2 (dose level 3) every 28 days, and intraperitoneal 24 mg / m 2 (dose level 1) or 36 mg / m 2GEN-1 (at dose levels 2 and 3). The cycle was repeated every 28 days until disease progression. Clinical benefit was seen in 57.1% of 14 patients with measurable disease (PR = 21.4%; SD = 35.7%). The highest amounts of partial responses (28.6%) and disease stabilizations (57.1%) were seen at dose level 3. The maximum tolerated dose was not reached. After treatment with GEN-1, increased levels of IL-12, IFN-γ, and TNF-α were found in peritoneal fluid.

[0162] Ovarian cancer is the fifth most common form of cancer affecting women and the most lethal gynecologic malignancy, ranking fourth among cancer deaths in women. First-line chemotherapy regimens for treating ovarian cancer typically are platinum-based combination therapies that are administered intravenously (IV) every 21 to 28 days for 4 to 6 treatments. Although nearly 90% of women initially respond to platinum-based therapies, 55%–75% of women will develop recurrent ovarian cancer within 2 years. Second-line and third-line therapies are often ineffective and toxic, including immune checkpoint inhibitors, which have a response rate of 11%–15% in recurrent patients with platinum resistance. There is an urgent need for safer and more effective therapies for treating ovarian cancer.

[0163] Cancer can evade detection and destruction by the immune system, despite the fact that many tumors elicit a marked robust immune response in the lymphocytic infiltration of the primary lesion. Tumor immune escape can be classified into induction of immune tolerance and resistance to activation of immune effector cell killing. The “immune editing” hypothesis suggests that tumors manipulate their microenvironment by creating a complex local and regional immunosuppressive network that contains various tumor-derived cytokines and other soluble factors. Thus, by the time a tumor becomes clinically detectable, the tumor has evolved mechanisms to escape the immune response mounted against it by the host. This resistance mechanism must be overcome to generate an effective and durable anti-tumor immunity. One of the most promising strategies for enhancing the patient's anti-tumor response appears to be the use of antibodies that block immune regulatory mechanisms, which can inhibit the host response to tumor-associated antigens. To date, CTLA4 and PD1 / PDL1 blocking monoclonal antibodies have shown significant results. However, a substantial number of patients with solid tumors do not respond to these agents. In these cases, the tumor microenvironment (TME) is considered to be less immunogenic, and pro-inflammatory cytokines such as IL-12 may play an important role in converting the TME to synergize with CTLA-4 blockade and PD-1 blockade.

[0164] GEN-1 nanoparticles contain a DNA plasmid encoding the IL-12 gene and a synthetic polymer that facilitates plasmid delivery. GEN-1 is designed for local delivery (e.g., intraperitoneal) to provide the potential for cytokine expression specifically in the tumor microenvironment, with the goal of achieving increased efficacy while minimizing potential systemic toxicity. GEN-1 has been studied as a single agent or in combination with standard chemotherapy in subjects with recurrent ovarian cancer. A phase I single-agent trial evaluated 13 platinum-resistant EOC patients who received IP GEN-1 treatments of 0.6 mg / m 2 、3 mg / m 2 、12 mg / m 2 and 24 mg / m 2 weekly, and safety and tolerability were evaluated every 4 weeks. The most common adverse events (AEs) were fever and abdominal pain. A significant increase in IFN-γ was observed in peritoneal fluid but not in serum. Another phase I trial was conducted in platinum-sensitive EOC patients who received intravenous (IV) carboplatin and docetaxel and escalating doses of IP GEN-1 as follows: 12 mg / m 2 、18 mg / m 2 or 24 mg / m 2 × 4 doses or 24 mg / m 2 × 6–8 doses. Similar AEs and a dose-dependent increase in IFN-γ concentrations were observed. No dose-limiting toxicity (DLT) was observed, and the MTD was not reached. Adding GEN-1 to chemotherapy did not attenuate efficacy or exacerbate side effects.

[0165] The GOG trial evaluated GEN-1 in 16 patients with persistent or recurrent platinum-resistant EOC. In this study, higher doses of GEN-1 were evaluated in combination with intravenous pegylated liposomal doxorubicin (PLD) 40 mg / m 2 (dose levels 1 and 2) or 50 mg / m 2 (dose level 3) every 28 days, and GEN-1 at 24 mg / m 2 (dose level 1) or 36 mg / m 2 intraperitoneally on days 1, 8, 15, and 22 of a 28-day cycle (dose levels 2 and 3). Cycles were repeated every 28 days until disease progression. Clinical benefit was found in 57.1% of 14 patients with measurable disease (PR = 21.4%; SD = 35.7%). The highest amount of partial responses (28.6%) and disease stability (57.1%) were found at dose level 3. The maximum tolerated dose was not reached. After GEN-1 treatment, increased levels of IL-12, IFN-γ, and TNF-α were found in peritoneal fluid.

[0166] Translational research results indicate that GEN-1 has biological activity in ovarian cancer subjects and promotes the dynamics of pre-immune T cell populations and the conversion of tumor naive T cells to cytotoxic effector T cells in the tumor microenvironment. To date, more than 100 ovarian cancer patients have received GEN-1 administration. The most common adverse events attributable to higher doses of GEN-1 are: abdominal pain, nausea, fatigue, fever, vomiting, and diarrhea.

[0167] In some aspects, the GEN-1 DNA plasmid is delivered using a synthetic polymer that promotes plasmid delivery, and the synthetic polymer is a lipid polymer.

[0168] In some aspects, the lipid polymer comprises polyethyleneimine (PEI) covalently linked independently to cholesterol and polyethylene glycol (PEG) groups.

[0169] The present disclosure provides a polymer system, PEG-PEI-cholesterol (PPC), which differs from WSLP (PEI-cholesterol) in that it contains a PEG moiety and produces significantly higher transfection efficiency in tumors. The addition of PEG is designed to enhance the stability of the nucleic acid / polymer complex in the biological environment to circumvent this defect in the prior art (WSLP). In addition, the addition of PEG chains allows ligands to be incorporated onto the PPC chain to improve tissue selectivity of delivery. For example, the cholesterol moiety directly linked to the PEI backbone in the prior art (WSLP) can extend further from the PEI backbone to produce a more flexible geometry for cell receptor interaction. Controlling the number of PEG molecules per unit of the PEI backbone is important for achieving optimal enhancement of transfection activity. At a fixed cholesterol content, the preferred composition ranges from a PEG:PEI molar ratio of 2-4. The optimal ratio between PEI and cholesterol is 1:0.5 to 1:1.

[0170] Certain aspects of the present disclosure relate to a combination therapy that includes: (i) a nucleic acid carrier (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer (e.g., a nanoparticle); and (ii) an immune checkpoint inhibitor.

[0171] Certain aspects of the present disclosure relate to a method of treating a subject having cancer, the method comprising administering to the subject a combination therapy that includes: (i) a nucleic acid carrier (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer (e.g., a nanoparticle); and (ii) an immune checkpoint inhibitor.

[0172] In some aspects, the polynucleotide encodes human IL-12. In some aspects, the polynucleotide encodes the p35 subunit of IL-12 and the p40 subunit of IL-12.

[0173] In some aspects, a nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL-12.

[0174] In some aspects, human IL-12p35 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to SEQ ID NO:85. In some aspects, human IL-12p40 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to SEQ ID NO:86.

[0175] In some aspects, human IL-12p35 comprises the following sequence: MGPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO:85).

[0176] In some aspects, human IL-12p40 comprises the following sequence: MGHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO:86).

[0177] In some aspects, the polynucleotide encoding human IL-12p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to SEQ ID NO:87. In some aspects, the polynucleotide encoding human IL-12p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identity to SEQ ID NO:88.

[0178] In some aspects, the polynucleotide encoding human IL-12p35 comprises the following sequence: atgggtccagcgcgcagcctcctccttgtggctaccctggtcctcctggaccacctcagtttggccagaaacctccccgtggccactccagacccaggaatgttcccatgccttcaccactcccaaaacctgctgagggccgtcagcaacatgctccagaaggccagacaaactctagaattttacccttgcacttctgaagagattgatcatgaagatatcacaaaagataaaaccagcacagtggaggcctgtttaccattggaattaaccaagaatgagagttgcctaaattccagagagacctctttcataactaatgggagttgcctggcctccagaaagacctcttttatgatggccctgtgccttagtagtatttatgaagacttgaagatgtaccaggtggagttcaagaccatgaatgcaaagcttctgatggatcctaagaggcagatctttctagatcaaaacatgctggcagttattgatgagctgatgcaggccctgaatttcaacagtgagactgtgccacaaaaatcctcccttgaagaaccggatttttataaaactaaaatcaagctctgcatacttcttcatgctttcagaattcgggcagtgactattgatagagtgatgagctatctgaatgcttcctaa (SEQ ID NO:87).

[0179] In some aspects, the polynucleotide encoding human IL-12p35 comprises the following sequence:

[0180] Atgggtcaccagcagttggtcatctcttggttttccctggtttttctggcatctcccctcgtggccatatgggaactgaagaaagatgtttatgtcgtagaattggattggtatccggatgcccctggagaaatggtggtcctcacctgtgacacccctgaagaagatggtatcacctggaccttggaccagagcagtgaggtcttaggctctggcaaaaccctgaccatccaagtcaaagagtttggagatgctggccagtacacctgtcacaaaggaggcgaggttctaagccattcgctcctgctgcttcacaaaaaggaagatggaatttggtccactgatattttaaaggaccagaaagaacccaaaaataagacctttctaagatgcgaggccaagaattattctggacgtttcacctgctggtggctgacgacaatcagtactgatttgacattcagtgtcaaaagcagcagaggctcttctgacccccaaggggtgacgtgcggagctgctacactctctgcagagagagtcagaggggacaacaaggagtatgagtactcagtggagtgccaggaggacagtgcctgcccagctgctgaggagagtctgcccattgaggtcatggtggatgccgttcacaagctcaagtatgaaaactacaccagcagcttcttcatcagggacatcatcaaacctgacccacccaagaacttgcagctgaagccattaaagaattctcggcaggtggaggtcagctgggagtaccctgacacctggagtactccacattcctacttctccctgacattctgcgttcaggtccagggcaagagcaagagagaaaagaaagatagagtcttcacggacaagacctcagccacggtcatctgccgcaaaaatgccagcattagcgtgcgggcccaggaccgctactatagctcatcttggagcgaatgggcatctgtgccctgcagttagac(SEQ IDNO: 88)

[0181] In some aspects, the nucleic acid vector (e.g., plasmid) contains introns, 3'UTR (e.g., hGH 3'UTR), antibiotic resistance genes, or any combination thereof (e.g., Figure 1 elements).

[0182] In some aspects, the lipid polymer contains polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups (e.g., Figure 2 lipid polymers).

[0183] In some aspects, the nanoparticles disclosed herein contain a DNA plasmid encoding human IL-12.

[0184] In some aspects, wherein the nanoparticles contain a synthetic polymer that promotes plasmid delivery, and the synthetic polymer is a lipid polymer.

[0185] In some aspects, the lipid polymer further contains polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups.

[0186] In some aspects, the gene delivery polymer is a cationic polymer or a non-condensing polymer. The cationic polymers are selected from the group consisting of: polylysine, polyethyleneimine, polyethyleneimine (PEI) functionalized derivatives, polypropyleneimine, aminoglycoside-polyamines, bisdeoxy-diamino-b-cyclodextrin, spermine, and spermidine. An example of a cationic gene delivery polymer suitable for the present disclosure is a PEI derivative that contains a PEI backbone, a lipid, and a hydrophilic polymer spacer, wherein the lipid is directly bound to the polyethyleneimine backbone or covalently bound to a polyethylene glycol spacer, which in turn is bound to the PEI via a biocompatible bond.

[0187] The cationic gene delivery polymers of the present disclosure may further contain targeting moieties, including antibodies or antibody fragments, cell receptors, growth factor receptors, cytokine receptors, folic acid, transferrin, epidermal growth factor (EGF), insulin, asialoserylglycoprotein, mannose-6-phosphate (monocytes), mannose (macrophages, some B cells), Lewis X and sialyl Lewis X(Endothelial cells), N-acetyl lactosamine (T cells), galactose (colon cancer cells), and thrombomodulin (mouse lung endothelial cells), fusogenic agents such as polymyxin B and hemagglutinin HA2, lysosome tropic agents, nuclear localization signals (NLS) such as T antigen, etc. Another gene delivery polymer is a non-aggregating polymer selected from the group consisting of: polyvinylpyrrolidone, polyvinyl alcohol, poly(lactide-co-glycolide) (PLGA), and triblock copolymers of PLGA and PEG. The gene delivery polymer can also be a non-aggregating polymer. Examples of such non-aggregating polymers include polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, polyglutamate, gelatin, polyphosphate ester, elastin-like hydrogel, agarose hydrogel, lipid microtubules, poly(lactide-co-glycolide), and polyethylene glycol-linked poly(lactide-co-glycolide).

[0188] The gene delivery polymer is a cationic polymer or a non-aggregating polymer. Cationic polymers are selected from the group consisting of: polylysine, polyethyleneimine, polyethyleneimine-functionalized derivatives, polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-β-cyclodextrin, spermine, and spermidine. An example of a cationic gene delivery polymer suitable for the present invention is a polyethyleneimine derivative that includes a polyethyleneimine (PEI) backbone, a lipid, and a polyethylene glycol spacer, where the lipid is directly bound to the polyethyleneimine backbone or covalently bound to the polyethylene glycol spacer, and the polyethylene glycol spacer is in turn bound to the PEI via a biocompatible bond.

[0189] In some aspects, the gene delivery polymer includes a lipopolyamine having the following formula:

[0190]

[0191] In some aspects, the gene delivery polymer includes a mixture of a lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl)methacrylamide, or polydivinyl ether maleic anhydride. In some aspects, the alkylated derivative of the lipopolyamine has the following formula:

[0192]

[0193] (Methoxypolyethylene glycol (mPEG)-modified Staramine),

[0194] where n represents an integer of 10 to 100 repeating units each containing 2 to 5 carbon atoms. In some aspects, the alkylated derivative of the lipopolyamine has the following formula:

[0195]

[0196] where n = 11 (Staramine-mPEG515). In some aspects, the alkylated derivative of the lipid polyamine has the following formula:

[0197]

[0198] In some aspects, the ratio of the lipid polyamine to the alkylated derivative of the lipid polyamine in the mixture is from 1:1 to 10:1. In some aspects, the lipid polyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipid polyamine to phosphate in the nucleic acid carrier of from about 0.01:1 to about 50:1 (e.g., from about 0.01:1 to about 40:1; from about 0.01:1 to about 30:1; from about 0.01:1 to about 20:1; from about 0.01:1 to about 10:1, or from about 0.01:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipid polyamine to phosphate in the nucleic acid carrier is from about 0.1:1 to about 50:1 (e.g., from about 0.1:1 to about 40:1; from about 0.1:1 to about 30:1; from about 0.1:1 to about 20:1; from about 0.1:1 to about 10:1, or from about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipid polyamine to phosphate in the nucleic acid carrier is from about 1:10 to about 10:1.

[0199] In some aspects, the gene delivery polymer comprises a lipid polyamine having the following formula:

[0200]

[0201] In some aspects, the gene delivery polymer comprises a mixture of a lipopolyamine and an alkylated derivative of the lipopolyamine. In some aspects, the alkylated derivative of the lipopolyamine is poly(oxyalkylene), polyvinylpyrrolidone, polyacrylamide, poly(dimethylacrylamide), polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl)methacrylamide, or polydivinylether maleic anhydride. In some aspects, the ratio of the lipopolyamine to the alkylated derivative of the lipopolyamine in the mixture is from 1:1 to 10:1. In some aspects, the lipopolyamine is present in an amount sufficient to produce a ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid carrier of from about 0.01:1 to about 50:1 (e.g., from about 0.01:1 to about 40:1; from about 0.01:1 to about 30:1; from about 0.01:1 to about 20:1; from about 0.01:1 to about 10:1, or from about 0.01:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid carrier is from about 0.1:1 to about 50:1 (e.g., from about 0.1:1 to about 40:1; from about 0.1:1 to about 30:1; from about 0.1:1 to about 20:1; from about 0.1:1 to about 10:1, or from about 0.1:1 to about 5:1). In some aspects, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid carrier is from about 1:10 to about 10:1.

[0202] In some aspects, the gene delivery polymer comprises a poloxamer backbone having a metal chelator covalently coupled to at least one end of the poloxamer backbone. In some aspects, the metal chelator is coupled to at least two ends of the poloxamer backbone. In some aspects, the poloxamer backbone is the poloxamer backbone disclosed in U.S. Publication No. 2010 / 0004313, which is incorporated herein by reference in its entirety. In some aspects, the metal chelator is the metal chelator disclosed in U.S. Publication No. 2010 / 0004313. In some aspects, the gene delivery polymer comprises a polymer having the following formula:

[0203]

[0204] and pharmaceutically acceptable salts thereof, wherein:

[0205] A represents an integer from 2 to 141;

[0206] B represents an integer from 16 to 67;

[0207] C represents an integer from 2 to 141;

[0208] RA and RC are the same or different and are R'-L- or H, where at least one of RA and RC is R'-L-;

[0209] L is a backbone, —CO—, —CH2—O—, or —O—CO—; and

[0210] R' is a metal chelator.

[0211] In some aspects, the metal chelator is RNNH—, RN2N—, or (R”—(N(R”)—CH2CH2)x)2—N—CH2CO—, where each x is independently 0-2, and where R” is HO2C—CH2—. In some aspects, the metal chelator is a crown ether selected from the group consisting of: 12-crown-4 ether, 15-crown-5 ether, 18-crown-6 ether, 20-crown-6 ether, 21-crown-7 ether, and 24-crown-8 ether. In some aspects, the crown ether is a substituted crown ether, where the substituted crown ether has:

[0212] (1) one or more crown ether oxygens independently replaced by NH or S,

[0213] (2) one or more crown ether —CH2—CH2— moieties replaced by —C6H4—, —C10H6—, or —C6H10—.

[0214] (3) one or more crown ether —CH2—O—CH2— moieties replaced by —C4H2O— or —C5H3N—, or

[0215] (4) any combination thereof.

[0216] In some aspects, the metal chelator is a cryptand, where the cryptand is selected from the group consisting of: (1,2,2) cryptand, (2,2,2) cryptand, (2,2,3) cryptand, and (2,3,3) cryptand.

[0217] In some aspects, the cryptand is a substituted cryptand, where the substituted cryptand has:

[0218] (1) one or more cryptand ether oxygens independently replaced by NH or S,

[0219] (2) one or more crown ether —CH2—CH2— moieties replaced by —C6H4—, —C10H6—, or —C6H10—.

[0220] (3) one or more crown ether —CH2—O—CH2— moieties replaced by —C4H2O— or —C5H3N—, or

[0221] (4) any combination thereof.

[0222] In some aspects, the gene delivery polymer is Crown Poloxamer (aza crown ether-linked poloxamer), where Crown Poloxamer comprises a polymer having the formula:

[0223]

[0224] or a pharmaceutically acceptable salt thereof, wherein:

[0225] a represents an integer of about 10 units; and

[0226] b represents an integer of about 21 units; and

[0227] wherein the total molecular weight of the polymer is from about 2,000 Da to about 2,200 Da.

[0228] In some aspects, the gene delivery polymer is present in a solution containing a nucleic acid carrier at about 0.1% - about 5% or about 0.5% - about 5%.

[0229] In some aspects, the gene delivery polymer is a β-amino ester. In some aspects, the polymer is present in a solution containing a nucleic acid carrier at about 0.1% - about 5% or about 0.5% - about 5%.

[0230] In some aspects, the gene delivery polymer is polyinosinic acid - polycytidylic acid. In some aspects, polyinosinic acid - polycytidylic acid is present in a solution containing a nucleic acid carrier at about 0.1% – about 5% or about 0.5% – about 5%.

[0231] In some aspects, the gene delivery polymer further comprises benzalkonium chloride.

[0232] In some aspects, the gene delivery polymer comprises BD15 - 12. In some aspects, the ratio of nucleotide to BD15 - 12 polymer (N:P) is 5:1.

[0233] In some aspects, the gene delivery polymer comprises Omnifect. In some aspects, the ratio of nucleotide to Omnifect polymer (N:P) is 10:1.

[0234] In some aspects, the gene delivery polymer comprises Crown Poloxamer (azacrown ether - linked poloxamer). In some aspects, the ratio of nucleotide to Crown Poloxamer (N:P) is 5:1. In some aspects, the gene delivery polymer comprises Crown Poloxamer and PEG - PEI - cholesterol (PPC) lipid polymer. In some aspects, the gene delivery polymer comprises Crown Poloxamer and benzalkonium chloride. In some aspects, the gene delivery polymer comprises Crown Poloxamer and Omnifect. In some aspects, the gene delivery polymer comprises Crown Poloxamer and linear polyethyleneimine (LPEI). In some aspects, the gene delivery polymer comprises Crown Poloxamer and BD15 - 12.

[0235] In some aspects, the gene delivery polymer comprises Staramine and mPEG-modified Staramine. In some aspects, the mPEG-modified Staramine is Staramine-mPEG515. In some aspects, the mPEG-modified Staramine is Staramine-mPEG11. In some aspects, the ratio of Staramine to mPEG-modified Staramine is 10:1. In some aspects, the ratio of nucleotide to polymer (N:P) is 5:1. In some aspects, the gene delivery polymer comprises Staramine, mPEG-modified Staramine, and Crown Poloxamer. In some aspects, the gene delivery polymer comprises Staramine, Staramine-mPEG515, and Crown Poloxamer. In some aspects, the gene delivery polymer comprises Staramine, Staramine-mPEG11, and Crown Poloxamer.

[0236] In some aspects, the gene delivery polymer comprises a poloxamer backbone as disclosed in WO 2022 / 072910 A1, which is incorporated herein by reference in its entirety.

[0237] In some aspects, intraperitoneal delivery comprises nanoparticles of a DNA plasmid encoding interleukin-12 (IL-12) and a synthetic polymer that facilitates plasmid delivery.

[0238] In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 40 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 45 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 50 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 55 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 60 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 65 mg / m 2 to about 80 mg / m 2 In some aspects, the nanoparticles are administered at a dose of about 70 mg / m 2 to about 80 mg / m2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 75 mg / m 2 to about 80 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 75 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 70 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 65 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 60 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 55 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 50 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 45 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 40 mg / m 2 administered at a dose of.

[0239] In some aspects, the nanoparticles are administered at the following doses: about 35 mg / m 2 , about 40 mg / m 2 , about 45 mg / m 2 , about 50 mg / m 2 , about 55 mg / m 2 , about 60 mg / m 2 , about 65 mg / m 2 , about 70 mg / m 2 , about 75 mg / m 2 or about 80 mg / m 2 .

[0240] In some aspects, the nanoparticles are administered at a dose of about 60 mg / m 2 administered at a dose of.

[0241] III. Immune checkpoint inhibitors

[0242] Immune checkpoint proteins interact with specific ligands that send signals inhibiting T cell function into T cells. Cancer cells take advantage of this by driving high-level expression of checkpoint proteins on their surface, thereby suppressing the anti-cancer immune response.

[0243] Immune checkpoint inhibitors comprise any compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes attenuation of function as well as complete blockade. In some aspects, the immune checkpoint protein is a human checkpoint protein.

[0244] In some aspects, the immune checkpoint inhibitor is an antagonist of the immune checkpoint protein. In some aspects, the immune checkpoint inhibitor is an agonist of the immune checkpoint protein.

[0245] In some aspects, the immune checkpoint protein is selected from the group consisting of: CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO-1, CEA, PVRIG, GARP, STING, Siglec-15, CD20, CD27, CD38, CD39, CD47, CD66a (CEACAM1), CD73, CD80, CD86, CD93, CD96, and / or CD161.

[0246] In some aspects, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0247] In some aspects, the immune checkpoint inhibitor is a small molecule inhibitor.

[0248] In some aspects, the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to PD-1. In some aspects, the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to PD-L1. In some aspects, the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to CTLA-4. In some aspects, the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to LAG-3.

[0249] In some aspects, the immune checkpoint inhibitor is an antibody. In some aspects, the immune checkpoint inhibitor comprises an antibody or a fragment thereof that specifically binds to an immune checkpoint protein. In some aspects, the immune checkpoint inhibitor is a monoclonal antibody, a fully human antibody, a chimeric antibody, a humanized antibody, or a fragment thereof that is capable of at least partially antagonizing the immune checkpoint protein.

[0250] In some aspects, the immune checkpoint inhibitor comprises the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence disclosed in Table 1. In some aspects, the immune checkpoint inhibitor comprises the heavy chain (HC) amino acid sequence and the light chain (LC) amino acid sequence disclosed in Table 2. In some aspects, the immune checkpoint inhibitor comprises VH complementarity determining region (CDR) 1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 as disclosed in Table 3.

[0251] Table 1 - VH and VL Sequences of Immune Checkpoint Inhibitor Antibodies

[0252]

[0253]

[0254]

[0255] Table 2 - HC and LC Sequences of Immune Checkpoint Inhibitor Antibodies

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264] Table 3 - VH CDR and VL CDR Sequences of Immune Checkpoint Inhibitor Antibodies

[0265]

[0266]

[0267]

[0268]

[0269]

[0270] In some aspects, the immune checkpoint inhibitor is ipilimumab. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 1 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 1.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 2 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 2.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 3 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 3.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 4 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 4 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 3 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 2.5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 2 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 1.5 mg / kg.

[0271] In some aspects, ipilimumab is administered at the following doses: about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg or about 5 mg / kg.

[0272] In some aspects, ipilimumab is administered at a dose of about 1 mg / kg.

[0273] In some aspects, ipilimumab is administered once every 1, 2, 3, 4, 5, 6, 7 or 8 weeks.

[0274] In some aspects, ipilimumab is administered once every 2 - 8 weeks (e.g., every 6 weeks) during treatment.

[0275] In some aspects, ipilimumab is administered intravenously.

[0276] In some aspects, the immune checkpoint inhibitor is nivolumab. In some aspects, nivolumab is administered at a dose of about 120 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 140 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 160 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 180 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 200 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 220 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 240 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 340 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 320 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 300 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 280 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 260 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 220 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 200 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 180 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 160 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 140 mg. In some aspects, nivolumab is administered at a dose of about 240 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 140 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 160 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 180 mg to about 240 mg.In some aspects, nivolumab is administered at a dose of about 200 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 220 mg to about 240 mg.

[0277] In some aspects, nivolumab is administered at a dose of about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, or about 360 mg.

[0278] In some aspects, nivolumab is administered at a dose of about 240 mg.

[0279] In some aspects, nivolumab is administered intravenously.

[0280] In some aspects, nivolumab is administered once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0281] In some aspects, nivolumab is administered once every 1-4 weeks (eg, every 2 weeks) during the treatment period.

[0282] PD-1

[0283] Human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity have been disclosed in U.S. Patent Nos. 8,008,449 and 8,779,105. Other anti-PD-1 mAbs have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication No. WO 2012 / 145493. Each of the anti-PD-1 HuMAbs disclosed in U.S. Patent No. 8,008,449 has been shown to exhibit one or more of the following characteristics: (A) 1 x 10 -7 M or smaller K D Binds to human PD-1 as determined by surface plasmon resonance using a Biacore biosensor system; (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon gamma production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates an Ab response; and (j) inhibits tumor cell growth in vivo. In some aspects, the anti-PD-1 antibodies of the combination therapy disclosed herein include mAbs that specifically bind to human PD-1 and exhibit at least one of the foregoing characteristics.

[0284] In some aspects, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as BMS-936558; formerly named 5C4, BMS-936558, MDX-1106 or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks the interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In some aspects, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In some aspects, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0285] Anti-PD-1 antibodies useful in the disclosed compositions also include isolated antibodies that specifically bind human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105; International Publication No. WO 2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that specific epitope region. These cross-competing antibodies are expected to have functional properties very similar to nivolumab due to their binding to the same epitope region of PD-1. In standard PD-1 binding assays such as Biacore analysis, ELISA assays or flow cytometry, cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab (see, e.g., International Publication No. WO 2013 / 173223).

[0286] In certain embodiments, the antibody or antigen-binding fragment thereof that cross-competes with nivolumab for binding to human PD-1 or that binds to the same epitope region of nivolumab that binds to human PD-1 is an mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art.

[0287] In some aspects, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as pabolizumab and MK-3475) is a humanized monoclonal IgG4 (S228P) antibody that directly targets the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587. Pembrolizumab has been approved by the FDA for the treatment of recurrent or refractory melanoma.

[0288] In some aspects, the anti-PD-1 antibody is REGN2810. In some aspects, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011). In some aspects, the anti-PD-1 antibody is MEDI0608 (formerly known as AMP-514), which is a monoclonal antibody. MEDI0608 is described, for example, in U.S. Patent No. 8,609,089. In some aspects, the anti-PD-1 antibody or an antigen-binding fragment thereof is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publication No. 2015 / 0079109. Antibodies or antigen-binding fragments thereof that bind the same epitope or have the same CDRs as any of these antibodies can be used.

[0289] Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for the uses disclosed herein can be generated using methods well known in the art. Alternatively, anti-PD-1 antibodies recognized in the art can be used.

[0290] In various aspects, the anti-PD-1 antibody is selected from the group consisting of: nivolumab (also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as Pembrolizumab and MK-3475 (see WO2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), Cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHIPHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Yuheng Pharmaceutical; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics, see WO 2017 / 19846) and IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540), the references of which are incorporated herein by reference.

[0291] Other anti-PD-1 monoclonal antibodies have been described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Publication No. 2016 / 0272708, and PCT Publication Nos. WO 2012 / 145493, WO 2008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 2017 / 020858, WO 2016 / 197367, WO 2017 / 024515, WO 2017 / 025051, WO 2017 / 123557, WO 2016 / 106159, WO 2014 / 194302, WO 2017 / 040790, WO 2017 / 133540, WO 2017 / 132827, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 106061, WO 2017 / 19846, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540, each of which is incorporated herein by reference.

[0292] Anti-PD-1 antibodies useful in the combination therapies of the present invention also include antigen-binding portions of the above-described antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of the VH and CH1 domains; and (iv) Fv fragment consisting of the VL and VH domains of a single arm of the antibody.

[0293] An anti-PD-1 antibody suitable for the disclosed combination therapies is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In certain embodiments, the anti-PD-1 antibody or its antigen-binding portion cross-competes with nivolumab for binding to human PD-1. In some aspects, the anti-PD-1 antibody or its antigen-binding portion is a chimeric, humanized or human monoclonal antibody or a portion thereof. In some aspects, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3 or IgG4 isotype can be used.

[0294] In some aspects, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain constant region of the human IgG1 or IgG4 isotype. In some aspects, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or its antigen-binding fragment contains an S228P mutation that replaces the serine residue in the hinge region with a proline residue normally found at the corresponding position in IgG1 isotype antibodies. This mutation present in nivolumab prevents Fab arm exchange with endogenous IgG4 antibodies while retaining the low affinity for activating Fc receptors associated with wild-type IgG4 antibodies (Wang et al., 2014). In some aspects, the antibody comprises a light chain constant region that is a human κ or λ constant region. In other embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an mAb or its antigen-binding portion. In certain embodiments, any of the therapeutic methods described herein includes administering an anti-PD-1 antibody, and the anti-PD-1 antibody is nivolumab.

[0295] In some aspects, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dostarlimab.

[0296] PD-L1

[0297] An anti-human PD-L1 antibody (or VH and / or VL domains derived therefrom) suitable for the combination therapies disclosed herein can be generated using methods known in the art. Examples of anti-PD-L1 antibodies that can be used in the methods of the present disclosure include the antibodies disclosed in U.S. Patent No. 9,580,507, which is incorporated herein by reference. The anti-PD-L1 human monoclonal antibodies disclosed in U.S. Patent No. 9,580,507 have been shown to exhibit one or more of the following characteristics: (a) a K -7 of 1 x 10 DBinding to human PD-L1, as determined by surface plasmon resonance using a Biacore biosensor system; (b) increasing T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increasing interferon γ production in an MLR assay; (d) increasing IL-2 secretion in an MLR assay; (e) stimulating antibody responses; and (f) reversing the effects of regulatory T cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies useful in the methods disclosed herein include monoclonal antibodies that specifically bind human PD-L1 and exhibit at least one of the foregoing characteristics.

[0298] Well-known anti-PD-L1 antibodies can be used. For example, the human anti-PD-L1 antibodies disclosed in U.S. Patent No. 7,943,743, the content of which is incorporated herein by reference, can be used. Such anti-PD-L1 antibodies include 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Other well-known anti-PD-L1 antibodies in the art that can be used include, for example, those described in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009 / 0317368, and PCT Publication Nos. WO 2011 / 066389 and WO 2012 / 145493, the teachings of which are also incorporated herein by reference. Other examples of anti-PD-L1 antibodies include atezolizumab (TECENTRIQ; RG7446) or durvalumab (IMFINZI; MEDI4736) or avelumab (Bavencio). Antibodies or antigen-binding fragments thereof that compete with any of these well-known antibodies or inhibitors for binding to PD-L1 can also be used.

[0299] In some aspects, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO 2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al. 2013 J Clin Oncol 31(suppl):3000; U.S. Patent No. 8,217,149), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013; September 27 - October 1, 2013; Amsterdam, The Netherlands. Abstract 802) or MSB0010718C (also known as avelumab; see US2014 / 0341917). In some aspects, an antibody that cross-competes with the PD-L1 antibody described above for binding to human PD-L1 or binds to the same epitope region of human PD-L1 as the PD-L1 antibody described above is an mAb. For administration to a human subject, these cross-competing antibodies can be chimeric antibodies, or can be humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art. In some aspects, the anti-PD-L1 antibody is selected from the group consisting of: BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; also known as MPDL3280A, RG7446; see US 8,217,149; also see, Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; also known as IMFINZI TM , MEDI-4736; see WO 2011 / 066389), avelumab (Pfizer; also known as MSB-0010718C (see WO 2013 / 079174), STI-1014 (Sorrento; see WO2013 / 181634), CX-072 (Cytomx; see WO2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, for example, WO 2017 / 034916) and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (Apr 2016)).

[0300] In some aspects, the PD-L1 antibody is atezolizumab Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0301] In some aspects, the PD-L1 antibody is durvalumab (IMFINZI TM ). Durvalumab is a human IgG1κ monoclonal anti-PD-L1 antibody.

[0302] In some aspects, the PD-L1 antibody is avelumab Avelumab is a human IgG1λ monoclonal anti-PD-L1 antibody.

[0303] In some aspects, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1 and any combination thereof.

[0304] In some aspects, the anti-PD-L1 antibody of the disclosed method comprises an isolated antibody that specifically binds human PD-L1 and cross-competes for binding to human PD-L1 with any anti-PD-L1 antibody disclosed herein (e.g., atezolizumab, durvalumab, and / or avelumab). In some embodiments, the anti-PD-L1 antibody binds the same epitope as any anti-PD-L1 antibody described herein (e.g., atezolizumab, durvalumab, and / or avelumab). In a standard PD-L1 binding assay such as Biacore analysis, ELISA assay, or flow cytometry, cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab and / or avelumab (see, for example, WO 2013 / 173223).

[0305] In some aspects, antibodies that cross-compete with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1, or that bind to the same epitope region of antibodies that bind human PD-L1 as atezolizumab, durvalumab, and / or avelumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0306] Anti-PD-L1 antibodies useful in the methods disclosed herein also include antigen-binding portions of the above-described antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be carried out by fragments of the full-length antibody.

[0307] CTLA-4

[0308] Monoclonal antibodies that specifically bind CTLA-4 include, but are not limited to, ipilimumab ( BMS) and tremelimumab (AstraZeneca / MedImmune), as well as the antibodies disclosed in U.S. Patent Application Publication Nos. 2005 / 0201994, 2002 / 0039581, and 2002 / 0086014, the respective contents of which are incorporated herein by reference, and the antibodies disclosed in U.S. Patent Nos. 5,811,097; 5,855,887; 6,051,227; 6,984,720; 6,682,736; 6,207,156; 5,977,318; 6,682,736; 7,109,003; 7,132,281; and 8,491,895, the respective contents of which are incorporated herein by reference, or antibodies comprising the heavy and light chain variable regions of any of these antibodies.

[0309] Human monoclonal antibodies that specifically bind CTLA-4 with high affinity have been disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described, for example, in U.S. Patent No. 7,034,121 and International Publication Nos. WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237, and WO 2000 / 037504. In some aspects, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some aspects, the CTLA-4 antagonist is selected from the group consisting of ipilimumab and tremelimumab. In some aspects, the CTLA-4 antagonist is ipilimumab.

[0310] In some aspects, the anti-CTLA-4 antibodies of the disclosed methods comprise isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any of the anti-CTLA-4 antibodies disclosed herein (e.g., ipilimumab) for binding to human CTLA-4. In some embodiments, the anti-CTLA-4 antibody binds to the same epitope as any of the anti-CTLA-4 antibodies described herein (e.g., ipilimumab). In standard CTLA-4 binding assays such as Biacore assays, ELISA assays, or flow cytometry, cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab (see, e.g., WO2013 / 173223).

[0311] In some aspects, antibodies that cross-compete with ipilimumab for binding to human CTLA-4 or that bind to the same epitope region of CTLA-4 as ipilimumab. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0312] The anti-CTLA-4 antibodies useful in the methods disclosed herein also include antigen-binding portions of the above-described antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be carried out by fragments of the full-length antibody.

[0313] LAG-3

[0314] Pathways involving LAG-3, BTLA, B7-H3, B7-H4, TIM-3, and KIR constitute 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).

[0315] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for the combination therapies disclosed herein can be generated using methods well known in the art. Alternatively, anti-LAG-3 antibodies recognized in the art can be used.

[0316] For example, an anti-human LAG-3 antibody described in US2011 / 0150892A1 and referred to as monoclonal antibody 25F7 (also referred to as "25F7" and "LAG3.1") can be used, the teachings of which are incorporated herein by reference. Other well-known anti-LAG-3 antibodies in the art that can be used include IMP731 (H5L7BW) described in US2011 / 007023, MK-4280 (28G-10) described in WO2016028672, REGN3767 described in Journal for ImmunoTherapy of Cancer, (2016) Vol.4, Supp.Supplement 1 Abstract Number: P195, BAP050, IMP-701 (LAG-525), IMP321 (eftilagimod alpha)), Sym022, TSR-033, MGD013, BI754111, FS118, AVA-017, and GSK2831781 described in WO2017 / 019894. These and other anti-LAG-3 antibodies that can be used in the claimed invention can be found, for example, in the following documents: WO2016 / 028672, WO2017 / 106129, WO2017 / 062888, WO2009 / 044273, WO2018 / 069500, WO2016 / 126858, WO2014 / 179664, WO2016 / 200782, WO2015 / 200119, WO2017 / 019846, WO2017 / 198741, WO2017 / 220555, WO2017 / 220569, WO2018 / 071500, WO2017 / 015560, WO2017 / 025498, WO2017 / 087589, WO2017 / 087901, WO2018 / 083087, WO2017 / 149143, WO2017 / 219995, US2017 / 0260271, WO2017 / 086367, WO / 2017 / 086419, WO2018 / 034227, and WO2014 / 140180. The content of each of these references is incorporated herein by reference.

[0317] Antibodies that compete with any of the above reference antibodies for binding to LAG-3 can also be used.

[0318] An exemplary anti-LAG-3 antibody is BMS-986016, as described in U.S. Patent No. 9,505,839, which is incorporated herein by reference. In some aspects, the anti-LAG-3 antibody is BMS-986016.

[0319] In some aspects, the antibody has the heavy chain CDR and light chain CDR or variable regions of BMS-986016. In some aspects, the antibody competes for binding and / or binds to the same epitope on LAG-3 as the antibodies mentioned above. In some aspects, the antibody binds to an epitope of human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO:82). In some aspects, the antibody binds to an epitope of human LAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO:83) or PAAPSSWG (SEQ ID NO:84).

[0320] IV. Anticancer Agents

[0321] In some aspects of the combination therapies or methods disclosed herein, the anticancer agent is a chemotherapeutic agent selected from the group consisting of: taxanes, platinum compounds, anthracyclines, cyclophosphamide, topotecan, carmustine (BCNU) or combinations thereof. In some aspects, the anticancer therapy is selected from the group consisting of: paclitaxel, carboplatin, docetaxel, albumin-bound paclitaxel, doxorubicin and any combination thereof.

[0322] In some aspects, the chemotherapeutic agent is selected from the group consisting of: topoisomerase inhibitors (e.g., irinotecan, topotecan, doxorubicin, epirubicin, idarubicin), antimicrotubule agents (e.g., paclitaxel, docetaxel), alkylating agents (e.g., cyclophosphamide, dacarbizine), platinum drugs (cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., gemcitabine, methotrexate, 5-fluorouracil) or combinations thereof.

[0323] In some aspects, the anticancer agent is doxorubicin.

[0324] In some aspects, the anticancer agent comprises paclitaxel.

[0325] In some aspects, the anticancer agent comprises carboplatin.

[0326] In some aspects, the anticancer agent comprises docetaxel.

[0327] In some aspects, the anticancer agent comprises albumin-bound paclitaxel.

[0328] In some aspects, the anticancer agent comprises.

[0329] In some aspects, the anticancer agent is administered once every three weeks before interval cytoreductive surgery for about 12 to about 18 weeks.

[0330] In some aspects, the anticancer agent is administered once every three weeks before interval cytoreductive surgery for about 12 to about 18 weeks.

[0331] In some aspects, the anti-cancer agent is administered at least about 28 days after interval cytoreductive surgery, once every three weeks for about 9 weeks.

[0332] In some aspects, the anti-cancer agent is selected from the group consisting of paclitaxel, carboplatin, docetaxel, albumin-bound paclitaxel, and any combination thereof.

[0333] In some aspects, a nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the anti-cancer agent.

[0334] In some aspects, the anti-cancer agent (e.g., the first) is administered, followed by the administration of a nucleic acid carrier formulated with a lipid polymer (e.g., the second), followed by the administration of an immune checkpoint inhibitor (e.g., the third).

[0335] In some aspects, the anti-cancer agent (e.g., the first) is administered, followed by the administration of a nucleic acid carrier formulated with a lipid polymer (e.g., the second), followed by an immune checkpoint inhibitor (e.g., the third), and then surgery is performed to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) (e.g., the fourth).

[0336] In some aspects, surgery is performed to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) (e.g., the first), followed by the administration of a nucleic acid carrier formulated with a lipid polymer (e.g., the second), and then the administration of an immune checkpoint inhibitor (e.g., the third, fourth, etc., depending on how many immune checkpoint inhibitors are administered). In some aspects, the anti-cancer agent is administered, followed by the administration of a DNA plasmid, followed by an immune checkpoint inhibitor, followed by interval cytoreductive surgery.

[0337] In some aspects, the anti-cancer agent is administered once every three weeks for about 12 to about 18 weeks before interval cytoreductive surgery.

[0338] In some aspects, the anti-cancer agent is administered at least about 28 days after interval cytoreductive surgery (e.g., once every three weeks for about 9 weeks).

[0339] In some aspects, the administration of the anti-cancer agent includes administering paclitaxel at a dose of about 25 - 250 mg / m 2 and optionally, subsequently administering carboplatin IV at a dose of about AUC 4 - 6.

[0340] In some aspects, the administration of the anti-cancer agent includes administering paclitaxel at the following doses: about 25 - 250 mg / m 2 about 50 - 250 mg / m 2 about 75 - 250 mg / m 2 about 100 - 250 mg / m 2, about 125 - 250 mg / m 2 , about 150 - 250 mg / m 2 , about 175 - 250 mg / m 2 , about 200 - 250 mg / m 2 , about 225 - 250 mg / m 2 , about 25 - 225 mg / m 2 , about 25 - 200 mg / m 2 , about 25 - 175 mg / m 2 , about 25 - 150 mg / m 2 , about 25 - 125 mg / m 2 , about 25 - 250 mg / m 2 , about 25 - 100 mg / m 2 , about 25 - 75 mg / m 2 or about 25 - 50 mg / m 2 , optionally, followed by IV administration of carboplatin at a dose of about AUC 4 - 6.

[0341] In some aspects, the administration of the anti - cancer agent includes administering paclitaxel at the following doses: about 25 mg / m 2 , about 50 mg / m 2 , about 75 mg / m 2 , about 100 mg / m 2 , about 125 mg / m 2 , about 150 mg / m 2 , about 175 mg / m 2 , about 200 mg / m 2 , about 225 mg / m 2 or about 250 mg / m 2 , optionally, followed by IV administration of carboplatin at a dose of about AUC 4 - 6.

[0342] In some aspects, the administration of the anti - cancer agent includes administering docetaxel at a dose of 25 - 250 mg / m 2 , optionally, followed by IV administration of carboplatin at a dose of about AUC 4 - 6.

[0343] In some aspects, the administration of the anti - cancer agent includes administering docetaxel at the following doses: about 25 - 250 mg / m 2 , about 50 - 250 mg / m 2 , about 75 - 250 mg / m 2 , about 100 - 250 mg / m 2 , about 125 - 250 mg / m 2 , about 150 - 250 mg / m 2 , about 175 - 250 mg / m 2, about 200 - 250 mg / m 2 , about 225 - 250 mg / m 2 , about 25 - 225 mg / m 2 , about 25 - 200 mg / m 2 , about 25 - 175 mg / m 2 , about 25 - 150 mg / m 2 , about 25 - 125 mg / m 2 , about 25 - 250 mg / m 2 , about 25 - 100 mg / m 2 , about 25 - 75 mg / m 2 or about 25 - 50 mg / m 2 , optionally, subsequently administer carboplatin IV at a dose of about AUC 4 - 6.

[0344] In some aspects, the administration of the anti - cancer agent includes administering docetaxel at the following doses: about 25 mg / m 2 , about 50 mg / m 2 , about 75 mg / m 2 , about 100 mg / m 2 , about 125 mg / m 2 , about 150 mg / m 2 , about 175 mg / m 2 , about 200 mg / m 2 , about 225 mg / m 2 or about 250 mg / m 2 , optionally, subsequently administer carboplatin IV at a dose of about AUC 4 - 6.

[0345] In some aspects, the administration of the anti - cancer agent includes administering nab - paclitaxel at a dose of 25 - 350 mg / m 2 , optionally, subsequently administer carboplatin IV at a dose of about AUC 4 - 6.

[0346] In some aspects, the administration of the anti - cancer agent includes administering nab - paclitaxel at the following doses: about 25 - 250 mg / m 2 , about 50 - 250 mg / m 2 , about 75 - 250 mg / m 2 , about 100 - 250 mg / m 2 , about 125 - 250 mg / m 2 , about 150 - 250 mg / m 2 , about 175 - 250 mg / m 2 , about 200 - 250 mg / m 2 , about 225 - 250 mg / m 2 , about 25 - 225 mg / m2 , about 25 - 200 mg / m 2 , about 25 - 175 mg / m 2 , about 25 - 150 mg / m 2 , about 25 - 125 mg / m 2 , about 25 - 250 mg / m 2 , about 25 - 100 mg / m 2 , about 25 - 75 mg / m 2 or about 25 - 50 mg / m 2 , optionally, followed by intravenous administration of carboplatin at a dose of about AUC 4 - 6.

[0347] In some aspects, the administration of the anti - cancer agent includes administering nab - paclitaxel at the following doses: about 25 mg / m 2 , about 50 mg / m 2 , about 75 mg / m 2 , about 100 mg / m 2 , about 125 mg / m 2 , about 150 mg / m 2 , about 175 mg / m 2 , about 200 mg / m 2 , about 225 mg / m 2 or about 250 mg / m 2 , optionally, followed by intravenous administration of carboplatin at a dose of about AUC 4 - 6.

[0348] In some aspects, the administration of the nanoparticles before interval cytoreductive surgery starts 15 days after the first administration of the anti - cancer agent, once a week, for at least about 12 to about 18 weeks.

[0349] In some aspects, the administration of the anti - cancer agent includes administering paclitaxel at a dose of about 175 mg / m 2 , followed by intravenous administration of carboplatin at a dose of about AUC4 - 6.

[0350] In some aspects, the administration of the anti - cancer agent includes administering docetaxel at a dose of about 75 mg / m 2 , followed by intravenous administration of carboplatin at a dose of about AUC4 - 6.

[0351] In some aspects, the administration of the anti - cancer agent includes administering nab - paclitaxel at a dose of about 260 mg / m 2 , followed by intravenous administration of carboplatin at a dose of about AUC 4 - 6.

[0352] V. Treatment methods

[0353] Certain aspects of the present disclosure relate to methods of treating a subject having cancer, which include administering to the subject a combination of: (i) a nanoparticle comprising an IL-2 encoding sequence (e.g., the nucleic acid is a plasmid-based gene expression system containing a DNA sequence encoding interleukin-12), and (ii) an immune checkpoint inhibitor. In some aspects, the method further includes administering a chemotherapeutic agent to the subject. In some aspects, the method further includes surgery. In some aspects, the nanoparticle comprises a PEG-PEI-cholesterol (PPC) lipid polymer.

[0354] The present disclosure also provides methods for treating mammalian cancer or a proliferative disorder by intratumoral, intraperitoneal, intravenous, intracapsular, intratracheal, intracranial, or systemic administration of a pharmaceutical composition comprising a plasmid-based gene expression system and a gene delivery polymer, without chemotherapeutic drugs. Mammalian cancers are selected from the group consisting of primary or metastatic ovarian tumors. In some aspects, the nucleic acid is a plasmid-based gene expression system containing a DNA sequence encoding human interleukin-12.

[0355] Treatment of tumors with the pharmaceutical composition (nucleic acid plus gene delivery polymer and one or more chemotherapeutic agents) results in tumor shrinkage and increased lifespan. According to the methods of the present disclosure, the combination of gene therapy (nucleic acid and gene delivery polymer) and chemotherapy (chemotherapeutic agent) produces additive and / or synergistic efficacy. The efficacy of the methods of the present invention is defined as, but not limited to, shrinkage of tumor size or reduction of tumor density, increase in lymphocyte count or neutrophil count or increase in survival rate, or all of the above. In addition, according to the methods of the present invention, the combination of gene therapy (nucleic acid and gene delivery polymer) and chemotherapy (chemotherapeutic agent) reduces the toxicity of the chemotherapeutic agent and reverses tumor resistance to chemotherapy. Toxicity herein is defined as any treatment-related adverse effect on clinical observation, including but not limited to abnormal hematology or serum chemistry or organ toxicity. In addition, according to the methods of the present invention, the combination of gene therapy (nucleic acid and gene delivery polymer) and suboptimal doses of chemotherapy (chemotherapeutic agent) enhances the anti-cancer effect to a level equal to or higher than that achieved with optimal doses of chemotherapeutic agents, but with less toxicity.

[0356] New cancer treatment strategies focus on delivering macromolecules carrying genetic information rather than the therapeutic protein itself, allowing the exogenously delivered genes to be expressed in the tumor environment. Compared with viral delivery systems, methods using non-viral gene delivery systems are considered safer, but due to poor efficiency, the practical applications of current polymer systems are not satisfactory. A strategy has recently been disclosed in which the gene transfection efficiency of low molecular weight PEI is enhanced by covalently linking cholesterol to form a water-soluble lipid polymer (WSLP). See, Mol. Ther., 2001, 4, 130. Transferring the IL-12 gene into solid tumors using WSLP was significantly superior to unmodified PEI and resulted in more significant tumor suppression.

[0357] Recognizing that due to the multifactorial nature of this disease, single treatment strategies for cancer are often ineffective. The methods disclosed herein provide the benefits of a combination from more than one drug or therapeutic agent to maximize the anti-cancer response. In some aspects, combined herein are the gene delivery of a chemotherapeutic agent and an immune checkpoint inhibitor and an anti-cancer IL-12 expression construct (e.g., GEN-1). Combining the safe and effective delivery of an anti-cancer gene with a standard chemotherapeutic agent and an immune checkpoint inhibitor can enhance the anti-cancer response. Such combination therapies can be used to reduce the chemotherapy dose and increase tumor sensitivity to chemotherapy. In some aspects, the compositions disclosed herein can comprise an anti-cancer gene, an immune checkpoint inhibitor, and at least one adjuvant chemotherapeutic drug complexed with a gene delivery polymer, wherein the combination is more effective than the gene therapy, immune checkpoint inhibitor, or chemotherapy treatment administered alone. Additionally, the combination therapies disclosed herein can be effectively targeted to multiple tumors and administered via different routes of administration.

[0358] CTLA4 blockade can significantly increase CD8 T cell infiltration in the tumor microenvironment (TME). The CTLA4 and PD1 blockade mechanisms are non-redundant, resulting in a combination of enhanced immune and clinical responses. However, most patients with solid tumors do not respond to these agents, in which the TME is considered to be less immunogenic, and in which pro-inflammatory cytokines such as IL-12 may play an important role in converting the TME that synergizes with CTLA4 blockade and PD1 blockade. IL-12 has been shown to enhance the homeostatic proliferation of CD8+ T cells in vivo, and CD8+ T cells are significantly increased in biopsy samples obtained from cancer patients treated with IL-12.

[0359] IL-12 is a potent pro-inflammatory cytokine that promotes the differentiation of CD4+ cells into Th1 cells and has a co-proliferative effect on pre-activated NK and T cells. IL-12 also independently and / or synergistically enhances the cytolytic capabilities of both NK and CD8+ T cells. The anti-tumor activity of systemic or local administration of IL-12 has been established in preclinical studies against various tumor cell lines. However, the potent pro-inflammatory anti-tumor immune response mediated by IL-12 can still be counteracted by tumor-mediated immunosuppressive mechanisms, in which the PD-1:PD-L interaction plays a key role. The induction of IFN-γ and other pro-inflammatory cytokines by IL-12 has been well demonstrated. IFNγ is a pleiotropic cytokine that has been reported to promote tumor immunity and tumor rejection in many models. In these models, IFNγ has anti-proliferative and pro-apoptotic functions and inhibits tumor angiogenesis. Many studies have shown the importance of IFNγ in the efficacy of tumor vaccines and IL-12 therapy. In clinical trials, IFNγ therapy has been associated with melanoma regression in some patients. However, clinical trials of patients with melanoma treated with IFNγ alone or in combination with IFNα have not shown improved survival compared to other therapies. Consistent with these findings, many studies have demonstrated that IFNγ stimulation can induce upregulation of PD-L1 in monocytes, tumor cells, and other cell types. In one study, the expression of PD-L1 increased in each cell line and primary cells incubated with IFNγ.

[0360] Certain aspects of the present disclosure relate to combination therapies that include: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer (e.g., a nanoparticle); and (ii) an immune checkpoint inhibitor (e.g., an antibody).

[0361] Certain aspects of the present disclosure relate to methods of treating a subject having cancer, the method comprising administering to the subject a combination therapy that includes: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer (e.g., a nanoparticle); and (ii) an immune checkpoint inhibitor (e.g., an antibody).

[0362] In some aspects, the polynucleotide encodes human IL-12.

[0363] In some aspects, the nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL12.

[0364] In some aspects, the nucleic acid carrier (e.g., plasmid) contains introns, 3'UTR (e.g., hGH 3'UTR), antibiotic resistance genes, or any combination thereof (e.g., Figure 1 elements).

[0365] In some aspects, the lipid polymer is composed of polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups (e.g., Figure 2 lipid polymer).

[0366] In some aspects, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0367] In some aspects, the immune checkpoint inhibitor is an antibody.

[0368] In some aspects, the immune checkpoint inhibitor is a small molecule inhibitor.

[0369] In some aspects, the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and cemiplimab.

[0370] In some aspects, the immune checkpoint inhibitor is a PD-1 antagonist, wherein the PD-1 antagonist is nivolumab.

[0371] In some aspects, the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0372] In some aspects, the immune checkpoint inhibitor is a CTLA-4 antagonist, which is ipilimumab.

[0373] In some aspects, the immune checkpoint inhibitor is a LAG-3 antagonist, wherein the LAG-3 antagonist is relatlimab.

[0374] In some aspects, the combination further includes an anti-cancer agent.

[0375] In some aspects, the anti-cancer agent is a chemotherapeutic agent.

[0376] In some aspects, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, albumin-bound paclitaxel, olaparib, and any combination thereof.

[0377] In some aspects, the anti-cancer agent is doxorubicin.

[0378] In some aspects, the anti-cancer agent is paclitaxel.

[0379] In some aspects, the anti-cancer agent is carboplatin.

[0380] In some aspects, the anti-cancer agent is docetaxel.

[0381] In some aspects, the anti-cancer agent is albumin-bound paclitaxel.

[0382] In some aspects, the anti-cancer agent is olaparib.

[0383] In some aspects, the method further comprises performing surgery (e.g., interval debulking surgery) in a subject to remove all or part of the tissue or tumor.

[0384] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered intratumorally or intraperitoneally.

[0385] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered intravenously.

[0386] In some aspects, the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, intracystically, or any combination thereof.

[0387] In some aspects, the immune checkpoint inhibitor is administered intratumorally or intraperitoneally.

[0388] In some aspects, the immune checkpoint inhibitor is administered intravenously.

[0389] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the immune checkpoint inhibitor.

[0390] In some aspects, the nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the anti-cancer agent.

[0391] In some aspects, the anti-cancer agent (e.g., the first) is administered, followed by the administration of the nucleic acid carrier formulated with a lipid polymer (e.g., the second), followed by the administration of the immune checkpoint inhibitor (e.g., the third).

[0392] In some aspects, the anti-cancer agent (e.g., the first) is administered, followed by the administration of the nucleic acid carrier formulated with a lipid polymer (e.g., the second), followed by the immune checkpoint inhibitor (e.g., the third), and then surgery is performed to remove all or part of the tissue or tumor (e.g., interval debulking surgery) (e.g., the fourth).

[0393] In some aspects, surgery is performed to remove all or part of the tissue or tumor (e.g., interval debulking surgery) (e.g., the first), followed by the administration of the nucleic acid carrier formulated with a lipid polymer (e.g., the second), and then the administration of the immune checkpoint inhibitor (e.g., the third, fourth, etc., depending on how many immune checkpoint inhibitors are administered).

[0394] In some aspects, an anti-cancer agent is administered, followed by administration of a DNA plasmid, followed by an immune checkpoint inhibitor, followed by intermediate cytoreductive surgery.

[0395] In some aspects, the anti-cancer agent is administered once every three weeks for about 12 to about 18 weeks before intermediate cytoreductive surgery.

[0396] In some aspects, the anti-cancer agent is administered at least about 28 days after intermediate cytoreductive surgery (e.g., once every three weeks for about 9 weeks).

[0397] In some aspects, administration of the anti-cancer agent includes administering paclitaxel at a dose of about 25 - 250 mg / m 2 and, optionally, subsequently administering carboplatin IV at a dose of about AUC 4 - 6.

[0398] In some aspects, administration of the anti-cancer agent includes administering paclitaxel at the following doses: about 25 - 250 mg / m 2 、about 50 - 250 mg / m 2 、about 75 - 250 mg / m 2 、about 100 - 250 mg / m 2 、about 125 - 250 mg / m 2 、about 150 - 250 mg / m 2 、about 175 - 250 mg / m 2 、about 200 - 250 mg / m 2 、about 225 - 250 mg / m 2 、about 25 - 225 mg / m 2 、about 25 - 200 mg / m 2 、about 25 - 175 mg / m 2 、about 25 - 150 mg / m 2 、about 25 - 125 mg / m 2 、about 25 - 250 mg / m 2 、about 25 - 100 mg / m 2 、about 25 - 75 mg / m 2 or about 25 - 50 mg / m 2 and, optionally, subsequently administering carboplatin IV at a dose of about AUC 4 - 6.

[0399] In some aspects, administration of the anti-cancer agent includes administering paclitaxel at the following doses: about 25 mg / m 2 、about 50 mg / m 2 、about 75 mg / m 2 、about 100 mg / m 2 、about 125 mg / m 2 、about 150 mg / m 2, about 175 mg / m 2 , about 200 mg / m 2 , about 225 mg / m 2 or about 250 mg / m 2 , optionally, followed by intravenous administration of carboplatin at a dose of about AUC 4-6.

[0400] In some aspects, the administration of the anti-cancer agent includes administering docetaxel at a dose of 25-250 mg / m 2 , optionally, followed by intravenous administration of carboplatin at a dose of about AUC 4-6.

[0401] In some aspects, the administration of the anti-cancer agent includes administering docetaxel at the following doses: about 25-250 mg / m 2 , about 50-250 mg / m 2 , about 75-250 mg / m 2 , about 100-250 mg / m 2 , about 125-250 mg / m 2 , about 150-250 mg / m 2 , about 175-250 mg / m 2 , about 200-250 mg / m 2 , about 225-250 mg / m 2 , about 25-225 mg / m 2 , about 25-200 mg / m 2 , about 25-175 mg / m 2 , about 25-150 mg / m 2 , about 25-125 mg / m 2 , about 25-250 mg / m 2 , about 25-100 mg / m 2 , about 25-75 mg / m 2 or about 25-50 mg / m 2 , optionally, followed by intravenous administration of carboplatin at a dose of about AUC 4-6.

[0402] In some aspects, the administration of the anti-cancer agent includes administering docetaxel at the following doses: about 25 mg / m 2 , about 50 mg / m 2 , about 75 mg / m 2 , about 100 mg / m 2 , about 125 mg / m 2 , about 150 mg / m 2 , about 175 mg / m 2 , about 200 mg / m 2 , about 225 mg / m 2 or about 250 mg / m2 , optionally, carboplatin is then administered IV at a dose of about AUC 4-6.

[0403] In some aspects, the administration of the anti-cancer agent includes administering albumin-bound paclitaxel at a dose of 25-350 mg / m 2 , optionally, followed by administering carboplatin IV at a dose of about AUC 4-6.

[0404] In some aspects, the administration of the anti-cancer agent includes administering albumin-bound paclitaxel at the following doses: about 25-250 mg / m 2 , about 50-250 mg / m 2 , about 75-250 mg / m 2 , about 100-250 mg / m 2 , about 125-250 mg / m 2 , about 150-250 mg / m 2 , about 175-250 mg / m 2 , about 200-250 mg / m 2 , about 225-250 mg / m 2 , about 25-225 mg / m 2 , about 25-200 mg / m 2 , about 25-175 mg / m 2 , about 25-150 mg / m 2 , about 25-125 mg / m 2 , about 25-250 mg / m 2 , about 25-100 mg / m 2 , about 25-75 mg / m 2 or about 25-50 mg / m 2 , optionally, followed by administering carboplatin IV at a dose of about AUC 4-6.

[0405] In some aspects, the administration of the anti-cancer agent includes administering albumin-bound paclitaxel at the following doses: about 25 mg / m 2 , about 50 mg / m 2 , about 75 mg / m 2 , about 100 mg / m 2 , about 125 mg / m 2 , about 150 mg / m 2 , about 175 mg / m 2 , about 200 mg / m 2 , about 225 mg / m 2 or about 250 mg / m 2 , optionally, followed by administering carboplatin IV at a dose of about AUC 4-6.

[0406] In some aspects, the administration of the nanoparticles begins 15 days after the first administration of the anti-cancer agent, once a week for at least about 12 weeks to about 18 weeks, prior to intermediate cytoreductive surgery.

[0407] In some aspects, the nanoparticles are administered at least about 28 days after intermediate cytoreductive surgery, and the administration begins 15 days after the first administration of the anti-cancer agent, once a week for at least about 9 weeks.

[0408] In some aspects, interleukin-12 (IL-12) formulated with a lipid polymer (e.g., nanoparticles) is administered at a dose of about 35 mg / m 2 to about 80 mg / m 2 of body surface area.

[0409] In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 40 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 45 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 50 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 55 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 60 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 65 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 70 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 75 mg / m 2 to about 80 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 75 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 70 mg / m 2 of body surface area. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 65 mg / m 2administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 60 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 55 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 50 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 45 mg / m 2 administered at a dose of. In some aspects, the nanoparticles are administered at a dose of about 35 mg / m 2 to about 40 mg / m 2 administered at a dose of.

[0410] In some aspects, the nanoparticles are administered at the following doses: about 35 mg / m 2 about 40 mg / m 2 about 45 mg / m 2 about 50 mg / m 2 about 55 mg / m 2 about 60 mg / m 2 about 65 mg / m 2 about 70 mg / m 2 about 75 mg / m 2 or about 80 mg / m 2 .

[0411] In some aspects, the nanoparticles are administered at a dose of about 60 mg / m 2 administered at a dose of.

[0412] In some aspects, the immune checkpoint inhibitor is ipilimumab. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 1 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 1.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 2 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 2.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 3 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 3.5 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 4 to about 5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 4 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 3 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 2.5 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 2 mg / kg. In some aspects, ipilimumab is administered at a dose of about 0.5 to about 1.5 mg / kg.

[0413] In some aspects, ipilimumab is administered at the following doses: about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, or about 5 mg / kg.

[0414] In some aspects, ipilimumab is administered at a dose of about 1 mg / kg.

[0415] In some aspects, ipilimumab is administered once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0416] In some aspects, ipilimumab is administered once every 2 - 8 weeks (e.g., every 6 weeks) during treatment.

[0417] In some aspects, ipilimumab is administered intravenously.

[0418] In some aspects, the immune checkpoint inhibitor is nivolumab. In some aspects, nivolumab is administered at a dose of about 120 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 140 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 160 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 180 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 200 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 220 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 240 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 340 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 320 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 300 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 280 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 260 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 220 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 200 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 180 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 160 mg. In some aspects, nivolumab is administered at a dose of about 120 mg to about 140 mg. In some aspects, nivolumab is administered at a dose of about 240 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some aspects, nivolumab is administered at a dose of about 140 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 160 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 180 mg to about 240 mg.In some aspects, nivolumab is administered at a dose of about 200 mg to about 240 mg. In some aspects, nivolumab is administered at a dose of about 220 mg to about 240 mg.

[0419] In some aspects, nivolumab is administered at the following doses: about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, or about 360 mg.

[0420] In some aspects, nivolumab is administered at a dose of about 240 mg.

[0421] In some aspects, nivolumab is administered intravenously.

[0422] In some aspects, nivolumab is administered once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

[0423] In some aspects, nivolumab is administered once every 1 - 4 weeks (e.g., every 2 weeks) during treatment.

[0424] In some aspects, the method further comprises administering a second immune checkpoint inhibitor.

[0425] In some aspects, the second immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA - 4, PD - 1 (and its ligands PD - L1 and PD - L2), and / or LAG - 3.

[0426] In some aspects, the second immune checkpoint inhibitor is an antibody.

[0427] In some aspects, the second immune checkpoint inhibitor is a small molecule inhibitor.

[0428] In some aspects, the second immune checkpoint inhibitor is a PD - 1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and cemiplimab.

[0429] In some aspects, the second immune checkpoint inhibitor is a PD - 1 antagonist, wherein the PD - 1 antagonist is nivolumab.

[0430] In some aspects, the second immune checkpoint inhibitor is a PD - L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0431] In some aspects, the second immune checkpoint inhibitor is a CTLA - 4 antagonist, which is ipilimumab.

[0432] In some aspects, the second immune checkpoint inhibitor is a LAG-3 antagonist, wherein the LAG-3 antagonist is relatlimab.

[0433] In some aspects, the first immune checkpoint inhibitor is nivolumab, and the second immune checkpoint inhibitor is ipilimumab.

[0434] In some aspects, nivolumab is administered at about 240 mg once every two weeks, and ipilimumab is administered at about 1 mg / kg once every six weeks.

[0435] In some aspects, the immune checkpoint inhibitor is administered once a week for at least about 12 weeks to at most about 18 weeks, starting at least about 22 days after the first administration of the anti-cancer agent and before interval cytoreductive surgery.

[0436] In some aspects, the immune checkpoint inhibitor is administered once a week for at least about 9 weeks, starting at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0437] In some aspects, interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-cancer agent.

[0438] In some aspects, interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid.

[0439] In some aspects, interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid.

[0440] In some aspects, interval cytoreductive surgery (ICS) is performed at least about 28 days before administration of the immune checkpoint inhibitor.

[0441] In some aspects, interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the immune checkpoint inhibitor.

[0442] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer (e.g., glioblastoma), lung cancer, any combination thereof, and metastases of any of the foregoing cancers.

[0443] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0444] In some aspects, the subject is a human.

[0445] In some aspects, the cancer is ovarian cancer.

[0446] Ovarian cancer (OC) is the most lethal gynecologic cancer in the United States. The five-year overall survival rate for advanced OC is 20%-30%, and more than 50% of patients who respond to current therapies experience recurrence of their disease. Recurrent, platinum-resistant OC is characterized by minimal response to chemotherapy (<10%-15%) and poor prognosis, with an estimated overall survival of <12 months. Therefore, there is a need to improve the prognosis of these patients. The impact of immune checkpoint inhibitors (ICIs) has been significant, providing durable response rates in several cancers. However, the response rate in ovarian cancer is low, ranging from 11%-15% in the platinum-resistant, recurrent setting. Dual immunotherapy with nivolumab and ipilimumab is associated with a higher objective response rate (31% vs. 12%) compared to nivolumab alone, but with limited durability (3.9 vs. 2 months).

[0447] The following examples are illustrative and do not limit the scope of the claimed aspects. Example

[0448] Example 1. Combination Therapy of GEN-1 with Anti-CTLA-4 in a Mouse Model of Ovarian Cancer

[0449] The ability of GEN-1 to synergize with dual ICIs (anti-PD1 + anti-CTLA4) was tested in a preclinical ID8-VEGF immunocompetent mouse model of ovarian cancer (OC). Our results showed that the combination of IL12 and dual ICIs led to enhanced and durable antitumor activity and survival of mice bearing ID8-VEGF tumors compared to mice treated with single therapies (results not shown). The improved antitumor activity was associated with increased T cell tumor infiltration and downregulation of the phenotypic signature of myeloid cells known to mediate immunosuppression, resulting in enhanced effector function of infiltrating T cells. These results provide preclinical data supporting the use of IL12 as a strategy in combination with dual anti-PD1 + anti-CTLA4 therapy in the treatment of OC.

[0450] Example 2. GEN-1 Combination Therapy with Immune Checkpoint Inhibitors (Predictive)

[0451] A combination strategy including IL-12 gene therapy (GEN-1) and CTLA4-PD-1 blockade will be tested. The mechanisms of action of the known sites of activity of IL-12 and CTLA4-PD-1 blockade are non-redundant. Therefore, the antitumor activity of combination therapy with rhIL-12 and immune checkpoint inhibitors (ipilimumab and nivolumab) will be tested. In addition, the study will evaluate the enhanced efficacy of current PD1 / PD-L1-based immunotherapies by targeting pathways that mediate resistance to ICIs.

[0452] Proposed dose levels of IL-12 and the potential of IL-12 to enhance the biological and clinical activity of the ipilimumab-nivolumab (Ipi-Nivo) combination

[0453] IL-12 has been shown to enhance the homeostatic proliferation of CD8+ T cells in vivo. CD8+ T cells are increased in biopsy samples obtained from regressing lesions of cancer patients treated with IL-12. Correlative assays in a Phase I study of IL-12 showed that immune cells from treated patients continuously produced IFN-γ. IFN-γ, a major inducer of PD-L1 expression, was detected at the interface of PD-L1(+) tumors and TILs, but not in PD-L1(−) tumors. PD-L1 expression is associated with a higher likelihood of response to anti-PD1 monoclonal antibodies. Thus, the effect of IL-12 to upregulate TILs in tumors and enhance IFN-γ secretion will be tested to increase PD-L1 expression and improve response.

[0454] Previous studies using rhIL-12 have demonstrated the biological and anti-tumor activity of IL-12. This study escalated the dose of the IL-12 component to 30, 100, 300, or 500 ng / kg in a cohort of three patients. Evaluation of dose-limiting toxicity and biological endpoints indicated that the 300 ng / kg dose was both the maximum tolerated dose and the optimal biological dose of IL-12.

[0455] Traditional chemotherapy regimens are designed to inhibit tumor growth by cytotoxic mechanisms, while immunocytokine therapies are designed to elicit tumor killing by enhancing the immune system against cancer cells. Interleukin-12 (IL-12) is one of the more active immunocytokines in inducing anti-cancer immunity. It is associated with immunomodulatory properties such as T lymphocyte and natural killer (NK) cell proliferation, activation of cytotoxic T lymphocytes (i.e., CD8+ lymphocytes), secretion of IFN-γ, and inhibition of immunosuppressive regulatory T cells. IL-12 also inhibits the process of tumor angiogenesis, which results in tumor death by starvation. These multiple and pleiotropic anti-cancer properties make IL-12 a potentially important immunotherapy.

[0456] The GEN-1 nanoparticle contains a DNA plasmid encoding the IL-12 gene and a synthetic polymer that facilitates plasmid delivery. GEN-1 is designed for local delivery (e.g., intraperitoneal) to provide the potential for cytokine expression specifically in the tumor microenvironment, with the goal of achieving increased efficacy while minimizing potential systemic toxicity. GEN-1 has been studied as a single agent or as a combination agent with standard chemotherapy in subjects with recurrent ovarian cancer. In addition, a Phase I clinical trial in subjects with newly diagnosed ovarian cancer has been completed, in which GEN-1 was evaluated as a combination agent with neoadjuvant chemotherapy.

[0457] Test the IL-12 produced by GEN-1 to evaluate whether CTLA4 and / or PD1 / PDL1 blocking monoclonal antibodies can overcome the immunosuppressive characteristics of peritoneal tumors by inducing an inflamed tumor microenvironment, thereby enhancing the efficacy of these checkpoint inhibitors.

[0458] Objectives

[0459] Phase I: Investigate the safety, tolerability (MTD, DLT), and recommended Phase 2 dose (P2RD) of the combination of GEN-1 and anti-PD1 / CTLA4 (dual ICI) in subjects with recurrent or persistent ovarian tumors.

[0460] Phase II: Determine the overall objective tumor response rate of the proposed combination regimen.

[0461] Measurement of primary endpoints

[0462] Phase I: The safety, tolerability, and recommended Phase 2 dose of the regimen are evaluated by the Data and Safety Monitoring Board (DSMB). Details of their responsibilities will be set out in the DSMB charter, which will be drafted prior to the start of the study for DSMB approval.

[0463] Phase II: The overall objective response rate is determined using RECIST 1.1.

[0464] In addition, the following will be evaluated: progression-free survival (PFS), overall survival (OS), objective tumor response rate (ORR), overall toxicity, and clinical benefit rate.

[0465] PFS will be measured as the time from enrollment to the first day of the occurrence of one of the following: i) death from any cause; or ii) confirmed progression of target lesions, non-target lesions, and / or new lesions according to the Response Evaluation Criteria in Solid Tumors (RECIST).

[0466] OS will be measured as the time from enrollment to the date of death.

[0467] ORR will be measured according to the response evaluation criteria of the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The protocol allows subjects with measurable and non-measurable disease to participate and will therefore deviate slightly from the standard RECIST, as described later in this protocol.

[0468] Design overview

[0469] This is a single-arm, multi-center Phase I / II trial designed to evaluate the safety, dosing, and preliminary efficacy of adding GEN-1 to a fixed-dose combination of two ICIs in patients with recurrent or persistent ovarian cancer. The dual-ICI regimen will be anti-PD1 nivolumab and anti-CTLA4 ipilimumab. Nivolumab (240 mg IV) will be administered with GEN-1 (starting dose 60 mg / m 2 ) every two weeks. Ipilimumab IV will be administered at 1 mg / kg every six weeks. Dosing will continue until disease progression, unacceptable toxicity, or up to 2 years in patients without disease progression.

[0470] Maximum Tolerated Dose (MTD): A standard 3+3 design will be used to determine the MTD of GEN-1 for this regimen. The starting dose of Gen-1 at 60 mg / m 2 will be combined with a fixed-dose combination of two ICIs. If more than 1 / 6 of the patients are determined to have dose-limiting toxicity (DLT), the DSMB may recommend reducing the dose level of GEN-1 to 45 mg / m2 for evaluation. The study can only proceed to a higher dose level if 0 out of 3 patients or <1 out of 6 patients show no evidence of DLT. The dose escalation schedule is presented in Table 1. If more than 1 / 6 of the patients show DLT at a dose level, the previous dose level is determined as the MTD. Confirmation of the MTD in at least 6 patients must be obtained before entering Phase II.

[0471] The Phase II part of the study is a two-stage design to evaluate whether the treatment will produce an objective response rate (ORR) of 45% or higher. In the first stage, twenty (20) patients will be enrolled and evaluated to determine if six (6) or more subjects achieve CR or PR. If not, the study will be stopped for futility. If the ORR target value is reached in the first stage, an additional 20 subjects will be enrolled to detect if at least 14 out of 40 subjects achieve CR or PR to confirm the response rate of the Phase II design.

[0472] Follow up all subjects for safety, disease progression, clinical benefit rate (objective response and durable stable disease at 12 weeks), and survival.

[0473] Subject Selection Criteria

[0474] The following inclusion criteria are designed to select subjects considered suitable for participation in the study. All relevant medical and non-medical conditions should be considered when determining whether this protocol is appropriate for a particular subject. The investigator may not waive the inclusion criteria, and the criteria are subject to review in the event of a cGCP or regulatory agency audit.

[0475] Study Treatment and Administration

[0476] Determination of MTD and Recommended Dose for Phase 2

[0477] A 3+3 dose escalation will be adopted. The safety of all subjects will be monitored starting from the time of signing the informed consent form. The safety data of subjects who have completed 2 cycles of the protocol treatment need to meet the conditions for dose escalation review. Generally, if 0 / 3 DLT, the dose is escalated; if >1 / 3 DLT, the dose needs to be reduced; if 1 / 3 DLT, it needs to be expanded to 6 patients. The highest dose level with <2 / 6 DLT observed will be determined as the MTD. The data will be reviewed by the study DSMB, and dose adjustments and the Phase II recommended dose will be recommended. During the Phase I part, the dual ICI will be given at a fixed dose at all dose levels, and the Gen-1 dose will be escalated according to the following table.

[0478] Table 4. Dose Escalation Plan for Phase I Part

[0479]

[0480]

[0481] Dual ICI (anti-PD1 and anti-CTLA4) and GEN-1

[0482] Nivolumab 240 mg IV will be administered together with GEN-1 (at the dose recommended by the DSMB), once every 2 weeks until disease progression, unacceptable toxicity, or up to 2 years in patients without disease progression.

[0483] Ipilimumab 1 mg / kg will be administered IV once every 6 weeks until disease progression, unacceptable toxicity, or up to 2 years in patients without disease progression.

[0484] GEN-1 (Study Drug)

[0485] Preparation

[0486] The human IL-12 plasmid (phIL-12-005) will be formulated with the lipid polymer PEG-PEI-cholesterol (PPC) in 10% lactose.

[0487] Human IL-12 plasmid

[0488] The phIL-12-005 plasmid contains an hIL-12 gene expression cassette within a plasmid containing the Kanr gene. The hIL-12 gene expression cassette of phIL-12-005 contains an immediate early enhancer and promoter derived from cytomegalovirus (CMV), a 5' untranslated region (UTR), a synthetic intron, the p35 gene, the human growth hormone (hGH) 3' UTR and polyadenylation signal sequence, the CMV promoter, a 5' UTR, a synthetic intron, the p40 gene, the hGH 3' UTR and polyadenylation signal sequence. The two hIL-12 subunits are each controlled by two separate CMV promoters. See Figure 1 。

[0489] PEG-PEI-cholesterol

[0490] The PPC is composed of a PEI backbone, which is independently linked to polyethylene glycol and cholesterol via covalent bonds. The molecular weights of PEI, PEG and cholesterol carbonyl are 1800, 550 and 414, respectively. See Figure 2 。

[0491] Route of administration & Administration of GEN-1

[0492] Infused into the peritoneal cavity via an IP catheter.

[0493] 25 mL of 0.9% normal saline is flushed through the implantable port (IP port) to check catheter patency. Heparin is not used to flush the catheter during sample collection or drug infusion.

[0494] Reconstituted GEN-1 (in 50 mL glass bottles or IV bags) is stable at room temperature for up to 24 hours. After confirming catheter patency, an IV bag containing GEN-1 will be administered via the patient's IP catheter. GEN-1 will be infused via the catheter by gravity from the IV bag while the valve is always open and flowing freely. A typical administration may take approximately 1 hour.

[0495] After GEN-1 infusion, at least 25 mL of 0.9% injectable normal saline is flushed a second time to ensure clearance of the study drug from the catheter.

[0496] Intraperitoneal catheter

[0497] Subcutaneous implantable IP silicone catheters can be used to administer GEN-1 into the peritoneal cavity. In previous preclinical compatibility studies and previous Phase I studies, GEN-1 has been shown to be compatible with silicone catheters. Port-A-Cath catheters (Deltec, Inc., St. Paul, MN) have been successfully used for IP delivery of GEN-1, with few catheter-related serious complications observed. Any other approved catheter with a subcutaneous port for IP delivery can also be used if suitable for aspirating biological samples for translational research. The Bard 9.6Fr silicone single-lumen venous catheter from Bard Access Systems (West Amelia Earhart Drive, Salt Lake City, Utah) or its equivalent (with or without cuff) can be used. Catheter compatibility studies using Port-A-Cath catheters have shown that exposure of the catheter to GEN-1 does not significantly affect the physicochemical properties or transfection activity of GEN-1. Similar compatibility studies are conducted using Bard catheters. Subjects can be treated via pre-existing IP catheters provided they have properties similar to the Port-A-Cath catheter device and are functional. If there are concerns about catheter patency or function, a catheterogram can be obtained to verify intraperitoneal infusion.

[0498] Catheterization

[0499] The IP catheter will be implanted according to hospital standard procedures; the procedures and risks associated with the placement of the IP catheter must be explained to the subjects, and surgical consent will be obtained from the subjects prior to catheter placement. Subjects will undergo IP catheter insertion at least 7 days prior to the planned study drug administration to allow for adequate healing and sealing around the catheter site. According to current institutional clinical practice, a semi-permanent subcutaneous access port such as the Port-A-Cath catheter (SIMS Deltec, Inc., St. Paul, MN Inc., 55112) or an equivalent device will be used.

[0500] During the course of the study, the study drug will be infused through this port. Approximately 25 mL of normal saline will be flushed through the catheter to check catheter patency prior to each infusion of the study drug; heparin should not be used to flush the catheter during sample collection or drug infusion. At the end of the study, the catheter can be removed at the discretion of the clinician upon completion of GEN-1 administration.

[0501] Subjects should be monitored for study drug-related toxicities.

[0502] Body weight changes

[0503] The calculated dose of GEN-1 is based on the protocol-specified dose (mg / m2) and the subject's body BSA calculated at baseline. The subject's weight should be monitored during treatment (e.g., typically monitored / recorded on the same day of treatment). If the subject's weight changes by ≥10%, the BSA is recalculated and the adjusted dose is administered during subsequent treatment cycles. The new BSA becomes the baseline for future weights and any dose adjustments.

[0504] Dose adjustments for dual ICI (see nivolumab prescribing information)

[0505] When nivolumab is administered in combination with ipilimumab, for adverse reactions meeting these dose adjustment guidelines, both ipilimumab and nivolumab are discontinued or permanently discontinued.

[0506] ICI and GEN-1 treatment

[0507] Each study treatment cycle will be defined as 6 weeks. On Day 1 of each cycle, all subjects will receive a fixed dose of dual ICI (nivolumab and ipilimumab), followed by the prescribed dose of Gen-1. Dosing by cycle is as shown in the following table:

[0508] Table 5 - Treatment cycles

[0509]

[0510] Safety

[0511] Subjects' safety will be monitored at each treatment visit (using physical examination and AE assessment) from the signing of the informed consent form until at least 30 days after the last dose of the study drug. Any suspected drug-related adverse event can be reported at any time during the follow-up period until it resolves to ≤Grade 2 (CTCAE v5.0).

[0512] Efficacy assessment

[0513] The primary endpoint is an objective tumor response (complete or partial) that occurs before progression, according to RECIST, version 1.1, which is used as the method to evaluate efficacy.

[0514] Disease parameters

[0515] Measurable disease. Measurable lesions are defined as lesions that can be accurately measured by chest x-ray as ≥20 mm in at least one dimension (the longest diameter to be recorded), by CT scan as ≥10 mm, or by clinical examination using calipers as ≥10 mm. All tumor measurements must be recorded in millimeters (or decimals of a centimeter).

[0516] Note: Tumor lesions located in a previously irradiated area will be considered non-measurable unless progression is documented or a biopsy is obtained to confirm persistence at least 90 days after completion of radiotherapy.

[0517] Malignant lymph node. When evaluated by CT scan (slice thickness of CT scan is recommended to be no greater than 5 mm), the short axis of a lymph node must be ≥15 mm to be considered pathologically enlarged and measurable. At baseline and during follow-up, only the short axis will be measured and followed.

[0518] Non-measurable disease. All other lesions (or disease sites), including small lesions (longest diameter <10 mm or pathologically enlarged lymph nodes with ≥10 to <15 mm short axis), are considered non-measurable disease. Leptomeningeal disease, ascites, pleural / pericardial effusion, cutaneous lymphangitis / pneumonitis, inflammatory breast disease, and abdominal masses (identified by physical examination rather than CT or MRI) are considered non-measurable.

[0519] Bone lesion: Osteolytic bone lesions or mixed osteolytic-osteoblastic lesions with identifiable soft tissue components that can be evaluated by CT or MRI are considered measurable lesions if the soft tissue component meets the above definition of measurability. Osteoblastic bone lesions are non-measurable.

[0520] Cystic lesions that meet the criteria for simple cysts according to the radiological definition should not be considered malignant lesions (neither measurable nor non-measurable) because by definition they are simple cysts. If "cystic lesions" that are considered to represent cystic metastases meet the above definition of measurability, they can be considered measurable lesions. However, if non-cystic lesions are present in the same patient, these are preferably selected as target lesions.

[0521] Target lesion. All measurable lesions (up to 2 lesions per organ, up to 5 in total) representing all involved organs should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected based on their size (the lesion with the longest diameter), representing all involved organs, but in addition, they should be those that allow them to be measured reproducibly. Sometimes, the largest lesion may not allow it to be measured reproducibly, in which case the next largest lesion that can be measured reproducibly should be selected. Calculate the sum of the diameters of all target lesions (longest for non-nodular lesions, short axis for nodular lesions), and report it as the baseline total diameter. If lymph nodes are included in the sum, only the short axis is added to the sum. The baseline total diameter will be used as a reference to further characterize any objective tumor regression in the measurable dimension of the disease.

[0522] Non-target lesion.All other lesions (or disease sites), including any measurable lesions outside of the five or more target lesions, should be identified as non-target lesions and recorded at baseline. These lesions do not need to be measured, but the presence, absence, or in rare cases, clear progression of each lesion should be noted throughout the follow-up period.

[0523] Study endpoint

[0524] Primary endpoint: Objective response rate

[0525] Phase I: Safety (DLT and MTD), 3 + 3 design in 12 to 18 patients.

[0526] Phase II: ORR (proportion of subjects achieving CR or PR. The same analysis method as for the primary endpoint will be used) will be evaluated in a two-stage design. N = 40.

Claims

1. A combination therapy, comprising: (a) A nucleic acid carrier comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer; and (b) An immune checkpoint inhibitor.

2. The combination therapy according to claim 1, wherein the polynucleotide encodes human IL-12.

3. The combination therapy according to claim 1 or 2, wherein the nucleic acid carrier comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL-12.

4. The combination therapy according to any one of claims 1-3, wherein the nucleic acid comprises an intron, 3'UTR, an antibiotic resistance gene, or any combination thereof (e.g., the elements of Figure 1).

5. The combination therapy according to any one of claims 1-4, wherein the lipid polymer comprises polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups (e.g., the lipid polymer of Figure 2).

6. The combination therapy according to any one of claims 1-5, wherein the combination further comprises an anti-cancer agent.

7. The combination therapy according to claim 6, wherein the anti-cancer agent is a chemotherapeutic agent.

8. The combination therapy according to claim 6, wherein the anti-cancer agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, albumin-bound paclitaxel, olaparib, and any combination thereof.

9. The combination therapy according to claim 6, wherein the anti-cancer agent is paclitaxel.

10. The combination therapy according to claim 6, wherein the anti-cancer agent is carboplatin.

11. The combination therapy according to claim 6, wherein the anti-cancer agent is docetaxel.

12. The combination therapy according to claim 6, wherein the anti-cancer agent is albumin-bound paclitaxel.

13. The combination therapy according to claim 6, wherein the anti-cancer agent is olaparib.

14. The combination therapy according to any one of claims 1-13, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), LAG-3, and any combination thereof.

15. The combination therapy according to any one of claims 1-14, wherein the immune checkpoint inhibitor is an antibody.

16. The combination therapy according to any one of claims 1-14, wherein the immune checkpoint inhibitor comprises an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or any combination thereof.

17. The combination therapy according to any one of claims 1-15, wherein the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, cemiplimab, and any combination thereof.

18. The combination therapy according to claim 17, wherein the PD-1 antagonist is nivolumab.

19. The combination therapy according to any one of claims 1-15, wherein the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

20. The combination therapy according to any one of claims 1-15, wherein the immune checkpoint inhibitor is a CTLA-4 antagonist, and the CTLA-4 antagonist is ipilimumab.

21. The combination therapy according to any one of claims 1-15, wherein the immune checkpoint inhibitor is a LAG-3 antagonist, and the LAG-3 antagonist is relatlimab.

22. The combination therapy according to any one of claims 1-21, wherein the combination therapy comprises two or more immune checkpoint inhibitors.

23. The combination therapy according to claim 22, wherein the combination therapy comprises two, three, or four immune checkpoint inhibitors.

24. The combination therapy according to claim 23, wherein the immune checkpoint inhibitor comprises an inhibitor of two or more immune checkpoint proteins selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and LAG-3.

25. The combination therapy according to claim 22 or 24, wherein the second of the two or more checkpoint inhibitors is an antibody.

26. The combination therapy according to claim 22 or 24, wherein at least one immune checkpoint inhibitor is a small molecule inhibitor.

27. The combination therapy according to any one of claims 22-25, wherein the immune checkpoint inhibitor comprises a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostarlimab, and cemiplimab.

28. The combination therapy according to claim 27, wherein the checkpoint inhibitor comprises nivolumab.

29. The combination therapy according to any one of claims 22-25, wherein the checkpoint inhibitor comprises a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

30. The combination therapy according to any one of claims 22-25, wherein the immune checkpoint inhibitor comprises a CTLA-4 antagonist.

31. The combination therapy according to claim 30, wherein the CTLA-4 antagonist is ipilimumab.

32. The combination therapy according to any one of claims 22-25, wherein the immune checkpoint inhibitor comprises a LAG-3 antagonist.

33. The combination therapy according to claim 32, wherein the LAG-3 antagonist is relatlimab.

34. The combination therapy according to any one of claims 4-33, wherein the 3'UTR is the hGH 3'UTR.

35. The combination therapy according to any one of claims 1-34, wherein the nucleic acid carrier is a plasmid.

36. The combination therapy according to any one of claims 1-35, wherein the lipid polymer is a nanoparticle.

37. A method of treating a subject having cancer, the method comprising administering to the subject the combination therapy according to any one of claims 1-36.

38. The method according to claim 37, wherein the nucleic acid carrier formulated with a lipid polymer is administered intratumorally or intraperitoneally.

39. The method according to claim 37 or 38, wherein the nucleic acid carrier formulated with a lipid polymer is administered intravenously.

40. The method according to any one of claims 37-39, wherein the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, intravenously, intracystically, or any combination thereof.

41. The method according to any one of claims 37-40, wherein the immune checkpoint inhibitor is administered intravenously.

42. The method according to any one of claims 37-41, wherein the nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the immune checkpoint inhibitor.

43. The method according to any one of claims 37-42, wherein the nucleic acid carrier formulated with a lipid polymer is administered before, simultaneously with, or after the anti-cancer agent.

44. The method according to any one of claims 37-43, wherein an anti-cancer agent is administered, followed by the administration of the nucleic acid carrier formulated with a lipid polymer, and then followed by the administration of the immune checkpoint inhibitor.

45. The method according to any one of claims 37-43, wherein an anti-cancer agent is administered, followed by the administration of the nucleic acid carrier formulated with a lipid polymer, followed by one or more immune checkpoint inhibitors, and then followed by surgery to remove all or part of the tissue or tumor.

46. The method according to any one of claims 37-43, wherein surgery is performed to remove all or part of the tissue or tumor, followed by the administration of the nucleic acid carrier formulated with a lipid polymer, and then followed by the administration of the immune checkpoint inhibitor.

47. The method according to any one of claims 45-46, wherein the surgery is intermediate cytoreductive surgery.

48. The method according to any one of claims 37-43, wherein the anti-cancer agent is administered, followed by the administration of the DNA nucleic acid carrier, followed by the immune checkpoint inhibitor, optionally followed by intermediate cytoreductive surgery.

49. The method according to any one of claims 37-48, wherein the administration of the anti-cancer agent comprises administering paclitaxel at a dose of about 25-250 mg / m 2 and optionally subsequently administering carboplatin IV at a dose of about AUC 4-6. The method according to any one of claims 37 - 48, wherein the administration of the anti - cancer agent comprises administering docetaxel at a dose of 25 - 250 mg / m 2 , optionally followed by intravenous administration of carboplatin at a dose of about AUC 4 - 6.

51. The method according to any one of claims 37 - 48, wherein the administration of the anti-cancer agent comprises administering albumin-bound paclitaxel at a dose of 25 - 350 mg / m 2 , and optionally subsequently administering carboplatin IV at a dose of about AUC 4 - 6. The method according to any one of claims 37-51, wherein the nucleic acid vector comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipid polymer is administered at a dose of about 35 mg / m 2 to about 80 mg / m 2 .

53. The method according to any one of claims 37-52, wherein the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer, lung cancer, any combination thereof, and metastases of any of the foregoing cancers.

54. The method according to claim 53, wherein the brain cancer is glioblastoma.

55. The method according to any one of claims 37-54, wherein the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

56. The method according to any one of claims 37-55, wherein the subject is a human.

57. The method according to any one of claims 37 - 56, wherein the lipid polymer is administered at a dose of about 60 mg / m 2 of.

58. The method according to any one of claims 37-57, wherein the immune checkpoint inhibitor comprises nivolumab, optionally wherein nivolumab is administered at about 240 mg.

59. The method according to claim 58, wherein nivolumab and the lipid polymer are administered once every 1 - 4 weeks during the treatment.

60. The method according to claim 59, wherein nivolumab and the lipid polymer are administered once every 2 weeks during the treatment.

61. The method according to any one of claims 37 - 60, wherein the second inhibitor is ipilimumab, optionally, wherein ipilimumab is administered at about 1 mg / kg.

62. The method according to claim 61, wherein ipilimumab is administered once every 2 - 4 weeks during the treatment.

63. The method according to claim 62, wherein ipilimumab is administered once every 6 weeks during the treatment.

64. The method according to any one of claims 37 - 63, wherein the nucleic acid vector is a plasmid.

65. The method according to any one of claims 37 - 64, wherein the lipid polymer is a nanoparticle.

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