Combination of IL-12 gene therapy and anti-VEGF for treatment of cancer

Abstract

The present disclosure relates to a combination therapy comprising an anti-VEGF antibody, a nanoparticle formulated plasmid comprising an IL-12 encoding nucleic acid, and optionally at least one adjuvant chemotherapeutic drug; and 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 to U.S. Provisional Application No. 63 / 374,900, filed September 7, 2022, which is incorporated herein by reference in its entirety.

[0003] Citations of sequence listings submitted electronically via EFS-WEB

[0004] The contents of the electronically submitted .XML file Sequence Listing (Name: 2437_081PC01_SequenceListing_ST26; Size: 9,811 bytes; and Creation Date: September 6, 2023) submitted with this application are incorporated herein by reference in their entirety. Technical Field

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

[0006] Ovarian cancer is the fifth most deadly cancer type among women in the United States, causing an estimated 14,000 deaths annually. Approximately 22,000 new cases of ovarian cancer are diagnosed annually, with the majority (approximately 70%) diagnosed at the advanced stages III and IV. Epithelial ovarian cancer (EOC) is characterized by tumor spread within the peritoneal cavity and carries a high risk of recurrence (75%, stages III and IV) after seemingly successful surgery and chemotherapy.

[0007] IL-12 is one of the most active cytokines for stimulating the immune response against cancer. However, when administered as a recombinant protein, the pharmacokinetics of IL-12 require that it be administered by frequent, large amounts of boluses, resulting in severe toxicity and limiting 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), providing the potential for specific expression of cytokines in the tumor microenvironment, with the goal of achieving enhanced efficacy while minimizing potential systemic toxicity. GEN-1 has been studied in subjects with recurrent ovarian cancer as a single agent or in combination with standard chemotherapy.

[0008] Traditional chemotherapy regimens are designed to inhibit tumor growth through cytotoxic mechanisms, while immunocytokine therapies are designed to trigger tumor killing by boosting the immune system against cancer cells. GEN-1 mitigates the toxicity issues associated with IL-12. Its nanoparticle properties allow for cell transfection and subsequent sustained local secretion of IL-12 at therapeutic levels, while avoiding the toxicities associated with recombinant IL-12.

[0009] There remains a need for ovarian cancer treatments, including those diagnosed with EOC and those with recurrent ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown are the differences in tumor weight (mg) after administration of mGEN-1, various dose levels of bevacizumab, and mGEN-1 combined with various dose levels of bevacizumab compared to the control group.

[0011] Figure 2 Untreated naked-Foxn1 nu Mice, naked-Foxn1 treated with doxorubicin (Doxil) + bevacizumab nu mice and nude-Foxn1 treated with Doxil+bevacizumab+mGEN-1 nu Different tumor burden levels in mice after tumor implantation were quantified via IVIS signal quantification.

[0012] Figure 3 Whole-body images via IVIS show untreated nude-Foxn1 nu Mice, naked-Foxn1 treated with Doxil+bevacizumab nu mice and nude-Foxn1 treated with Doxil+bevacizumab+mGEN-1 nu Different tumor burden levels in mice after tumor implantation.

[0013] Figure 4 An exemplary hIL-12 expression plasmid is shown.

[0014] Figure 5 The PEG-PEI-cholesterol structure is shown.

[0015] Figure 6 The dosing schedules for the neoadjuvant chemotherapy (NACT) + bevacizumab arm and the NACT + bevacizumab + GEN-1 arm of the clinical protocol are shown.

[0016] Figure 7 An overview of the timelines for the NACT+bevacizumab arm and the NACT+bevacizumab+GEN-1 arm of the clinical protocol is shown. SUMMARY OF THE INVENTION

[0018] Certain aspects of the present disclosure relate to combination therapies comprising: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

[0019] Certain aspects of the present disclosure relate to methods of treating a subject having cancer, the methods comprising administering to the subject a combination therapy comprising: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

[0020] In some aspects, the polynucleotide encodes human IL-12.

[0021] In some aspects, the nucleic acid vector (eg, 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.

[0022] In some aspects, the promoter is a CMV promoter.

[0023] In some aspects, the nucleic acid vector (eg, plasmid) comprises an intron, a 3'UTR (eg, hGH 3'UTR), an antibiotic resistance gene, or any combination thereof (eg, Figure 4 components).

[0024] In some aspects, the lipopolymer comprises polyethyleneimine (PEI) (e.g., Figure 5 lipid polymer).

[0025] In some aspects, the combination further comprises an anti-cancer agent.

[0026] In some aspects, the anticancer agent is a chemotherapeutic agent.

[0027] In some aspects, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0028] In some aspects, the anticancer agent is doxorubicin.

[0029] In some aspects, the anticancer agent is paclitaxel.

[0030] In some aspects, the anticancer agent is carboplatin.

[0031] In some aspects, the anticancer agent is docetaxel.

[0032] In some aspects, the anticancer agent is nab-paclitaxel.

[0033] In some aspects, the anticancer agent is olaparib.

[0034] In some aspects, the anti-VEGF antibody is selected from the group consisting of bevacizumab (e.g., Avastin or a biosimilar thereof) or ranibizumab (e.g., Lucentis or a biosimilar thereof).

[0035] In some aspects, the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence at least about 85% identical to SEQ ID NO: 1 (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1) and a variable light chain (VL) comprising an amino acid sequence at least about 85% identical to SEQ ID NO: 2 (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2).

[0036] In some aspects, the method further comprises surgery to remove all or part of a tissue or tumor in the subject (eg, interval cytoreductive surgery).

[0037] In some aspects, nucleic acid vectors formulated with lipopolymers are administered intratumorally or intraperitoneally.

[0038] In some aspects, nucleic acid vectors formulated with lipopolymers are administered intravenously.

[0039] In some aspects, the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravenously, intrathecally, or any combination thereof.

[0040] In some aspects, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0041] In some aspects, the anti-VEGF antibody is administered intravenously.

[0042] In some aspects, the anti-VEGF antibody is administered intravesically.

[0043] In some aspects, the nucleic acid vector formulated with a lipopolymer is administered before, simultaneously with, or after the anti-VEGF antibody.

[0044] In some aspects, the nucleic acid vector formulated with the lipopolymer is administered before, simultaneously with, or after the anti-cancer agent.

[0045] In some aspects, an anti-cancer agent is administered (eg, first), followed by administration of a nucleic acid vector formulated with a lipopolymer (eg, second), and followed by administration of an anti-VEGF antibody (eg, third).

[0046] In some aspects, an anti-cancer agent is administered (e.g., first), followed by administration of a nucleic acid vector formulated with a lipopolymer (e.g., second), followed by an anti-VEGF antibody (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreduction) is performed (e.g., fourth).

[0047] In some aspects, an anti-cancer agent is administered, followed by administration of a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0048] In some aspects, the anticancer agent is administered prior to interval cytoreductive surgery once every three weeks for about 12 to about 18 weeks.

[0049] In some aspects, the anticancer agent is administered for at least about 28 days after interval cytoreductive surgery (eg, once every three weeks for about 8-10 weeks, such as 9 weeks).

[0050] In some aspects, administration of the anticancer agent comprises administration of paclitaxel at a dose of about 100-200 mg / m² (eg, about 175 mg / m²), optionally followed by IV administration of carboplatin at a dose of, eg, about AUC 5-6.

[0051] In some aspects, administration of the anticancer agent comprises administration of docetaxel at a dose of 50-100 mg / m² (eg, about 75 mg / m²), optionally followed by IV administration of carboplatin at a dose of, eg, about AUC 5-6.

[0052] In some aspects, administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of 200-300 mg / m² (eg, about 260 mg / m²), optionally followed by IV administration of carboplatin at a dose of, eg, about AUC 5-6.

[0053] In some aspects, administration of nanoparticles prior to interval cytoreduction begins 14-18 days (eg, 15 days) after the first administration of the anticancer agent, eg, once a week for at least about 12 weeks to about 18 weeks.

[0054] In some aspects, the nanoparticles are administered at least about 28 days after interval cytoreductive surgery, and administration begins 15 days after the first administration of the anticancer agent and continues weekly for at least about 9 weeks.

[0055] In some aspects, interleukin-12 (IL-12) formulated with a lipopolymer (e.g., nanoparticles) is administered at a dose of about 35 mg / m² to about 80 mg / m².

[0056] In such aspects, interleukin-12 (IL-12) plasmid formulated with a lipopolymer (e.g., nanoparticle) is administered at a dose of about 80 mg / m².

[0057] In some aspects, the anti-VEGF antibody is administered weekly for at least about 12 weeks up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anticancer agent.

[0058] In some aspects, the anti-VEGF antibody is administered weekly for at least about 9 weeks, at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0059] In some aspects, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV (eg, about 15 mg / kg IV).

[0060] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anticancer agent.

[0061] In some aspects, intermittent cytoreduction (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0062] In some aspects, intermittent cytoreduction (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0063] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the anti-VEGF antibody.

[0064] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody.

[0065] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer (e.g., glioblastoma), lung cancer, and any combination thereof, and metastasis of any cancer.

[0066] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0067] In some aspects, the subject is a human. Detailed Description of the Invention

[0069] I. Definition

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which this disclosure pertains. In the event of a conflict, the present application (including definitions) shall prevail. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. 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.

[0071] 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 are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0072] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural references. The term "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "a plurality."

[0073] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. Generally, the term "about" is used herein to modify a numerical value above and below the stated value by a variation of 10%, either upward or downward (higher or lower).

[0074] Throughout this disclosure, various aspects are presented in range format. It should be understood that the description of range format is merely for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present disclosure. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and each numerical value within the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as single digits within the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the scope. The listed numerical ranges include the numbers defining the range, and include each integer within the defined range.

[0075] Units, prefixes and symbols are expressed in the form accepted by their International System of Units (SI). Numerical ranges include the numbers defining the range. Where a numerical range is enumerated, it will be appreciated that each intermediate integer value and each fraction thereof, as well as each subrange between the upper and lower limits of the range, is also specifically disclosed. The upper and lower limits of any range may be independently included in the range or excluded from the range, and each range comprising any limit, not comprising a limit or comprising two limits simultaneously is also encompassed in the present disclosure. Therefore, the ranges enumerated herein are understood to be shorthand for all values within the range, including enumerated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0076] Where values are explicitly enumerated, it is understood that values of quantities or amounts that are substantially the same as the enumerated values are also within the scope of this disclosure. Where combinations are disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of a disclosure is disclosed as having multiple alternatives, examples of the disclosure are also disclosed herein, with each alternative being excluded, either individually or in any combination with other alternatives; more than one element of the disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0077] As used herein, the term "and / or" should be considered to specifically disclose each of the two specified features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases 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).

[0078] It is to be understood that whatever aspects are described herein using the language "comprising," other similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0079] As used herein, the term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount or quantity of a drug or pharmaceutically active substance sufficient to elicit a desired or desired therapeutic response, or in other words, an amount sufficient to elicit a significant biological response when administered to a patient.

[0080] "Transfection" or "transfection" shall mean the transport of nucleic acids from the external environment of a cell to the internal environment of a cell, particularly the cytoplasm and / or the nucleus. Without being bound by any particular theory, it is understood that nucleic acids can be delivered to cells after being encapsulated in or adhered to one or more cationic polymer / nucleic acid complexes or being entrained therewith. A specific transfection example delivers nucleic acids 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, hybridization sequences, or synthetic or semisynthetic sequences, and are of natural or artificial origin. In addition, the size of the nucleic acids 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 technology known to those skilled in the art.

[0081] As used herein, the term "pharmaceutical 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 the methods taught by the present disclosure, which induces a desired biological or pharmacological effect, which may include, but is not limited to, (1) having a prophylactic effect on an organism and preventing undesirable biological effects, such as preventing infection, (2) alleviating a condition 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 may be local, such as providing a local anesthetic effect, or it may be systemic.

[0082] As used herein, the term "biocompatible" or "biodegradable" is defined as the conversion of a material into less complex intermediates or end products by solubilization, hydrolysis, or by the action of biologically formed entities, which may be enzymes and other products of organisms.

[0083] As used herein, "effective amount" means an amount of a nucleic acid or biologically active agent sufficient to provide the desired local or systemic effects and performance as attendant to any medical treatment at a reasonable risk / benefit ratio.

[0084] As used herein, "peptide" means peptides of any length and includes proteins. The terms "polypeptide" and "oligopeptide" are used herein without any particular intended size limitation unless a specific size is indicated.

[0085] As used herein, "derivatives" of carbohydrates include, for example, acid forms of sugars, such as glucuronic acid; amines of sugars, such as galactosamine; phosphates of sugars, such as mannose-6-phosphate, and the like.

[0086] As used herein, "administering" and like terms mean delivering the composition to the individual being treated so 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 the individual, typically by subcutaneous, intramuscular, transdermal, intravenous, or intraperitoneal routes. Injections 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 liquids prior to injection, or as emulsions. Suitable excipients for administration include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and, if desired, small amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like.

[0087] As used herein, "efficacy" and like terms mean disappearance of a tumor or reduction in tumor size or decrease in tumor density or increase in lymphocyte count or increase in neutrophil count or improvement in survival, or all of the above.

[0088] As used herein, "toxicity" is defined as any treatment-related adverse effect on clinical observation, including but not limited to abnormal hematology or serum chemistry results or organ toxicity.

[0089] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product to which the promoter / regulatory sequence is operably linked. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in 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 results in the production of the gene product in a cell only when an inducer corresponding to the promoter is present in the cell.

[0090] As used herein, the term "expression" refers to the process by which a gene produces a biochemical substance, such as a polypeptide. This process includes any manifestation of the functional presence of a gene in a cell, including but not limited to gene knockdown, transient expression, and stable expression. It includes but is not limited to the transcription of a gene into messenger RNA (mRNA) and the translation of this mRNA into a polypeptide. The expression of a gene produces a "gene product."

[0091] As used herein, a gene product can be a nucleic acid, such as a messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. Gene products described herein also include nucleic acids with post-transcriptional modifications (e.g., polyadenylation), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, addition of lipids, association with other protein subunits, proteolytic cleavage, etc.).

[0092] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising 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 those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0093] As used herein, the term "operably linked" or "transcriptional control" refers to a functional connection 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. DNA sequences that are operably linked can be adjacent to each other, for example, in the case of linking two protein coding regions, the DNA sequences are in the same reading frame.

[0094] As used herein, the term "transfer vector" refers to a composition comprising an isolated nucleic acid and a substance that can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. The term transfer vector should also be interpreted 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.

[0095] As used herein, the term "host cell" can be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In particular aspects, the term "host cell" refers to a cell 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 parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the host cell genome.

[0096] " Percentage (%) amino acid sequence identity " about peptide sequences as described herein is defined as after aligning sequences and introducing gaps when necessary to realize maximum percentage sequence identity, and without considering any conservative substitution as a part for sequence identity, the percentage of the amino acid residue identical with the amino acid residue in the specific peptide sequence as described herein (for example, the specific peptide sequence characterized by the sequence identifier in the sequence table) in the candidate sequence of interest to be compared. The sequence alignment carried out for determining amino acid sequence identity percentage can be carried out according to programs known in the art, as for example described in EP 1 241 179 B1, which is incorporated herein by reference, particularly including page 9, line 35 to page 10, line 40, which has the definition used therein and table 1 about possible conservative substitutions. For example, the technical staff can use publicly available computer software. The computer program method for determining sequence identity includes but is not limited to BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. According to one embodiment, the software alignment program used can be BLAST. The technical staff can determine the appropriate parameters for measuring the comparison, including any algorithm required for maximum comparison on the full length of the sequence being compared. According to one embodiment, % identity values can be generated using the WU-BLAST-2 computer program (Altschul et al., 1996, Methods in Enzymology 266:460-480, which is incorporated herein by reference). According to one embodiment, when executing the WU-BLAST-2 computer program, the following parameters are used: Most WU-BLAST-2 search parameters are set to default values. 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 being searched. However, these values can be adjusted to increase sensitivity. The % sequence identity value can be determined by dividing by: (a) the number of identical amino acid residues that match between the specific amino acid sequence described herein for comparison (e.g., the specific polypeptide sequence represented by a sequence identifier in the sequence listing) and the candidate amino acid sequence of interest to be compared, such as 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 sequences described herein for comparison (e.g., the specific polypeptide sequence represented by a SEQ ID. NO. in the sequence listing).

[0097] "Percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences as described herein is defined as the percentage of nucleotides in the candidate sequence of interest being 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 the sequence listing), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0098] As used herein, the term "homology" or "identity" refers to the identity of the subunit sequence between two polymer molecules, such as between two nucleic acid molecules, such as between two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in the two molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, then the two sequences are 90% homologous.

[0099] In the context of two or more nucleic acids or peptide sequences, identity percentage refers to two or more identical sequences.When comparing and comparing maximum correspondence in a comparison window or a specified region, as measured by using one of the following sequence comparison algorithms or by manual comparison and visual inspection, if two sequences have the identical amino acid residues or nucleotides of specified percentage (for example, on specified region, or if not specified, 60% identity on the whole 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), then two sequences are "substantially identical". In some embodiments, the sequence of the present invention can be used to compare the sequence of the reference sequence of the test sequence.For example, the sequence of the reference sequence of the test sequence is used to compare the ...

[0100] A "coding sequence" or a sequence that "encodes" a particular molecule (e.g., a therapeutic molecule) is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when operably linked to appropriate regulatory sequences (e.g., a promoter). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Although the "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it is 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.

[0101] Coding sequences may include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.A transcription termination sequence is typically located 3' to the coding sequence.

[0102] As used herein, the term "recombinant DNA / RNA technology" refers to the manipulation of nucleic acid sequences outside an organism. This technology includes, but is not limited to: combining nucleic acid sequences derived from various 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 various sources into appropriate vectors (e.g., delivery vectors, expression vectors, integration vectors); modifying or altering nucleotide sequences (e.g., by mutagenesis, insertion of modified nucleotides, 5'-capping, polyadenylation); and synthesizing artificial nucleotide sequences. Various 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, prokaryotic and eukaryotic cell transformation or transduction, 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 in vitro (see, for example, Green & Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-3, 4th edition).

[0103] As used herein, the term "recombinant" refers to any nucleic acid (such as DNA or RNA), peptide (such as oligopeptides, polypeptide or protein), cell or organism prepared by combining the genetic material from two or more different sources. In some respects, recombinant nucleic acids, peptides, cells or organisms comprise a part for the genetic material from at least one source. In some respects, a "recombinant DNA" molecule can include a DNA molecule derived from a kind of organism and inserted into a host organism to produce a new genetic combination. In some respects, a "recombinant RNA" molecule (such as, a recombinant mRNA molecule) can include an RNA molecule derived from a kind of organism and inserted into a host organism to produce the expression of a desired genetic product in a host organism. In some respects, a "recombinant peptide" molecule can include an amino acid molecule derived from an organism or cell, which is expressed by a recombinant nucleic acid molecule.

[0104] As used herein, the term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from coexisting materials 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.

[0105] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0106] As used herein, the term "primary tumor" refers to the original or first tumor that forms in a subject.

[0107] As used herein, the terms "metastasis," "metastatic," "secondary tumor," or "metastatic tumor" refer to a cancer (e.g., a tumor) formed from cancer cells derived from a primary cancer (e.g., a tumor) that spreads to other locations or areas of the body.

[0108] As used herein, the term "specifically binds" refers to an antigen binding molecule that recognizes and binds to a protein binding partner (such as a tumor antigen) present in a sample, but does not substantially recognize or bind other molecules in the sample.

[0109] As used herein, the term "tumor heterogeneity" refers to the molecular or genetic changes exhibited by daughter cells of a tumor after multiple divisions and proliferations during tumor growth, resulting in differences in tumor growth rate, invasiveness, drug sensitivity, prognosis, etc. This is one of the hallmarks of malignant tumors.

[0110] As used herein, the term "cancer" refers to a large class of various diseases characterized by the uncontrolled growth of abnormal cells (e.g., malignant cells) in the body. Unregulated cell division and growth lead to the formation of malignant tumors, which invade adjacent tissues by local diffusion and can also be transferred to distant parts of the body by the lymphatic system or blood flow. In some aspects, the method of the present disclosure can be used to reduce the size of a primary tumor or a metastatic tumor, or to treat a primary tumor or a metastatic tumor. The patient's condition that can be treated or prevented by the method of the present disclosure includes, for example, various tumors, including benign or malignant tumors, various hyperplasias, etc. The method of the present disclosure can achieve the inhibition and / or reversal of the undesirable excessive proliferation cell growth involved in such a patient's condition. In some aspects, cancer can be ovarian cancer.

[0111] As used herein, "ovarian cancer" refers to a cancer that arises in or involves the ovaries (e.g., ovarian epithelium). As used herein, the term "cancer" or "tumor" refers to an uncontrolled cell growth that interferes with the normal function of the body's organs and systems. A subject suffering from cancer or a tumor is a subject with 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 by worsening the function of the affected organs. Ovarian cancer is typically treated by cytoreductive surgery (also referred to herein as "tumor reduction") followed by the administration of chemotherapy. As used herein, "cytoreductive surgery" refers to the surgical removal of at least a portion of ovarian cancer tissue from a subject. Cytoreductive surgery can remove varying amounts of tumor tissue from a subject, depending on the location and characteristics of the tumor tissue, the health of the subject, and complex factors that can be assessed by those skilled in the art. In some embodiments, cytoreductive surgery can remove at least 10% of 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.

[0112] As used herein, the term "transfected" or "transformed" or "transduced" refers 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 an exogenous nucleic acid. The cell includes a primary cell of the subject and its progeny.

[0113] As used herein, "refractory" refers to a disease, such as cancer, that does not respond to treatment. In one embodiment, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. Refractory cancers are also known as drug-resistant cancers. In some aspects, refractory or recurrent malignancies may be treated with the methods disclosed herein.

[0114] As used herein, "relapse" as used herein refers to the return of signs and symptoms of a disease (e.g., cancer) or the recurrence of a disease (e.g., cancer during a period of improvement), e.g., after treatment, e.g., after a previous treatment for cancer therapy.

[0115] As used herein, the term "combination therapy" means a therapy that includes at least gene therapy, an anticancer agent, and an antibody with binding specificity to VEGF, 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.

[0116] As used herein, the terms "treat," "treatment," and "treating" refer to therapeutic and prophylactic treatments or preventative measures, wherein the purpose is to reverse, alleviate, ameliorate, reduce, inhibit, slow the progression, development, severity, or recurrence of undesirable symptoms, complications, conditions, disorders, or biochemical markers of a disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to: relief of symptoms; a reduction in the extent of the condition, disorder, or disease; a stable (i.e., non-worsening) state of the condition, disorder, or disease; a delayed onset or slowing of the progression of the condition, disorder, or disease; an improvement or alleviation (whether partial or complete) of the condition, disorder, or disease state, whether detectable or undetectable; an improvement in at least one measurable physical parameter, which is not necessarily discernible by the patient; or an enhancement or improvement of the condition, disorder, or disease. In some aspects, treatment includes eliciting a clinically significant response without excessive levels of side effects. In some aspects, treatment includes prolonged survival compared to expected survival without treatment. As used herein, the term "amelioration" or "ameliorating" refers to a decrease in the severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or preventing 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., "prophylactic agent," "prophylactic treatment," "prophylactically 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.

[0117] As used herein, the terms "individual" and "subject" have the same meaning in this article and can be humans 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., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a human. In some aspects, the patient is a subject suffering from a disease, disorder or condition, or is at risk of suffering from a disease, disorder or condition, or otherwise needs the compositions and methods provided herein.

[0118] As used herein, the terms "therapeutically effective amount," "therapeutically effective," "effective amount," or "in an effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition that is effective to achieve a specific biological outcome as described herein (e.g., but not limited to, treating or reducing the growth of a cancer or tumor). When an "immunologically effective amount," "anti-tumor effective amount," "tumor suppression effective amount," or "therapeutically effective amount" is indicated, the precise amount of immune effector cells and therapeutic agents of the present disclosure to be administered can be determined by a physician taking into account the individual's age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). An effective amount of immune effector cells refers to, but is not limited to, an amount of immune effector cells that: can increase, enhance, or prolong the anti-tumor activity of immune effector cells; increase the number of anti-tumor immune effector cells or activated immune effector cells; promote tumor regression, tumor shrinkage, and / or tumor necrosis.

[0119] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, preparations and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0120] The term "excipient" refers to any substance that is not a therapeutic agent itself, but 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 promote the formation of a dosage unit of the composition. Excipients include, but are not limited to, solvents, penetration enhancers, wetting agents, antioxidants, lubricants, emollients, substances added to improve the appearance or texture of the composition, and substances for forming a hydrogel. Any such excipient can be used in any dosage form according to the present disclosure. The excipients of the aforementioned categories are not meant to be exhaustive, but merely illustrative, as those 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 the drug. Excipients can be inert substances, inactive substances, and / or non-pharmaceutically active substances. Excipients can be used for a variety of purposes.

[0121] Those skilled in the art can select one or more excipients according to specific desired characteristics by routine experiments and without excessive burden. The amount of each excipient used can vary within the scope of routine in the art. The technology and excipients that can be used for formulation 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).

[0122] As used herein, the term "immune response" refers to the biological response for foreign agents or abnormal cells (for example, tumor cells) in an organism, wherein the response protects an organism from such agents / cells and the disease caused by them. Immune response is mediated by the action of immune system cells (such as T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) and the soluble macromolecules (including antibodies, cytokines and complement) produced by these cells or liver, so as to selectively target, combine, damage, destroy and / or eliminate the invading pathogens in an organism, cells or tissues infected by pathogens, cancer cells or other abnormal cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation. In some aspects, immune response includes, for example, activation or suppression of T cells, such as effector T cells or Th cells, such as CD4+ or CD8+T cells, or suppression of regulatory T cells (Treg cells).

[0123] As used herein, the term "autologous" refers to any material derived from an individual that is subsequently reintroduced into the same individual.

[0124] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0125] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains (VH and VL, respectively), and in addition the constant domain of the light chain (CL) and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain containing the VL and CL domains and a heavy chain fragment containing the VH and CH1 domains.

[0126] 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 reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0127] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen that binds to 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.

[0128] 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 natural antibodies (VH and VL, respectively) generally have similar structures, with 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., WH Freeman and Co., page 91 (2007)).

[0129] As used interchangeably herein, "paratope" or "antigen binding site" refers to the portion of an antibody that recognizes and binds an antigen. The antigen binding site is formed by an arrangement of several individual amino acid residues in the variable regions of the antibody heavy and light chains, which are arranged in close spatial proximity in the tertiary structure of the Fv region. In one embodiment, the antigen binding site is defined as a set of six CDRs contained in a cognate VH / VL pair.

[0130] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain whose sequence is highly variable and contains antigen-contacting residues. Typically, an antibody comprises 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 indicated, the CDR residues and other residues (e.g., 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).

[0131] An "acceptor human framework" for the purposes herein is a framework comprising the 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.

[0132] As used herein, "framework" or "FR" refers to the variable domain amino acid residues other than the CDR residues. The framework of the variable domain is generally composed of four framework domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR amino acid sequences typically appear in the following sequence: (a) in 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.

[0133] Vascular endothelial growth factor (VEGF) is a homodimer member of the cystine knot family of growth factors. Generally, unless otherwise indicated, VEGF refers to any natural VEGF from any vertebrate source, including mammals such as primates (e.g., people) and rodents (e.g., mice and rats). The term encompasses "full length," unprocessed VEGF and any form of VEGF produced by processing in cells. The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants. VEGF-dimer refers to a homodimer of two identical VEGF-molecules. The complex formed by two identical antibody molecules bound to a VEGF-dimer is referred to herein as a VEGF-dimer-antibody-complex.

[0134] The "first and second antigen-binding sites" contained in the VEGF-dimer-antibody-complex refer to the antigen-binding sites contained in the VH / VL pair of each of the two antibodies contained in the VEGF-dimer-antibody-complex. For example, the antigen-binding site of one of the two anti-VEGF antibodies in the VEGF-dimer-antibody complex is the "first antigen-binding site," while the antigen-binding site of the other of the two anti-VEGF antibodies is automatically the "second antigen-binding site."

[0135] VEGF stimulates cellular responses by binding to tyrosine kinase receptors (VEGF-receptors, or "VEGFRs") on the cell surface, causing them to dimerize and be activated by transphosphorylation, although at different sites, times, and degrees. VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTKs). VEGF-A binds to VEGFR-1 (Flt-1), which interacts with domain 2 of VEGF-R1, and to VEGFR-2 (KDR / Flk-1), which interacts with domains 2 and 3 of VEGF-R2.

[0136] As used herein, the "VEGF-R1 binding region" and "VEGF-R2 binding region" of a VEGF molecule or VEGF-dimer refer to those amino acids on VEGF that interact with domain 2 of VEGF-R1 or domains 2 or 3 of VEGF-R2, respectively.

[0137] As used herein, the terms "anti-VEGF antibody" and "antibody that binds to VEGF" refer to an antibody or antigen-binding fragment thereof that is capable of binding to VEGF with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting VEGF. In one embodiment, the extent of binding of the anti-VEGF antibody to unrelated, non-VEGF proteins is less than about 10% of the binding of the antibody to VEGF, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antibody that binds to VEGF has a dissociation constant (KD) of <1 nM, or <0.15 nM. An antibody is said to "specifically bind" to VEGF when it has a KD of 1 μM or less.

[0138] "VEGFR blocking selectivity" is used herein as an abbreviated term to refer to the property of an anti-VEGF antibody that, when bound to a VEGF dimer, preferentially inhibits the binding of VEGF to VEGF-R2 rather than to VEGF-R1. An anti-VEGF antibody that is capable of completely blocking the binding of VEGF to VEGF-R2 but not to VEGF-R1 is considered to selectively block VEGF signaling through VEGF-R2 but not VEGF-R1, i.e., exhibits "VEGFR blocking selectivity."

[0139] As used herein, "affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a 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.

[0140] As used herein, the term "epitope" refers to a site on an antigen (protein antigen or non-protein antigen) to which an anti-VEGF antibody binds. An epitope can be formed by a continuous stretch of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, due to the folding of the antigen (i.e., by the tertiary folding of the protein antigen) in spatial proximity. Linear epitopes are generally still bound by anti-VEGF antibodies after exposure of the protein antigen to a denaturant, while conformational epitopes are generally destroyed after treatment with a denaturant. An epitope comprises 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.

[0141] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using routine 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 antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

[0142] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows for the classification of multiple monoclonal antibodies that specifically bind to VEGF based on the binding profiles of each of the multiple antibodies to chemically or enzymatically modified antigen surfaces (see, e.g., US 2004 / 0101920). The antibodies in each bin bind to the same epitope, which can be a unique epitope that is significantly different from or partially overlaps with the epitope represented by another bin. Competitive binding can also be used to readily determine whether an antibody binds to the same epitope of VEGF as a reference anti-VEGF antibody or competes for binding with the reference anti-VEGF antibody. For example, an "antibody that binds to the same epitope as a reference anti-VEGF antibody" means that, in a competition assay, the antibody blocks binding of the reference anti-VEGF antibody to its antigen by 50% or more, whereas, in a competition assay, the reference antibody blocks binding of the antibody to its antigen by 50% or more. Also, for example, to determine whether an antibody binds to the same epitope as a reference anti-VEGF antibody, the reference antibody is allowed to bind to VEGF under saturating conditions. After removing excess reference anti-VEGF antibody, the ability of the anti-VEGF antibody in question to bind VEGF is assessed. If the anti-VEGF antibody is able to bind to VEGF after saturation binding of the reference anti-VEGF antibody, it can be concluded that the anti-VEGF antibody in question binds to a different epitope than the reference anti-VEGF antibody. However, if the anti-VEGF antibody in question is unable to bind to VEGF after saturation binding of the reference anti-VEGF antibody, the anti-VEGF antibody in question may bind to the same epitope as the reference anti-VEGF antibody. To confirm whether the antibody in question binds to the same epitope or is simply hindered from binding due to steric factors, routine assays (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art) can be used. This assay should be performed in two settings, i.e., both antibodies are saturated. If, in both settings, only the first (saturating) antibody is able to bind to VEGF, it can be concluded that the anti-VEGF antibody in question and the reference anti-VEGF antibody compete for VEGF binding.

[0143] Sometimes, two antibodies are said to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are said to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0144] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested 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 survival, or an improvement in various physiological symptoms associated with cancerous conditions. An "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 tumor occurrence.

[0145] As used herein, the terms "chemotherapy" or "chemotherapeutic agent" refer to a variety of chemotherapeutic agents that can be used in accordance with embodiments of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer.

[0146] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient is effective and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. The composition may be sterile.

[0147] 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 formats well known in the art.

[0148] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art and are fully explained in the literature.

[0149] II.GEN-1 (Nanoparticle-formulated IL-12 DNA)

[0150] In animal models, recombinant IL-12 has been shown to induce potent T cell-mediated antitumor effects, leading to regression of established tumors, followed by systemic immune memory. See The Oncologist, 1996, vol. 1, 88. However, in several experimental trials and 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. A gene delivery method that could provide therapeutic levels of IL-12 locally at the tumor site would have the advantage of generating an anticancer response without causing systemic toxicity.

[0151] 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 serious practical limitations, mainly due to increased cancer incidence and strong immune responses of the host system to viral antigens. Since non-viral gene delivery systems are less toxic, there is considerable interest in their development. It has been demonstrated that polyvinylpyrrolidone (PVP), a non-viral gene delivery system, is used to deliver IL-12 to treat renal carcinoma (Renca) and colon cell carcinoma (CT26). See Gene Ther., 1999, Vol.6, 833. When tumors receive this gene therapy, they show 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 is well tolerated and highly effective in Renca and CT26 tumor-bearing animals. Tumor rejection mice are also protected from subsequent re-attacks, indicating the presence of lasting systemic immunity. Functionalized and less toxic water-soluble lipid polymers (WSLPs) have been tested for delivery of the IL-12 gene to CT26 colon cancer tumors. See Mahato et al, Mol. Ther., 2001, vol. 4, 130. Treatment with an IL-12 plasmid (pIL-12) and WSLP (pIL-12 / WSLP) resulted in higher levels of intratumoral gene expression than naked DNA.

[0152] Interleukin-12 (IL-12) is a proinflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16: 495-521 (1998). IL-12 primarily functions as a 70 kDa heterodimeric protein composed of two disulfide-linked subunits, p35 and p40. IL-12 p40 homodimers do exist, but other than acting as antagonists for binding to the IL-12 receptor, they do not appear to mediate biological responses. Same as above. The precursor form of the IL-12 p40 subunit (NM_002187; P29460; also known as IL-12B, natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2) is 328 amino acids long, while its mature form is 306 amino acids long. The precursor form of the IL-12 p35 subunit (NM_000882; P29459; also known as IL-12A, natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acids long, and its mature form is 197 amino acids long. Same as above. The genes for the IL-12 p35 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 in response to antigen stimulation. Active IL-12 heterodimers are formed after protein synthesis. Same as above.

[0153] Since 1994, IL-12 protein has been studied as a promising anticancer therapeutic agent because it can activate NK cells and cytotoxic T cells. See Nastala, CL 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 leads to an adaptive response and a progressive decrease in IL-12-induced interferon gamma (IFNγ) levels in the blood. Ibid. In addition, although it is recognized that the anticancer activity induced by IL-12 is mainly mediated by the secondary secretion of IFNγ, IL-12 also induces IFNγ and other cytokines (such as TNF-a) or chemokines (IP-10 or MIG), causing severe toxicity. Ibid.

[0154] In addition to negative feedback and toxicity, the marginal efficacy of IL-12 therapy in clinical settings may be caused by the strongly immunosuppressive environment in humans.

[0155] In addition, when compared to naked DNA, the secondary effects of cytokine IL-12 production, namely IFN-γ and nitric oxide (NO) levels, were also higher in WSLP-treated tumors. A single injection of the pIL-12 / WSLP complex produced suboptimal effects on tumor growth and animal survival, while repeated delivery produced better efficacy, suggesting that the system's delivery was insufficient. J. Control Release 2003, Vol. 87, 177. Similarly, intratumoral injection of an IL-12 plasmid in another polymer carrier, PAGA, only produced partial inhibition of CT26 tumors. See Gene Ther., 2002, Vol. 9, 1075. These results demonstrate the need for more effective delivery systems. Although they have shortcomings in early preclinical trials, the excellent molecular flexibility of polymeric gene carriers allows complex modifications and new functionalizations, which are necessary for the development of more effective gene delivery systems.

[0156] In order to obtain the desired results from the combination approach involving gene therapy, it is important to select an appropriate gene delivery system. The gene delivery system used in the above combination experiments (Molecular Therapy, 2004, Vol. 9, 829) was the water-soluble lipid polymer PEI-cholesterol (WSLP).

[0157] In some aspects, the DNA plasmid further encodes a synthetic polymer that facilitates plasmid delivery, which is a lipopolymer.

[0158] In some aspects, the lipopolymer further consists of polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups.

[0159] 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 nucleic acid / polymer complexes in biological environments to avoid this defect in the prior art (WSLP). In addition, the addition of PEG chains allows ligands to be incorporated into PPC chains to improve the tissue selectivity of delivery. For example, the cholesterol moiety directly attached 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 PEI backbone is important for achieving optimal enhancement of transfection activity. The preferred composition range is a PEG:PEI molar ratio of 2-4 at a fixed cholesterol content. The optimal ratio of PEI to cholesterol is 1:0.5 to 1:1.

[0160] Certain aspects of the present disclosure relate to combination therapies comprising: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

[0161] Certain aspects of the present disclosure relate to methods of treating a subject having cancer, the methods comprising administering to the subject a combination therapy comprising: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

[0162] In some aspects, the polynucleotide encodes human IL- 12. In some aspects, the polynucleotide encodes the p35 and p40 IL-12 subunits.

[0163] In some aspects, human IL-12 p35 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 3. In some aspects, human IL-12 p40 comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 4.

[0164] In some aspects, human IL-12 p35 comprises the following sequence:

[0165] MGPARSLLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCL ASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO: 3).

[0166] In some aspects, human IL-12 p40 comprises the following sequence:

[0167] MGHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 4).

[0168] In some aspects, the polynucleotide encoding human IL-12 p35 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 5. In some aspects, the polynucleotide encoding human IL-12 p35 is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 6.

[0169] In some aspects, the polynucleotide encoding human IL-12 p35 comprises the following sequence:

[0170] atgggtccagcgcgcagcctcctccttgtggctaccctggtcctcctggaccacctcagtttggccagaaacctccccgtggccactccagacccaggaatgttcccatgccttcaccactcccaaaacctgctgagggccgtcagcaacatgctccagaaggccagacaaactctagaattttacccttgcacttctgaagagattgatcatgaagatatcacaaaagataaaaccagcacagtggaggcctgtttaccattggaattaaccaagaatgagagttgcctaaattccagagagacctctttcataactaatgggagttgcctggcctccagaaagacctcttttatgatggccctgtgccttagtagtatttatgaagacttgaagatgtaccaggtggagttcaagaccatgaatgcaaagcttctgatggatcctaagaggcagatctttctagatcaaaacatgctggcagttattgatgagctgatgcaggccctgaatttcaacagtgagactgtgccacaaaaatcctcccttgaagaaccggatttttataaaactaaaatcaagctctgcatacttcttcatgctttcagaattcgggcagtgactattgatagagtgatgagctatctgaatgcttcctaa (SEQ ID NO: 5).

[0171] In some aspects, the polynucleotide encoding human IL-12 p35 comprises the following sequence:

[0172] Atgggtcaccagcagttggtcatctcttggttttccctggtttttctggcatctcccctcgtggccatatgggaactgaagaaagatgtttatgtcgtagaattggattggtatccggatgcccctggagaaatggtggtcctcacctgtgacacccctgaagaagatggtatcacctggaccttggaccagagcagtgaggtcttaggctctggcaaaaccctgaccatccaagtcaaagagtttggagatgctggccagtacacctgtcacaaaggaggcgaggttctaagccattcgctcctgctgcttcacaaaaaggaagatggaatttggtccactgatattttaaaggaccagaaagaacccaaaaataagacctttctaagatgcgaggccaagaattattctggacgtttcacctgctggtggctgacgacaatcagtactgatttgacattcagtgtcaaaagcagcagaggctcttctgacccccaaggggtgacgtgcggagctgctacactctctgcagagagagtcagaggggacaacaaggagtatgagtactcagtggagtgccaggaggacagtgcctgcccagctgctgaggagagtctgcccattgaggtcatggtggatgccgttcacaagctcaagtatgaaaactacaccagcagcttcttcatcagggacatcatcaaacctgacccacccaagaacttgcagctgaagccattaaagaattctcggcaggtggaggtcagctgggagtaccctgacacctggagtactccacattcctacttctccctgacattctgcgttcaggtccagggcaagagcaagagagaaaagaaagatagagtcttcacggacaagacctcagccacggtcatctgccgcaaaaatgccagcattagcgtgcgggcccaggaccgctactatagctcatcttggagcgaatgggcatctgtgccctgcagttagac (SEQ IDNO: 6).

[0173] In some aspects, the nucleic acid vector (eg, 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.

[0174] In some aspects, the nucleic acid vector (eg, plasmid) comprises an intron, a 3'UTR (eg, hGH 3'UTR), an antibiotic resistance gene, or any combination thereof (eg, Figure 4 components).

[0175] In some aspects, the lipopolymer comprises polyethyleneimine (PEI) (e.g., Figure 5 lipid polymer).

[0176] In some aspects, the nanoparticles disclosed herein comprise a DNA plasmid encoding human IL-12.

[0177] In some aspects, wherein the nanoparticle comprises a synthetic polymer that facilitates plasmid delivery, it is a lipopolymer.

[0178] In some aspects, the lipopolymer further comprises polyethyleneimine (PEI) independently covalently linked to cholesterol and polyethylene glycol (PEG) groups.

[0179] In some aspects, the gene delivery polymer is a cationic polymer or a non-condensing polymer. The cationic polymer is selected from the group comprising: polylysine, polyethyleneimine, functionalized derivatives of polyethyleneimine (PEI), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine and spermidine. An example of a cationic gene delivery polymer suitable for the present disclosure is a PEI derivative comprising 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 is in turn bound to the PEI via a biocompatible bond.

[0180] The cationic gene delivery polymers of the present disclosure may further comprise a targeting moiety, including antibodies or antibody fragments, cell receptors, growth factor receptors, cytokine receptors, folic acid, transferrin, epidermal growth factor (EGF), insulin, asialomucoid, mannose-6-phosphate (monocytes), mannose (macrophages, some B cells), Lewis X and sialyl Lewis X(endothelial cells), N-acetyllactosamine (T cells), galactose (colon cancer cells) and thrombomodulin (mouse lung endothelial cells), fusogens such as polymyxin B and hemagglutinin HA2, lysosomal tropism agents, nuclear localization signals (NL) such as T antigen, etc. Another gene delivery polymer is a non-condensing polymer selected from the group comprising: polyvinyl pyrrolidone, polyvinyl alcohol, poly (lactide-co-glycolide) (PLGA) and triblock copolymers of PLGA and PEG. The gene delivery polymer can also be a non-condensing polymer. Examples of such non-condensing polymers include polyvinyl pyrrolidone, polyvinyl alcohol, poloxamer, polyglutamic acid, gelatin, polyphosphate, silk elastin hydrogel, agarose hydrogel, lipid microtubules, poly (lactide-co-glycolide) and polyethylene glycol-linked poly (lactide-co-glycolide).

[0181] The gene delivery polymer is a cationic polymer or a non-condensing polymer. The cationic polymer is selected from the group consisting of polylysine, polyethyleneimine, functionalized derivatives of polyethyleneimine, polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, and spermidine. An example of a cationic gene delivery polymer suitable for use in the present invention is a polyethyleneimine derivative comprising a polyethyleneimine (PEI) backbone, a lipid, and a polyethylene glycol spacer, wherein the lipid is directly bound to the polyethyleneimine backbone or covalently bound to the polyethylene glycol spacer, which in turn is bound to the PEI via a biocompatible bond.

[0182] In some aspects, nanoparticles comprising a DNA plasmid encoding interleukin-12 (IL-12) and a synthetic polymer that facilitates plasmid delivery are delivered intraperitoneally.

[0183] In some aspects, the nanoparticles are administered at a dose of about 35 mg / m² to about 80 mg / m². In some aspects, the nanoparticles are administered at a dose of about 50 mg / m² to about 100 mg / m². In some aspects, the nanoparticles are administered at a dose of about 80 mg / m².

[0184] III. Anti-VEGF Antibodies

[0185] In another aspect of the foregoing combination therapies, the agent is an anti-VEGF antibody.

[0186] In some aspects, the anti-VEGF antibody is selected from bevacizumab (eg, Avastin or a biosimilar thereof) or ranibizumab (eg, Lucentis or a biosimilar thereof).

[0187] In some aspects, the antibody having binding specificity for vascular endothelial growth factor (VEGF) is bevacizumab or a biosimilar thereof.

[0188] In some aspects, the anti-VEGF antibody comprises a first amino acid sequence that is at least about 85% identical to SEQ ID NO: 1 (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1) and a second amino acid sequence that is at least about 85% identical to SEQ ID NO: 2 (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2).

[0189] In some aspects, the heavy chain of the anti-VEGF antibody comprises the sequence:

[0190] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:1).

[0191] In some aspects, the light chain of the anti-VEGF antibody comprises the sequence:

[0192] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2).

[0193] In some aspects, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0194] In some aspects, the anti-VEGF antibody is administered intravenously.

[0195] In some aspects, the nucleic acid vector formulated with a lipopolymer is administered before, simultaneously with, or after the anti-VEGF antibody.

[0196] In some aspects, the nucleic acid vector formulated with the lipopolymer is administered before, simultaneously with, or after the anti-cancer agent.

[0197] In some aspects, an anti-cancer agent is administered (eg, first), followed by administration of a nucleic acid vector formulated with a lipopolymer (eg, second), and followed by administration of an anti-VEGF antibody (eg, third).

[0198] In some aspects, an anti-cancer agent is administered (e.g., first), followed by administration of a nucleic acid vector formulated with a lipopolymer (e.g., second), followed by an anti-VEGF antibody (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreduction) is performed (e.g., fourth).

[0199] In some aspects, an anti-cancer agent is administered, followed by administration of a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0200] In some aspects, the anti-VEGF antibody is administered prior to interval cytoreductive surgery, eg, at least about 22 days after the first administration of the anticancer agent, eg, weekly for at least about 12 weeks up to about 18 weeks.

[0201] In some aspects, the anti-VEGF antibody is administered weekly for at least about 12 weeks up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anticancer agent.

[0202] In some aspects, the anti-VEGF antibody is administered weekly for at least about 9 weeks, at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0203] In some aspects, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV (e.g., about 15 mg / kg IV). In some aspects, the anti-VEGF antibody is administered at a dose of about 15 mg / kg IV.

[0204] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anticancer agent. In some aspects, interval cytoreductive surgery (ICS) is administered about 28 days after administration of the anticancer agent.

[0205] In some aspects, intermittent cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid. In some aspects, intermittent cytoreductive surgery (ICS) is administered about 7 days after administration of the DNA plasmid.

[0206] In some aspects, intermittent cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid. In some aspects, intermittent cytoreductive surgery (ICS) is administered about 7 days after administration of the DNA plasmid.

[0207] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the anti-VEGF antibody. In some aspects, interval cytoreductive surgery (ICS) is administered about 28 days prior to administration of the anti-VEGF antibody.

[0208] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody. In some aspects, interval cytoreductive surgery (ICS) is administered about 28 days after administration of the anti-VEGF antibody.

[0209] IV. Anticancer Agents

[0210] In one aspect of the aforementioned therapy, the anticancer agent is a chemotherapeutic drug selected from the group consisting of taxanes, platinums, doxorubicin, cyclophosphamide, topotecan, carmustine (BCNU), or a combination thereof. In some aspects, the anticancer therapy is selected from the group consisting of paclitaxel, carboplatin, docetaxel, nab-paclitaxel, doxorubicin, and any combination thereof.

[0211] In some aspects, the anticancer agent is doxorubicin.

[0212] In some aspects, the anticancer agent comprises paclitaxel.

[0213] In some aspects, the anticancer agent comprises carboplatin.

[0214] In some aspects, the anticancer agent comprises docetaxel.

[0215] In some aspects, the anticancer agent comprises nab-paclitaxel.

[0216] In some aspects, the anticancer agent comprises.

[0217] In some aspects, the anticancer agent is administered prior to interval cytoreductive surgery once every three weeks for about 12 to about 18 weeks.

[0218] In some aspects, the anticancer agent is administered prior to interval cytoreductive surgery once every three weeks for about 12 to about 18 weeks.

[0219] In some embodiments, the anticancer agent is administered for at least about 28 days after interval cytoreductive surgery, eg, once every 1-2 weeks (eg, every 3 weeks) for about 8-10 weeks (eg, 9 weeks).

[0220] In some aspects, the anticancer agent is administered at least about 28 days after interval cytoreductive surgery, and every three weeks for about 9 weeks.

[0221] In some aspects, the anticancer agent is selected from the group consisting of paclitaxel, carboplatin, docetaxel, nab-paclitaxel, and any combination thereof.

[0222] In some aspects, the nucleic acid vector formulated with the lipopolymer is administered before, simultaneously with, or after the anti-cancer agent.

[0223] In some aspects, an anti-cancer agent is administered (eg, first), followed by administration of a nucleic acid vector formulated with a lipopolymer (eg, second), and followed by administration of an anti-VEGF antibody (eg, third).

[0224] In some aspects, an anti-cancer agent is administered (e.g., first), followed by administration of a nucleic acid vector formulated with a lipopolymer (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreduction) is performed (e.g., fourth).

[0225] In some aspects, an anti-cancer agent is administered, followed by administration of a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0226] In some aspects, the anticancer agent is administered prior to interval cytoreductive surgery. In some aspects, the anticancer agent is administered prior to interval cytoreductive surgery, once every three weeks for about 12 weeks to about 18 weeks.

[0227] In some embodiments, the anticancer agent is administered for at least about 28 days after interval cytoreductive surgery (eg, once every three weeks for about 9 weeks).

[0228] In some aspects, administration of the anticancer agent comprises administration of paclitaxel at a dose of about 100-200 mg / m² (about 175 mg / m²), optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0229] In some aspects, administration of the anticancer agent comprises administration of docetaxel at a dose of 50-100 mg / m² (eg, about 75 mg / m²), optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0230] In some aspects, administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of 200-300 mg / m² (eg, about 260 mg / m²), optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0231] In some aspects, administration of the nanoparticles prior to interval cytoreduction begins 15 days after the first administration of the anticancer agent and continues weekly for at least about 12 weeks to about 18 weeks.

[0232] In some aspects, administration of the anticancer agent comprises administration of paclitaxel at a dose of about 150 mg / m² to about 200 mg / m² (e.g., about 175 mg / m²) followed by administration of carboplatin at a dose of about AUC 4-8 IV (e.g., about AUC 5-6 IV).

[0233] In some aspects, administration of the anticancer agent comprises administration of paclitaxel at a dose of about 175 mg / m² followed by IV administration of carboplatin at a dose of about AUC5-6.

[0234] In some aspects, administration of the anticancer agent comprises administration of docetaxel at a dose of about 50 mg / m² to about 100 mg / m² (e.g., about 75 mg / m²) followed by administration of carboplatin at a dose of about AUC 4-8 IV (e.g., about AUC 5-6 IV).

[0235] In some aspects, administration of the anticancer agent comprises administration of docetaxel at a dose of about 75 mg / m² followed by IV administration of carboplatin at a dose of about AUC5-6.

[0236] In some aspects, administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of about 240 mg / m² to about 300 mg / m² (e.g., about 260 mg / m²) followed by administration of carboplatin at a dose of about AUC 4-8 IV (e.g., about AUC 5-6 IV).

[0237] In some aspects, administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of about 260 mg / m² followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0238] V. Treatment Methods

[0239] Certain aspects of the present disclosure relate to methods of treating a subject having cancer comprising administering to the subject a combination of (i) nanoparticles, and (ii) an antibody having binding specificity for vascular endothelial growth factor (VEGF).

[0240] The present invention also provides a method for treating a mammalian cancer or hyperproliferative disorder by administering a pharmaceutical composition intratumorally, intraperitoneally, intravenously, intrathecally, intratracheally, intracranially, or systemically, wherein the pharmaceutical composition comprises a plasmid-based gene expression system and a gene delivery polymer, and does not comprise a chemotherapeutic agent. The mammalian cancer is selected from the group consisting of primary or metastatic tumors of the ovary. Preferably, the nucleic acid is a plasmid-based gene expression system comprising a DNA sequence encoding interleukin-12.

[0241] Treating tumors with the pharmaceutical composition (nucleic acid plus gene delivery polymer and one or more chemotherapeutic agents) results in tumor shrinkage and life extension. According to the method disclosed herein, 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 inventive method is defined as, but not limited to, a reduction in tumor size or a reduction in tumor density, an increase in lymphocyte count or an increase in neutrophil count or an improvement in survival, or all of the above. In addition, according to the method of the present invention, the combination of gene therapy (nucleic acid and gene delivery polymer) and chemotherapy (chemotherapeutic agent) reduces the toxicity of chemotherapeutic agents and reverses the resistance of tumors to chemotherapy. Toxicity herein is defined as any treatment-related adverse effects observed clinically, including but not limited to abnormal hematology or serum chemistry or organ toxicity. In addition, according to the method 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 the optimal dose of chemotherapeutic agent, but with less toxicity.

[0242] New cancer treatment strategies focus on delivering macromolecules that carry genetic information, rather than therapeutic proteins themselves, allowing exogenously delivered genes to be expressed in the tumor environment. Compared to viral delivery systems, methods utilizing non-viral gene delivery systems are considered safer, but due to poor efficiency, the practical application of current polymer systems is not satisfactory. Recently, a strategy has been disclosed in which the gene transfection efficiency of low molecular weight PEI is enhanced by covalently attaching cholesterol to form a water-soluble lipid polymer (WSLP). See Mol. Ther., 2001, 4, 130. Using WSLP to transfer IL-12 genes into solid tumors is significantly better than unmodified PEI and results in more significant tumor suppression.

[0243] It is widely recognized that due to the multifactorial nature of cancer, single treatment strategies for the disease are generally ineffective. It is increasingly recognized that the benefits of combining more than one drug to maximize the anti-cancer response. In the present disclosure, we have combined chemotherapeutics with gene delivery of anti-cancer genes that are locally administered to the tumor site to improve treatment safety and efficacy. The safe and effective local delivery of anti-cancer genes combined with standard chemotherapeutics will enhance anti-cancer response and patient survival without increasing toxicity. This combination therapy will reduce chemotherapy doses and increase the sensitivity of tumors to chemotherapy. In the present disclosure, it is demonstrated that a pharmaceutical composition comprising an anti-cancer gene compounded with a gene delivery polymer and at least one adjuvant chemotherapy drug is more effective than gene therapy or chemotherapy administered alone. In addition, when given by different routes of administration, combination therapy is effective for a variety of tumors and does not increase toxicity relative to monotherapy.

[0244] Certain aspects of the present disclosure relate to combination therapies comprising: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

[0245] Certain aspects of the present disclosure relate to methods of treating a subject having cancer, the methods comprising administering to the subject a combination therapy comprising: (i) a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

[0246] In some aspects, the polynucleotide encodes human IL-12.

[0247] In some aspects, the nucleic acid vector (eg, 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.

[0248] In some aspects, the nucleic acid vector (eg, plasmid) comprises an intron, a 3'UTR (eg, hGH 3'UTR), an antibiotic resistance gene, or any combination thereof (eg, Figure 4 components).

[0249] In some aspects, the lipopolymer comprises polyethyleneimine (PEI) (e.g., Figure 5 lipid polymer).

[0250] In some aspects, the combination further comprises an anti-cancer agent.

[0251] In some aspects, the anticancer agent is a chemotherapeutic agent.

[0252] In some aspects, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0253] In some aspects, the anticancer agent is doxorubicin.

[0254] In some aspects, the anticancer agent is paclitaxel.

[0255] In some aspects, the anticancer agent is carboplatin.

[0256] In some aspects, the anticancer agent is docetaxel.

[0257] In some aspects, the anticancer agent is nab-paclitaxel.

[0258] In some aspects, the anticancer agent is olaparib.

[0259] In some aspects, the anti-VEGF antibody is selected from the group consisting of bevacizumab (e.g., Avastin or a biosimilar thereof) or ranibizumab (e.g., Lucentis or a biosimilar thereof).

[0260] In some aspects, the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence at least about 85% identical to SEQ ID NO: 1 (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1) and a variable light chain (VL) comprising an amino acid sequence at least about 85% identical to SEQ ID NO: 2 (e.g., 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2).

[0261] In some aspects, the method further comprises surgery to remove all or part of a tissue or tumor in the subject (eg, interval cytoreductive surgery).

[0262] In some aspects, nucleic acid vectors formulated with lipopolymers are administered intratumorally or intraperitoneally.

[0263] In some aspects, nucleic acid vectors formulated with lipopolymers are administered intravenously.

[0264] In some aspects, the anti-VEGF antibody is administered intratumorally, intraperitoneally, intracystically, or any combination thereof.

[0265] In some aspects, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0266] In some aspects, the anti-VEGF antibody is administered intravenously.

[0267] In some aspects, the nucleic acid vector formulated with a lipopolymer is administered before, simultaneously with, or after the anti-VEGF antibody.

[0268] In some aspects, the nucleic acid vector formulated with the lipopolymer is administered before, simultaneously with, or after the anti-cancer agent.

[0269] In some aspects, an anti-cancer agent is administered (eg, first), followed by administration of a nucleic acid vector formulated with a lipopolymer (eg, second), and followed by administration of an anti-VEGF antibody (eg, third).

[0270] In some aspects, an anti-cancer agent is administered (e.g., first), followed by administration of a nucleic acid vector formulated with a lipopolymer (e.g., second), followed by an anti-VEGF antibody (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreduction) is performed (e.g., fourth).

[0271] In some aspects, an anti-cancer agent is administered, followed by administration of a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0272] In some aspects, the anticancer agent is administered prior to interval cytoreductive surgery once every three weeks for about 12 to about 18 weeks.

[0273] In some embodiments, the anticancer agent is administered for at least about 28 days after interval cytoreductive surgery (eg, once every three weeks for about 9 weeks).

[0274] In some aspects, administration of the anticancer agent comprises administration of paclitaxel at a dose of about 100-200 mg / m² (about 175 mg / m²), optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0275] In some aspects, administration of the anticancer agent comprises administration of docetaxel at a dose of 50-100 mg / m² (eg, about 75 mg / m²), optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0276] In some aspects, administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of 200-300 mg / m² (eg, about 260 mg / m²), optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

[0277] In some aspects, administration of the anticancer agent comprises administering olaparib PO twice at a dose of about 250 mg to about 350 mg (eg, about 300 mg).

[0278] In some aspects, administration of the nanoparticles prior to interval cytoreduction begins 15 days after the first administration of the anticancer agent and continues weekly for at least about 12 weeks to about 18 weeks.

[0279] In some aspects, the nanoparticles are administered at least about 28 days after interval cytoreductive surgery and are administered weekly for at least about 9 weeks starting 15 days after the first administration of the anticancer agent.

[0280] In some aspects, interleukin-12 (IL-12) formulated with a lipopolymer (e.g., nanoparticles) is administered at a dose of about 35 mg / m² to about 80 mg / m². In some aspects, interleukin-12 (IL-12) formulated with a lipopolymer (e.g., nanoparticles) is administered at a dose of about 50 mg / m² to about 100 mg / m². In some aspects, interleukin-12 (IL-12) formulated with a lipopolymer (e.g., nanoparticles) is administered at a dose of about 80 mg / m².

[0281] In some aspects, the anti-VEGF antibody is administered weekly for at least about 12 weeks up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anticancer agent.

[0282] In some aspects, the anti-VEGF antibody is administered weekly for at least about 9 weeks, at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0283] In some aspects, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV (e.g., about 15 mg / kg IV). In some aspects, the anti-VEGF antibody is administered at a dose of about 15 mg / kg IV.

[0284] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anticancer agent.

[0285] In some aspects, intermittent cytoreduction (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0286] In some aspects, intermittent cytoreduction (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0287] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the anti-VEGF antibody.

[0288] In some aspects, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody.

[0289] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer (e.g., glioblastoma), lung cancer, and any combination thereof, and metastasis of any cancer.

[0290] In some aspects, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0291] In some aspects, the subject is a human.

[0292] In some aspects, the subject is treated with one or more of: neoadjuvant chemotherapy (NACT), bevacizumab (BEV), GEN-1, interval cytoreductive surgery (ICS), minimal residual disease (MRD) detection by second laparoscopy (SLL), BEV+Olaparib, and BEV+GEN-1.

[0293] In some aspects, (a) neoadjuvant chemotherapy (NACT), bevacizumab (BEV), and GEN-1 are administered to the subject. In some aspects, in (a), the subject is treated with NACT for 4-6 cycles. In some aspects, in (a), BEV is administered to the subject in cycles 2, 3, 6, and 7. In some aspects, in (a), each cycle includes BEV except for the following cycles: (1) cycle 1, (2) the last cycle of neoadjuvant therapy immediately before ICS (which can be C4, C4+1, or C4+2), and (3) the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). In some aspects, in (a), GEN-1 is administered to the subject weekly starting on C1D15. In some aspects, each cycle is a 21-day cycle.

[0294] In some aspects, NACT is carboplatin and paclitaxel. In some aspects, NACT is administered once per cycle (e.g., once every three weeks). In some aspects, paclitaxel is administered IV at a dose of about 175 mg / m², followed by IV administration of carboplatin AUC5-6 on C1D1. In some aspects, BEV is administered IV on day 1 of the included cycle at a dose of about 15 mg / kg. In some aspects, GEN-1 is administered IP at a dose of about 80 mg / m².

[0295] In some aspects, (a) is followed by (b) interval cytoreduction. In some aspects, ICS will be performed at least 4 weeks after the last dose of NACT from (a).

[0296] In some aspects, subject (c) is administered neoadjuvant chemotherapy (NACT), bevacizumab (BEV), and GEN-1. In some aspects, in (c), the subject is treated with NACT for 3 cycles. In some aspects, in (c), BEV is administered to the subject in cycles 2, 3, 6, and 7. In some aspects, in (c), each cycle includes BEV except for the following cycles: (1) cycle 1, (2) the last cycle of neoadjuvant therapy immediately before ICS (which can be C4, C4+1, or C4+2), and (3) the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). In some aspects, in (c), GEN-1 is administered to the subject weekly. In some aspects, each cycle is a 21-day cycle.

[0297] In some aspects, NACT is carboplatin and paclitaxel. In some aspects, NACT is administered once per cycle (e.g., once every three weeks). In some aspects, paclitaxel is administered IV at a dose of about 175 mg / m², followed by IV administration of carboplatin AUC5-6 on C1D1. In some aspects, BEV is administered IV on day 1 of the included cycle at a dose of about 15 mg / kg. In some aspects, GEN-1 is administered IP at a dose of about 80 mg / m².

[0298] In some aspects, (b) is followed by (c).

[0299] In some aspects, (c) is followed by (d) detection of minimal residual disease (MRD) by a second laparoscopy (SLL).

[0300] In some aspects, during the maintenance phase, (e) BEV and Olaparib are administered to the subject. In some aspects, during the maintenance phase, if the subject is BRCA+ / homologous recombination deficiency positive (HRD+), (e) is administered to the subject. In some aspects, in (e), BEV is administered IV at approximately 15 mg / kg, once a day for 3 weeks, for a maximum of 18 cycles. In some aspects, in (e), Olaparib is administered PO twice at a dose of approximately 300 mg. In some aspects, (d) is followed by (e).

[0301] In some aspects, during the maintenance phase, the subject is administered (f) BEV and GEN-1. In some aspects, during the maintenance phase, if the subject is BRCA- / homologous recombination proficient (HRP), the subject is administered (f). In some aspects, in (f), BEV is administered IV at approximately 15 mg / kg every 3 weeks for up to 18 cycles. In some aspects, in (f), GEN-1 is administered IP at a dose of approximately 80 mg / m² every 21 days for up to an additional 18 cycles. In some aspects, (d) is followed by (f).

[0302] In some aspects, the subject is treated with one, two, three, four or more of (a), (b), (c), (d) and (e). In some aspects, the subject is treated with all of (a), (b), (c), (d) and (e).

[0303] In some aspects, the subject is treated with one, two, three, four or more of (a), (b), (c), (d), and (f).

[0304] In some aspects, the subject is treated with all of (a), (b), (c), (d), and (f).

[0305] The following examples are illustrative, and do not limit the scope of the claimed aspects. Example

[0306] Example 1. GEN-1 enhances the activity of anti-VEGF antibodies in a mouse model.

[0307] Figure 1 The potential for synergistic efficacy of VEGF level reduction and inhibition by concurrent administration of anti-VEGF antibodies (e.g., bevacizumab) with GEN-1 was demonstrated. nu Mice were injected intraperitoneally with SKOV-3 (human epithelial ovarian carcinoma) cells (7x10 6 Anti-VEGF antibodies were administered intravenously at different dose levels: 5 mg / kg (low), 10 mg / kg (medium), and 20 mg / kg (high).

[0308] Anti-VEGF antibodies (e.g., bevacizumab) are administered 9 days after the initial tumor implantation and continue weekly for 6 weeks. mGEN-1 (100 μg DNA) is then administered intraperitoneally starting on day 14 after the initial tumor implantation and continues weekly for 4 weeks.

[0309] Mice were euthanized 59 days after initial tumor implantation and tumors were removed and subsequently weighed. Low-dose anti-VEGF antibodies (e.g., bevacizumab) have shown improved efficacy when administered in combination with GEN-1, which improves the therapeutic index and cost. Figure 1 .

[0310] Example 2. GEN-1 enhances the activity of anti-VEGF antibodies in combination with anticancer agents in a mouse model.

[0311] Figure 2The potential for synergistic efficacy of VEGF level reduction and inhibition by concurrent administration of anti-VEGF antibodies (e.g., bevacizumab) and anticancer agents with GEN-1 was demonstrated. nu Mice were injected intraperitoneally with 500 μL SKOV-3-Luc (human ovarian epithelial carcinoma) cells (7 × 10 6 Starting 2 weeks after tumor implantation, Doxil was administered intraperitoneally at a dose of 7.5 mg / kg every other week. Ten days after tumor implantation, an anti-VEGF antibody (e.g., bevacizumab) was administered intravenously at 10 mg / kg weekly.

[0312] mGEN-1 (100 μg DNA) was then administered intraperitoneally weekly starting two weeks after the initial tumor implantation. Tumor burden in animals was then quantified using IVIS imaging. Figure 2 The whole body image of the mouse via IVIS imaging is shown in Figure 3 shown.

[0313] Example 3. Clinical combination of GEN-1+NACT+anti-VEGF antibody

[0314] This will be a 1:1 randomized, open-label, multicenter Phase II trial with a safety run-in to evaluate the safety, dosing, efficacy, and biologic activity of GEN-1 added to neoadjuvant chemotherapy (NACT) + an anti-VEGF antibody (e.g., bevacizumab) (BEV) compared to NACT + BEV alone. NACT will be the standard regimen of carboplatin + paclitaxel administered every three weeks for 7-9 cycles. The regimen will require at least 4 cycles of neoadjuvant chemotherapy and allow up to 2 additional cycles (C4+1 and C4+2) before ICS at the discretion of the principal investigator based on response and other clinical considerations. ICS will be administered after a 3-4 week rest following the last dose of NACT. After at least 4 weeks of recovery from ICS, 3 additional adjuvant cycles of study treatment will be administered.

[0315] In addition, BEV will be included in every cycle except for the following: Cycle 1, the last cycle of neoadjuvant therapy immediately before ICS, and the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). In the experimental arm, 80 mg / m² IP GEN-1 will be administered every 7 days starting on Cycle 1 Day 15 (C1D15) and continued weekly until the last cycle of adjuvant therapy. BEV will not be administered before or at any time within 30 days after surgery. The experimental arm will add GEN-1 weekly to each NACT + BEV cycle starting on Cycle 1 Day 15. FDA-approved BEV biosimilars are permitted.

[0316] The safety run-in phase will evaluate the safety of adding GEN-1 weekly to the NACT+BEV regimen in up to 12 subjects. This will be a standard 3+3 design, with the Data Safety Monitoring Board (DSMB) evaluating a cohort of three subjects who have received at least two cycles of NACT+BEV+GEN-1 prior to starting the main phase of the study. Run-in phase patients will also be randomized. Subjects must have received at least two cycles of chemotherapy + BEV + GEN-1 to be evaluated for safety. Prior to Phase II dosing of GEN-1, at least six subjects in the GEN-1 arm must be available for safety assessment.

[0317] Once the DSMB determines the recommended safe dose from the safety phase, the Phase II study can begin enrollment. The study will randomize approximately 50 subjects. At the completion of the NACT, all subjects will undergo a second laparoscopy (SLL) to determine if minimal residual disease (MRD) is positive. The SLL will be performed by a gynecologic oncologist using standardized surgical techniques.

[0318] Maintenance therapy will be determined by BRCA+ / HRD status. All participants will receive BEV, while only BRCA+ / HRD participants will receive olaparib in addition to BEV. Participants in the BRCA- / HRD arm of the trial will receive GEN-1 and BEV. All participants will be followed for disease progression and survival.

[0319] Research phase

[0320] Introduction period

[0321] To ensure that the combination of NACT+BEV+GEN-1 is safe, the study will enroll at least six subjects in the experimental arm prior to starting the main phase of the protocol. No more than two of the six subjects treated in the experimental arm may exhibit dose-limiting toxicity prior to starting the main phase of the study. The independent DSMB will review the safety data of subjects administered at least two cycles of NACT+BEV+GEN-1 and provide recommendations on dose adjustments, safety monitoring, and dosing for the main phase of the study. The DSMB charter will specify the definition of dose-limiting toxicity (DLT) during the safety phase and the responsibilities of the committee, including dose adjustments and recommending the Phase II dose of GEN-1.

[0322] Phase II

[0323] Once the DSMB determines the recommended safe dose from the safety phase, the main phase of the study can begin enrolling subjects. The study will randomize approximately 50 subjects (25 per arm) in the Phase II co-administration. All subjects will be randomized to either NACT+BEV+GEN-1 or NACT+BEV alone. Subjects will receive 4-6 cycles of treatment before interval cytoreductive surgery (ICS), followed by at least 2 cycles of treatment post-surgery. At the end of the last chemotherapy cycle for all subjects, SLL will be performed before starting the maintenance phase.

[0324] maintain

[0325] After SLL is complete, subjects will begin maintenance therapy, which will begin no earlier than 4 weeks (ideally 5-7 weeks) after the SLL date.

[0326] All subjects will receive BEV (or an FDA-approved biosimilar) every 21 days until disease progression or unacceptable toxicity or up to 15 months.

[0327] BRCA+ / HRD subjects only: Lynparza 300 mg PO twice daily will be administered to all BRCA+ / HRD subjects starting in the maintenance phase until disease progression or unacceptable toxicity or up to 24 months. In addition, BEV 15 mg / kg will be administered as a single agent every 3 weeks until disease progression or unacceptable toxicity for up to an additional 18 cycles.

[0328] BRCA- / HRP subjects only: GEN-1 with BEV 15 mg / kg will be administered in BRCA- / HRP subjects every 21 days until disease progression or unacceptable toxicity for up to an additional 18 cycles.

[0329] Study population

[0330] Approximately 50 subjects (including up to 12 for a safety run-in) with newly diagnosed advanced ovarian cancer will be randomized.

[0331] Inclusion criteria

[0332] 1. Subjects must be suspected of having high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer and undergo a pre-treatment biopsy to obtain histological confirmation via laparoscopy, interventional radiology, or CT- or ultrasound-guided core biopsy. A pathology report is required to provide histological documentation of the original primary tumor.

[0333] 2. Subjects must have International Federation of Gynecology and Obstetrics (FIGO) stage III or IV disease and have been determined to benefit from neoadjuvant therapy based on standard of care clinical considerations.

[0334] 3. Only subjects with high-grade serous adenocarcinoma histology epithelial cell type are eligible.

[0335] 4. The subject must have sufficient:

[0336] 1. Bone marrow function: Absolute neutrophil count (ANC) greater than or equal to 1,500 / mcL. This ANC is unlikely to be induced or supported by granulocyte colony-stimulating factor. Platelet count greater than or equal to 100,000 / mcL.

[0337] II. Renal function: GFR estimated by Cockcroft-Gault >= 50 ml / min. Urine dipstick showing proteinuria 1+ or less (patients with a urine dipstick reading of 2+ or more should undergo a 24-hour urine collection with less than 2 g protein / 24 hrs.)

[0338] III. Liver function: Bilirubin ≤1.5 x ULN. SGOT (AST) and SGPT (ALT) ≤3.0 x ULN, alkaline phosphatase ≤2.5 x ULN.

[0339] IV. Neurological function: Neuropathy (sensory and motor) less than or equal to Grade 1.

[0340] 5. Subjects should have no active infection requiring isolation, parenteral antibiotics, or serious uncontrolled medical illness or condition within four weeks of study entry.

[0341] 6. Any hormone therapy for malignant tumors must be stopped at least one week before the first treatment. Continuation of hormone replacement therapy is allowed.

[0342] 7. Subjects must have a performance status score of 0-1 according to the Eastern Cooperative Oncology Group (ECOG) criteria.

[0343] 8. Subjects of childbearing potential must have a negative serum pregnancy test within 14 days before the start of protocol therapy and use effective contraceptive measures. If applicable, subjects must stop breastfeeding before entering the study.

[0344] 9. Subjects must obtain satisfactory results in the baseline laboratory analyses and diagnostic procedures specified in the protocol.

[0345] 10. Subjects must sign an IRB / EC-approved informed consent and authorization form allowing the release of personal health information.

[0346] 11. Participants must be at least 18 years old.

[0347] 1. Subjects who have previously received GEN-1 treatment.

[0348] 2. History of allergic reaction to compounds with similar chemical or biological composition to GEN-1 or other agents used in this study.

[0349] 3. Subjects who have received oral or parenteral corticosteroids within 2 weeks of study entry, or have ongoing systemic immunosuppressive therapy, such as chronic steroid use (prednisone equivalents > 10 mg / day) unrelated to chemotherapy administration. Steroids are allowed to prevent IV contrast agent allergy.

[0350] 4. Subjects with autoimmune diseases who have required immunosuppressive therapy within the past 2 years. Examples of autoimmune diseases include systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis.

[0351] 5. Subjects with known human immunodeficiency virus (HIV) or human T-lymphotropic virus (HTLV) infection were excluded.

[0352] 6. Subjects with other invasive malignancies were excluded if there was any evidence of other invasive malignancies within the past three years. Subjects were also excluded if their previous cancer treatment was contraindicated for the regimen. Subjects with non-invasive malignancies such as non-melanoma skin cancer, melanoma in situ, etc. were eligible.

[0353] 7. Subjects who have received prior radiotherapy to any part of the abdomen or pelvis are excluded. Prior radiotherapy for localized breast, head and neck, or skin cancer is allowed, provided that it was completed more than three years before registration and the patient remains free of recurrent or metastatic disease.

[0354] 8. Subjects who have previously received chemotherapy for any abdominal or pelvic tumor are excluded. Subjects may have received prior adjuvant chemotherapy for localized breast cancer, provided that this chemotherapy was completed more than three years prior to enrollment and the patient remains free of recurrent or metastatic disease.

[0355] 9. Subjects known to have active hepatitis.

[0356] 10. Subjects known to have nephrotic syndrome (proteinuria grade 2 or higher).

[0357] 11. Subjects who also have serious medical problems unrelated to malignancy that would significantly limit full compliance with the study or would expose the subject to extreme risk or shorten life expectancy.

[0358] 12. Subjects with clinically significant cardiovascular disease. This includes:

[0359] a) Uncontrolled hypertension, defined as systolic blood pressure (BP) >150 mmHg or diastolic blood pressure >90 mmHg for at least two days. (Subjects with uncontrolled hypertension may become eligible once hypertension is controlled).

[0360] b) Myocardial infarction or unstable angina occurred within 6 months before registration.

[0361] c) A history of severe ventricular arrhythmia (i.e., ventricular tachycardia or ventricular fibrillation) or arrhythmia requiring antiarrhythmic drug treatment (except for atrial fibrillation well controlled by antiarrhythmic drugs).

[0362] d) Baseline ECG (electrocardiogram) QTc interval ≥450 ms.

[0363] e) Baseline ejection fraction ≤50% as assessed by echocardiography or MUGA.

[0364] f) New York Heart Association (NYHA) class II or higher congestive heart failure.

[0365] 13. Subjects of childbearing potential, subjects who have not taken appropriate contraceptive measures, pregnant subjects, or subjects who are breastfeeding are not eligible for this trial.

[0366] 14. Subjects with a history or physical examination evidence of CNS disease, including primary brain tumors, epileptic seizures not controlled by standard drug therapy, any brain metastases, or a history of cerebrovascular accident (CVA, stroke), transient ischemic attack (TIA), or subarachnoid hemorrhage within six months of the first treatment date of this study.

[0367] 15. Subjects with a history of diverticulitis. Diverticular disease is not excluded.

[0368] 16. Subjects who have had hemoptysis in the last month.

[0369] 17. Subjects with any condition / abnormality that would interfere with proper placement of an IP catheter for study drug administration, including abdominal surgery within 4 weeks of study entry (for reasons other than IP port placement), bowel dysfunction, fistula, or suspected extensive adhesions based on past medical history or laparoscopic findings.

[0370] Enrollment and withdrawal procedures

[0371] Newly diagnosed subjects with advanced ovarian cancer will be randomly assigned to receive either NACT+BEV+GEN-1 or NACT+BEV.

[0372] Study Treatment and Administration

[0373] Treatment allocation

[0374] Eligible subjects will be randomized in a 1:1 ratio to receive NACT+BEV+GEN-1 or NACT+BEV alone. As this is an open-label study, site staff, subjects, and the sponsor will be aware of each subject's treatment status.

[0375] Neoadjuvant chemotherapy (NACT)

[0376] control arm

[0377] NACT: Paclitaxel 175 mg / m² will be administered intravenously (IV) on C1D1, followed by carboplatin AUC 5-6 IV. This cycle will be repeated every 21 days, beginning on day 1 of the first four cycles of ICS and the first three cycles of ICS. Up to two additional cycles (C4+1 and C4+2) may be added before ICS at the physician's discretion. If a paclitaxel response occurs, docetaxel 75 mg / m² or nab-paclitaxel (Abraxane) 260 mg / m² may be substituted according to institutional guidelines. Body surface area (BSA) will be calculated according to local practice.

[0378] BEV will be included in every cycle except the following: Cycle 1, the last cycle of neoadjuvant therapy immediately before ICS, and the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). FDA-approved biosimilars of BEV may be used in this study.

[0379] In the experimental arm, GEN-1 will be initiated on C1D15 and continued weekly until the last cycle of adjuvant therapy.

[0380] Interval cytoreductive surgery (ICS) will be performed at least 28 days after the last cycle of neoadjuvant chemotherapy, and after recovery from ICS (at least 28 days), protocol therapy will continue as adjuvant chemotherapy for an additional 3 cycles. BEV should not be administered before or at any time within 28 days after surgery.

[0381] Prophylactic dexamethasone is permitted only at the initial dose of NACT to prevent hypersensitivity reactions. BEV 15 mg / kg will be administered IV on day 1 of cycles 2, 3, 6, and 7. During the maintenance phase, BEV 15 mg / kg will be administered as a single agent every 3 weeks until disease progression or unacceptable toxicity, for a maximum of 18 additional cycles. Overall, BEV can be administered for up to 24 cycles.

[0382] Hypersensitivity reaction prevention

[0383] All patients receiving paclitaxel should be pretreated with a corticosteroid such as dexamethasone on day 1 of cycle 1. Subsequent administration of corticosteroids (e.g., C2D1) during chemotherapy is contraindicated to avoid attenuating the effect of GEN-1. Patients who experience a hypersensitivity reaction can be treated with docetaxel or abraxane instead.

[0384] Dosage adjustments

[0385] Investigators should follow their institutional standards for dose adjustments due to adverse reactions to chemotherapy, BEV, or Olaparib. Any dose adjustment or skipped dose of GEN-1 should be approved by the study chair or study monitor. If necessary due to adverse events related to chemotherapy drugs, doses of GEN-1 can be skipped at any time for up to two weeks to maintain the treatment plan. However, delays in GEN-1 administration preferably do not delay or change the timing of chemotherapy administration. If grade 3 or 4 toxicity attributed to GEN-1 occurs, subsequent doses are skipped until recovery to ≤ grade 1. Generally, GEN-1 can be given as long as ANC>500 and PLT>50K, otherwise dose adjustments can be considered.

[0386] If a subject experiences grade 3 abdominal pain following the analgesic regimen, the dose of GEN-1 will be reduced to 60 mg / m² for all subsequent doses.

[0387] Refer to the package insert (label) and local institutional guidelines for carboplatin, paclitaxel, abraxane, bevacizumab (or biosimilar), and docetaxel for complete prescribing information.

[0388] GEN-1 (investigational medicinal product)

[0389] Human IL-12 plasmid (phIL-12-005) was formulated with the lipopolymer PEG-PEI-cholesterol (PPC) in 10% lactose.

[0390] Human IL-12 plasmid

[0391] The phIL-12-005 plasmid contains Kan r The plasmid of the gene contains the hIL-12 gene expression cassette. The hIL-12 gene expression cassette of phIL-12-005 contains the immediate early enhancer and promoter derived from cytomegalovirus (CMV), 5' untranslated region (UTR), synthetic introns, p35 gene, human growth hormone (hGH) 3'UTR and polyadenylation signal sequence, CMV promoter, 5'UTR, synthetic introns, p40 gene, hGH 3'UTR and polyadenylation signal sequence. The two hIL-12 subunits are controlled by two separate CMV promoters. (See Figure 4 ).

[0392] PEG-PEI-cholesterol

[0393] PEG-PEI-cholesterol (PPC) consists of a PEI backbone to which polyethylene glycol and cholesterol are independently attached via covalent bonds. The molecular weights of PEI, PEG, and cholesterol carbonyl groups are 1800, 550, and 414, respectively. (See Figure 5 ).

[0394] Route of Administration and Administration of GEN-1

[0395] GEN-1 was administered after peritoneal lavage and collection of transformed samples using the IP port.

[0396] Check catheter patency by flushing 25 mL of 0.9% saline through the IP port. Do not flush the catheter with heparin during sample collection or medication infusion.

[0397] Reconstituted GEN-1 (in a 50 mL glass vial or IV bag) is stable at room temperature for up to 24 hours. After confirming catheter patency, an IV bag containing GEN-1 will be administered through the patient's IP catheter. GEN-1 will be infused from the IV bag via the catheter by gravity, with the valve open and flowing freely. A typical administration may take approximately 1 hour.

[0398] After GEN-1 infusion, a second flush with at least 25 ml of 0.9% saline was administered to ensure clearance of the study drug from the catheter.

[0399] To date, no toxicities associated with systemic administration of recombinant IL-12 therapy have been detected with GEN-1. Phase 1 studies of GEN-1 have demonstrated minimal or no systemic uptake of GEN-1 or its downstream cytokines.

[0400] A subcutaneous implantable IP silicone catheter can be used to administer GEN-1 to the peritoneal cavity. In previous preclinical compatibility studies and previous Phase I studies, GEN-1 has been shown to be compatible with silicone catheters. The Port-A-Cath catheter (Deltec, Inc., St. Paul, MN) has been successfully used for IP delivery of GEN-1, with few serious catheter-related complications observed. Although the Port-A-Cath catheter is preferred, any other approved catheter with a subcutaneous port for IP delivery may also be used if suitable for aspirating biological samples for translational research. A Bard 9.6 Fr silicone single-lumen catheter for intravenous access from Bard Access Systems (West Amelia Earhart Drive, Salt Lake City, Utah) or an equivalent catheter with or without a cuff may be used. Catheter compatibility studies performed using the Port-A-Cath catheter showed that exposure of the catheter to GEN-1 did not significantly affect the physicochemical properties or transfection activity of GEN-1. Similar compatibility studies were performed using the Bard catheter. Subjects can be treated via pre-existing IP catheters, provided they have similar properties to the Port-A-Cath catheter device and are functional. If there are concerns about catheter patency or function, a catheter angiogram can be obtained to verify intraperitoneal infusion.

[0401] Catheter Insertion: An IP catheter will be implanted according to standard institutional procedures; the procedure and risks associated with IP catheter placement must be explained to the subject, and the subject will sign a procedural consent form prior to catheter placement. Subjects will have their IP catheter inserted at least 7 days prior to planned study drug administration to allow for adequate healing and sealing around the catheter site. A semi-permanent subcutaneous access port, such as a Port-A-Cath catheter (SIMS Deltec, Inc., St. Paul, MN 55112) or equivalent device, will be used according to current institutional clinical practice. The port will be located in the lower chest.

[0402] Study drug will be infused through this port during the study. Prior to each study drug infusion, at least 25 mL of normal saline will be flushed through the catheter to check for patency; heparin will not be used to flush the catheter during sample collection or drug infusion. Peritoneal / ascites lavage fluid should be obtained prior to GEN-1 infusion for translational studies. For patients enrolled in the NACT+BEV+GEN-1 arm, the catheter may be removed at the clinician's discretion upon completion of GEN-1 administration.

[0403] Study Procedure

[0404] Screening Overview (Day -21 to Cycle 1 Day 1)

[0405] Written informed consent must be obtained ≤ 21 days before the start of treatment and before any specifically protocol-included procedures are performed. All eligible subjects, regardless of randomized arm, will undergo a baseline assessment before dosing.

[0406] Screening assessments will be performed after informed consent is obtained and within 21 days prior to the start of treatment. For both treatment arms, screening procedures will include a medical history, laparoscopy / biopsy, physical examination, vital signs, Eastern Cooperative Oncology Group (ECOG) performance status, ECG (electrocardiogram), laboratory tests (including serum pregnancy test and CA-125), and radiographic imaging scans. Radiographic imaging scans may be completed within 21 days prior to the start of treatment. Screening laboratory procedures may be repeated during the screening period to assess eligibility parameters.

[0407] Prior to randomization, the Principal Investigator will collect, review, and determine the acceptability of the screening assessments, and sufficient time must be provided for the subject to have their IP catheter inserted. Any changes in health assessed by physical examination or vital signs must be acceptable to the Principal Investigator before the start of study treatment.

[0408] Eligible subjects will be randomized in a 1:1 ratio to receive NACT+BEV+GEN-1 or NACT+BEV.

[0409] NACT and interval cytoreduction

[0410] All subjects will receive 7-9 cycles of standard NACT every 21 days. (See Figure 6 ). BEV will be administered on C2D1, C3D1, and on C6D1 and C7D1 after ICS (additional neoadjuvant cycles will be administered if necessary, in addition to the cycle immediately before ICS). GEN-1 will be added to the regimen of subjects in the experimental arm weekly starting on C1D15. In subjects randomized to the experimental arm, an IP port must be placed at least 7 days before GEN-1 administration to allow healing. ICS will be administered 4 weeks after the last dose of the neoadjuvant chemotherapy cycle (based on response as judged by the investigator). The remaining 3 cycles of the adjuvant treatment regimen will be started after at least a 4-week recovery period from ICS.

[0411] maintain

[0412] All subjects will receive BEV every 21 days until tolerability or unacceptable toxicity occurs, for a maximum of 18 additional cycles.

[0413] The run-in phase to ensure the safety of the NACT+BEV+GEN-1 combination will evaluate at least six subjects randomized to the experimental arms using a 3+3 design. Subjects must receive at least two cycles of NACT+BEV+GEN-1 to be evaluated for safety. Before the DSMB can recommend a Phase II dose for GEN-1, at least six subjects from the GEN-1 arm must be available for safety evaluation. Generally, fewer than two of the six subjects are likely to experience dose-limiting toxicities to proceed to Phase II. The DSMB will review the safety data of the evaluable subjects and provide recommendations to the sponsor and study chair.

[0414] The safety of the subjects will be monitored at each treatment visit (using physical examination and AE assessment) from the time of signing the informed consent until at least 30 days after the last chemotherapy or GEN-1 administration. Any suspected drug-related AE can be reported at any time during the follow-up period until it resolves to ≤ Grade 2 (CTCAE v5.0).

[0415] At screening, before ICS, and approximately 4 weeks after completing all carboplatin + paclitaxel chemotherapy, subjects will be monitored for anti-tumor activity by clinical (CA-125) and CT or MRI scans. Subjects will then be monitored clinically by CA-125 and CT / MRI every 3 months until progression. Before starting alternative treatment, investigator-determined radiographic progression (according to RECIST v.1.1) will be observed and recorded.

[0416] SLL was performed approximately 6–8 weeks (± 1 week) after day 1 of the last adjuvant chemotherapy cycle. SLL was performed at least 4 weeks after the last dose of BEV.

[0417] Evaluate

[0418] The primary endpoint for evaluating efficacy will be histopathologically confirmed MRD in the presence of SLL. For the purpose of the primary endpoint, the presence of minimal residual disease (MRD) in the presence of SLL will be defined as any histopathological or cytological evidence of viable residual cancer in SLL-derived peritoneal biopsy or washings.

[0419] The MRD rate in SLL was defined as the proportion of subjects with MRD+ (microscopic or macroscopic disease only) in SLL.

[0420] For exploratory analyses, residual disease at the time of SLL was further categorized into three categories: a) complete remission (no evidence of disease on any biopsy or peritoneal washings); b) macroscopic MRD (visible or suspicious disease at surgery confirmed by histopathological diagnosis); and c) microscopic MRD (no clear visible residual disease at the time of SLL but positive microscopic biopsy or positive peritoneal washings cytology). Fisher's exact test was used to compare the difference in the incidence of MRD between the two groups at the time of SLL positivity (including partial remission and persistent disease). Eighty percent (80%) and 95% Clopper Pearson exact confidence intervals were generated for individual treatment rates and treatment rates adjusted for continuity differences in proportions.

[0421] Secondary endpoints will include progression-free survival (PFS) and overall survival (OS). PFS is defined as the duration from randomization to investigator-assessed progression or death, whichever occurs first. CT scans may also be collected. OS is defined as the time (in months) from the date of randomization to the date of death. In subjects without confirmed death or who are still alive as of the OS cutoff date, survival time will be censored at the last study follow-up date or the cutoff date, whichever comes first.

[0422] This protocol allowed participation of subjects with both measurable and non-measurable disease and therefore deviated slightly from the standard RECIST 1.1 language with respect to the use of CA-125 to define biochemical response and progression. Note: "Response" below refers to clinical response, not "biochemical response," unless otherwise noted.

Claims

1. Combination therapy comprising: (a) a nucleic acid vector comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with a lipopolymer; and (b) an antibody or an antigen-binding fragment thereof (anti-VEGF antibody) that specifically binds to vascular endothelial growth factor (VEGF).

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

3. The combination therapy of claim 1 or 2, wherein the nucleic acid vector 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 of any one of claims 1-3, wherein the nucleic acid vector comprises an intron, a 3'UTR, an antibiotic resistance gene, or any combination thereof (eg, elements of Figure 4).

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

6. The combination therapy of any one of claims 1-5, wherein the combination further comprises an anticancer agent.

7. The combination therapy of any one of claim 6, wherein the anticancer agent is a chemotherapeutic agent.

8. The combination therapy of claim 6, wherein the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

9. The combination therapy of claim 6, wherein the anticancer agent is paclitaxel.

10. The combination therapy of claim 6, wherein the anticancer agent is carboplatin.

11. The combination therapy of claim 6, wherein the anticancer agent is docetaxel.

12. The combination therapy of claim 6, wherein the anticancer agent is nab-paclitaxel.

13. The combination therapy of claim 6, wherein the anticancer agent is olaparib.

14. The combination therapy of any one of claims 1-13, wherein the anti-VEGF antibody is selected from the group consisting of bevacizumab or ranibizumab.

15. The combination therapy of claim 14, wherein the anti-VEGF antibody is bevacizumab.

16. The combination therapy of claim 15, wherein the anti-VEGF antibody is Avastin or a biosimilar thereof.

17. The combination therapy of claim 14, wherein the anti-VEGF antibody is ranibizumab.

18. The combination therapy of claim 17, wherein the anti-VEGF antibody is Luminox or a biosimilar thereof.

19. The combination therapy of any one of claims 1-18, wherein the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence at least about 85% identical to SEQ ID NO: 1 and a variable light chain (VL) comprising an amino acid sequence at least about 85% identical to SEQ ID NO:

2.

20. The combination therapy of claim 19, wherein the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 1, and a variable light chain (VL) comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:

2.

21. The combination therapy of any one of claims 1-20, wherein the nucleic acid vector comprises a plasmid.

22. The combination therapy of any one of claims 4-21, wherein the 3'UTR comprises hGH 3'UTR.

23. A method of treating a subject having cancer, the method comprising administering to the subject the combination therapy of any one of claims 1-22.

24. The method of claim 23, wherein the nucleic acid vector formulated with the lipopolymer is administered intratumorally or intraperitoneally.

25. The method of any one of claims 23-24, wherein the nucleic acid vector formulated with the lipopolymer is administered intravenously.

26. The method of any one of claims 23-25, wherein the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravenously, intrathecally, or any combination thereof.

27. The method of any one of claims 23-26, wherein the anti-VEGF antibody is administered intravenously.

28. The method of any one of claims 23-27, wherein the nucleic acid vector formulated with the lipopolymer is administered before, simultaneously with, or after the anti-VEGF antibody.

29. The method of any one of claims 23-28, wherein the nucleic acid vector formulated with the lipopolymer is administered before, simultaneously with, or after the anticancer agent.

30. The method of any one of claims 23-29, wherein an anti-cancer agent is administered, followed by administration of the nucleic acid vector formulated with the lipopolymer, and followed by administration of the anti-VEGF antibody.

31. The method of any one of claims 23-30, wherein an anti-cancer agent is administered, followed by administration of the nucleic acid vector formulated with the lipopolymer, followed by the anti-VEGF antibody, and then surgery is performed to remove all or part of the tissue or tumor.

32. The method of claim 31, wherein the surgery is interval cytoreductive surgery.

33. The method of any one of claims 23-32, wherein an anti-cancer agent is administered, followed by administration of a DNA plasmid, followed by the anti-VEGF antibody, followed by interval cytoreductive surgery.

34. The method of any one of claims 23-33, wherein the anticancer agent is administered once every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

35. The method of any one of claims 23-34, wherein the anticancer agent is administered at least about 28 days after the interval cytoreductive surgery.

36. The method of any one of claims 23-34, wherein the anticancer agent is administered once every three weeks for about 9 weeks after the interval cytoreductive surgery.

37. The method of any one of claims 23-35, wherein administration of the anticancer agent comprises administration of paclitaxel at a dose of about 100-200 mg / m², optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

38. The method of claim 37, wherein administration of the anticancer agent comprises administration of paclitaxel at a dose of about 175 mg / m², optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

39. The method of any one of claims 23-38, wherein administration of the anticancer agent comprises administration of docetaxel at a dose of 50-100 mg / m², optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

40. The method of claim 39, wherein administration of the anticancer agent comprises administration of docetaxel at a dose of about 75 mg / m², optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

41. The method of any one of claims 23-40, wherein administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of 200-300 mg / m², optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

42. The method of claim 41, wherein administration of the anticancer agent comprises administration of nab-paclitaxel at a dose of about 260 mg / m², optionally followed by IV administration of carboplatin at a dose of about AUC 5-6.

43. The method of any one of claims 23-42, wherein administration of the nanoparticles prior to the interval cytoreductive surgery begins 15 days after the first administration of the anticancer agent and continues weekly for at least about 12 weeks to about 18 weeks.

44. The method of any one of claims 23-43, wherein the nanoparticles are administered at least about 28 days after the interval cytoreductive surgery and administration begins 15 days after the first administration of the anticancer agent and continues weekly for at least about 9 weeks.

45. The method of any one of claims 23-44, wherein the interleukin-12 (IL-12) plasmid formulated with a lipopolymer is administered at a dose of about 35 mg / m² to about 80 mg / m².

46. The method of any one of claims 23-44, wherein the interleukin-12 (IL-12) plasmid formulated with a lipopolymer is administered at a dose of about 50 mg / m² to about 100 mg / m².

47. The method of any one of claims 23-44, wherein the interleukin-12 (IL-12) plasmid formulated with a lipopolymer is administered at a dose of about 80 mg / m².

48. The method of any one of claims 1-47, wherein the lipopolymer is a nanoparticle.

49. The method of any one of claims 23-48, wherein the anti-VEGF antibody is administered weekly for at least about 12 weeks up to about 18 weeks prior to the interval cytoreductive surgery, at least about 22 days after the first administration of the anticancer agent.

50. The method of any one of claims 23-49, wherein the anti-VEGF antibody is administered weekly for at least about 9 weeks at least about 28 days after the interval cytoreductive surgery and at least about 22 days after the first administration of the anticancer agent.

51. The method of any one of claims 23-50, wherein the anti-VEGF antibody is administered IV at a dose of about 10-20 mg / kg.

52. The method of claim 51, wherein the anti-VEGF antibody is administered IV at a dose of about 15 mg / kg.

53. The method of any one of claims 23-52, wherein the interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anticancer agent.

54. The method of any one of claims 23-53, wherein the intermittent cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

55. The method of any one of claims 23-54, wherein the intermittent cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

56. The method of any one of claims 23-55, wherein the interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the anti-VEGF antibody.

57. The method of any one of claims 23-56, wherein the interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody.

58. The method of any one of claims 23-57, wherein the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer, lung cancer, and any combination thereof, and metastases of any of said cancers.

59. The method of claim 58, wherein the brain cancer is glioblastoma.

60. The method of any one of claims 23-59, wherein the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

61. The method of any one of claims 23-60, wherein the subject is a human.

Images