Combining IL-12 gene therapy with anti-VEGF to treat cancer

Abstract

The present disclosure relates to combination therapies comprising anti-VEGF antibodies, nanoparticle-formulated plasmids containing IL-12-encoding nucleic acids, and, optionally, at least one adjuvant chemotherapeutic agent, as well as methods of treatment using such combination therapies and / or compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] References to sequence listings submitted electronically via EFS-WEB The .XML file submitted with this disclosure (Name: 2437_081PC01_SequenceListing_ST26; Size: 9,811 bytes; Creation Date: September 6, 2023) is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to the fields of cancer therapy, gene therapy and immunology. [Background technology]

[0004] Ovarian cancer is the fifth most deadly type of cancer among women in the United States, killing an estimated 14,000 women annually. There are approximately 22,000 new cases of ovarian cancer each year, with the majority of cases, approximately 70%, being diagnosed at advanced stages III and IV. Epithelial ovarian cancer (EOC) is characterized by tumor dissemination in the peritoneal cavity and carries a high risk of recurrence (75%, stages III and IV) after seemingly successful surgery and chemotherapy.

[0005] IL-12 is one of the most active cytokines for stimulating immune responses against cancer. However, when administered as a recombinant protein, the pharmacokinetics of IL-12 necessitate frequent bolus injections, resulting in severe toxicity that limits its use. GEN-1 is an IL-12 DNA plasmid vector formulated using a lipopolymer delivery system. GEN-1 can be delivered locally (e.g., intraperitoneally), offering the possibility of cytokine expression specifically in the tumor microenvironment, with the aim of achieving increased 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.

[0006] While traditional chemotherapy regimens are designed to inhibit tumor growth through cytotoxic mechanisms, immunocytokine therapy is designed to induce tumor death by enhancing the immune system against cancer cells. GEN-1 mitigates the toxicity issues associated with IL-12. Its nanoparticle profile allows for cell transfection and subsequent sustained local secretion of IL-12 at therapeutic levels while avoiding the toxicity associated with recombinant IL-12.

[0007] There remains a need for treatments for ovarian cancer, including those diagnosed with EOC and recurrent ovarian cancer. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the difference in tumor weight (mg) compared to the control group after administration of mGEN-1, various dose levels of bevacizumab, and mGEN-1 in combination with various dose levels of bevacizumab.

[0009] [Figure 2]FIG. 11 shows different tumor burden levels after tumor implantation in untreated nude Foxn1nu mice, nude Foxn1nu mice treated with doxorubicin (Doxil) + bevacizumab, and nude Foxn1nu mice treated with Doxil + bevacizumab + mGEN-1, plotted by IVIS signal quantification.

[0010] [Figure 3] FIG. 10 shows different tumor burden levels after tumor implantation in untreated nude Foxn1nu mice, nude Foxn1nu mice treated with Doxil+bevacizumab, and nude Foxn1nu mice treated with Doxil+bevacizumab+mGEN-1, as shown by IVIS whole-body imaging.

[0011] [Figure 4] FIG. 1 shows an exemplary hIL-12 expression plasmid.

[0012] [Figure 5] FIG. 1 shows the PEG-PEI-cholesterol structure.

[0013] [Figure 6] FIG. 1 shows the administration schedules for the neoadjuvant chemotherapy (NACT) + bevacizumab group and the NACT + bevacizumab + GEN-1 group of the clinical protocol.

[0014] [Figure 7] FIG. 1 shows an outline of the schedule for the NACT + bevacizumab group and the NACT + bevacizumab + GEN-1 group of the clinical protocol. Summary of the Invention

[0015] 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 in a lipopolymer (e.g., a nanoparticle), and (ii) an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0016] Certain aspects of the present disclosure relate to methods of treating a subject suffering from cancer, 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 in a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0017] In some embodiments, the polynucleotide encodes human IL-12.

[0018] In some embodiments, the nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL12.

[0019] In some embodiments, the promoter is a CMV promoter.

[0020] In some embodiments, the nucleic acid vector (eg, a plasmid) comprises an intron, a 3'UTR (eg, an hGH 3'UTR), an antibiotic resistance gene, or any combination thereof (eg, elements of Figure 4).

[0021] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to cholesterol and polyethylene glycol (PEG) groups (eg, the lipopolymer of Figure 5).

[0022] In some embodiments, the combination further comprises an anti-cancer agent.

[0023] In some embodiments, the anti-cancer agent is a chemotherapeutic agent.

[0024] In some embodiments, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0025] In some embodiments, the anti-cancer agent is doxorubicin.

[0026] In some embodiments, the anti-cancer agent is paclitaxel.

[0027] In some embodiments, the anti-cancer agent is carboplatin.

[0028] In some embodiments, the anti-cancer agent is docetaxel.

[0029] In some embodiments, the anti-cancer agent is nab-paclitaxel.

[0030] In some embodiments, the anti-cancer agent is olaparib.

[0031] In some embodiments, 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).

[0032] In some embodiments, the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO:1 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO:1), and a variable light chain (VL) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO:2 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO:2).

[0033] In some embodiments, the method further comprises surgery to remove all or part of the tissue or tumor in the subject (eg, interval cytoreductive surgery).

[0034] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered intratumorally or intraperitoneally.

[0035] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered intravenously.

[0036] In some embodiments, the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravenously, intravesically, or any combination thereof.

[0037] In some embodiments, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0038] In some embodiments, the anti-VEGF antibody is administered intravenously.

[0039] In some embodiments, the anti-VEGF antibody is administered intravesically.

[0040] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

[0041] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered prior to, concurrently with, or after the anti-cancer agent.

[0042] In some embodiments, an anti-cancer agent is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third).

[0043] In some embodiments, an anti-cancer drug is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third), followed by surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) (e.g., fourth).

[0044] In some embodiments, an anti-cancer drug is administered, followed by a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0045] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0046] In some embodiments, the anti-cancer agent is administered at least about 28 days after interval cytoreductive surgery (eg, every 3 weeks for about 8-10 weeks, eg, 9 weeks).

[0047] In some embodiments, the administration of the anticancer agent comprises paclitaxel at a dose of about 100 to 200 mg / m 2 (e.g., about 175 mg / m 2 ) optionally followed by carboplatin, for example at a dose of about AUC 5-6 IV.

[0048] In some embodiments, the administration of the anticancer drug is docetaxel at a dose of about 50 to 100 mg / m 2 (e.g., about 75 mg / m 2 ) optionally followed by carboplatin, for example at a dose of about AUC 5-6 IV.

[0049] In some embodiments, the administration of the anticancer drug is nab-paclitaxel at a dose of about 200-300 mg / m 2 (e.g., about 260 mg / m 2 ) optionally followed by carboplatin, for example at a dose of about AUC 5-6 IV.

[0050] In some embodiments, administration of nanoparticles prior to interval cytoreductive surgery begins 14 to 18 days (e.g., 15 days) after the first administration of the anticancer agent, and occurs weekly for at least about 12 weeks to about 18 weeks.

[0051] In some embodiments, the nanoparticles are administered at least about 28 days after interval cytoreductive surgery, and administration begins 15 days after the first administration of the anti-cancer agent and occurs weekly for at least about 9 weeks.

[0052] In some embodiments, interleukin-12 (IL-12) formulated in lipopolymers (e.g., nanoparticles) is administered at a concentration of about 35 mg / m 2 ~about 80mg / m 2 is administered at a dose of

[0053] In this embodiment, the interleukin-12 (IL-12) plasmid formulated in lipopolymer (e.g., nanoparticles) is administered at a concentration of about 80 mg / m 2 is administered at a dose of

[0054] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 12 weeks and up to about 18 weeks, beginning at least about 22 days after the first administration of the anti-cancer agent, prior to interval cytoreductive surgery.

[0055] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 9 weeks, beginning at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0056] In some embodiments, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV (eg, about 15 mg / kg IV).

[0057] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-cancer agent.

[0058] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid.

[0059] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid.

[0060] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days prior to administration of the anti-VEGF antibody.

[0061] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-VEGF antibody.

[0062] In some embodiments, 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, as well as metastasis of any cancer.

[0063] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0064] In some embodiments, the subject is a human. DETAILED DESCRIPTION OF THE INVENTION

[0065] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0066] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this 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 invention will be apparent from the detailed description and claims.

[0067] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one," can be used interchangeably herein. In certain embodiments, the term "a" or "an" means "single." In other embodiments, the term "a" or "an" includes "two or more" or "plural."

[0068] 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 that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10% above or below (even higher or lower).

[0069] Throughout this disclosure, various aspects are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose 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 individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. The numerical ranges described are inclusive of the numbers defining the range and include each integer within the defined range.

[0070] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is understood that each intervening integer and each fractional integer between the stated upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can be independently included in or excluded from the range, and each range in which either, neither, or both limits are included is also encompassed within the present disclosure. Thus, ranges described herein are understood to be shorthand for all values ​​within the range, including the stated 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.

[0071] Where a value is explicitly recited, it is understood that values ​​of approximately the same quantity or amount as the recited value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and within the scope of the present disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of the present disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded alone or in any combination with other alternatives are also hereby disclosed. More than one element of the present disclosure may have such an exclusion, and all combinations of elements having such an exclusion are disclosed herein.

[0072] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of 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 encompass each of the following embodiments: 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).

[0073] Whenever an embodiment is described herein using the phrase "comprising," it is understood that other similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0074] As used herein, the term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount or quantity of a drug or pharmaceutically active substance sufficient to elicit the necessary or desired therapeutic response, in other words, an amount sufficient to elicit an appreciable biological response when administered to a patient.

[0075] The term "transfect" or "transfection" refers to the transport of nucleic acids from the external environment of a cell to the intracellular environment, particularly with respect to the cytoplasm and / or nucleus. Without being bound by any particular theory, it should be understood that nucleic acids can be delivered to a cell after being encapsulated within, attached to, or incorporated into one or more cationic polymer / nucleic acid complexes. A specific transfection example delivers nucleic acids to the nucleus of a cell. Nucleic acids include DNA and RNA and their synthetic analogs. Such nucleic acids include missense, antisense, nonsense, and proteinogenic nucleotides, as well as on / off rate-regulating nucleotides that control the production of proteins, peptides, and nucleic acids. In particular, but not limited to, they can be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences, or synthetic or semisynthetic sequences, and can be of natural or artificial origin. Furthermore, nucleic acids can vary in size, ranging from oligonucleotides to chromosomes. These nucleic acids can be of human, animal, plant, bacterial, viral, or synthetic origin. They can be obtained by any technique known to those skilled in the art.

[0076] As used herein, the term "pharmaceutical agent" or "drug" or any other similar term means any chemical or biological material or compound suitable for administration by methods previously known in the art and / or taught in this disclosure that induces a desired biological or pharmacological effect, including, but not limited to, (1) having a prophylactic effect on an organism and preventing an undesired biological effect, e.g., preventing infection; (2) alleviating a condition caused by disease, e.g., alleviating pain or inflammation caused as a result of disease; and / or (3) alleviating, mitigating, or completely eliminating disease from an organism. The effect may be local, such as providing a local anesthetic effect, or may be systemic.

[0077] As used herein, the term "biocompatible" or "biodegradable" is defined as the conversion of a material into simpler intermediate or end products by solubilizing hydrolysis or by the action of biologically formed entities, which may be enzymes and other products of organisms.

[0078] As used herein, "effective amount" means that amount of nucleic acid or bioactive agent sufficient to provide the desired local or systemic effect and performance at a reasonable risk / benefit ratio such as is consistent with any medical treatment.

[0079] As used herein, "peptide" means a peptide of any length and includes proteins. The terms "polypeptide" and "oligopeptide" are used herein without any particular intended size limitation unless a particular size is otherwise specified.

[0080] As used herein, a "derivative" of a carbohydrate is, for example, an acid form of a sugar, such as glucuronic acid; an amine of a sugar, such as galactosamine; a phosphate of a sugar, such as mannose-6-phosphate; and the like.

[0081] As used herein, " administering " and similar terms refer to delivering the composition to the individual being treated so that the composition can circulate systemically, bind to target cells, and be taken up by endocytosis.Therefore, the composition is preferably administered to the individual systemically, typically by subcutaneous, intramuscular, transdermal, intravenous or intraperitoneal route.The injection for such use can be prepared in a conventional form, as a liquid solution or suspension, or as a solid form suitable for preparing as a liquid solution or suspension before injection, or as an emulsion.Suitable excipients that can be used for administration include, for example, water, saline, dextrose, glycerol, ethanol, etc., and optionally include small amounts of auxiliary substances such as wetting agents or emulsifiers, buffers, etc.

[0082] As used herein, "efficacy" and similar terms mean tumor disappearance or tumor shrinkage or reduction in tumor density or increase in lymphocyte count or increase in neutrophil count or improved survival, or all of the above.

[0083] As used herein, "toxicity" is defined as any treatment-related adverse effect on clinical findings, including but not limited to abnormal hematology or serum chemistry results or organ toxicity.

[0084] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary to express a gene product operably linked to the promoter / regulatory sequence. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies 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, when operably linked to a polynucleotide that encodes a particular gene product, essentially means that an inducer corresponding to the promoter will result in the production of the gene in a cell only when the nucleotide sequence of the product is present in the cell.

[0085] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, e.g., a polypeptide. This process includes any expression of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. It includes, but is not limited to, the transcription of a gene into messenger RNA (mRNA) and the translation of such mRNA into a polypeptide. Expression of a gene results in a "gene product."

[0086] As used herein, a gene product can be either a nucleic acid, e.g., messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcription product. Gene products as described herein further include nucleic acids that have post-transcriptional modifications, e.g., polyadenylation, or polypeptides that have post-translational modifications, e.g., methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, etc.

[0087] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An 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 a recombinant polynucleotide.

[0088] As used herein, the term "operably linked" or "transcriptional control" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which 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 placed in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operable linked DNA sequences can be adjacent to each other; for example, when two protein coding regions need to be linked, the DNA sequences are in the same reading frame.

[0089] As used herein, the term "transfer vector" refers to a composition containing 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 ionic or amphiphilic compounds, plasmids, and viruses. The term transfer vector should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes.

[0090] As used herein, the term "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In certain 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 due, for example, to mutations or environmental influences that may occur in subsequent generations, or to integration of the nucleic acid molecule into the host cell genome.

[0091] "Percent (%) amino acid sequence identity" with respect to the polypeptide sequences described herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific polypeptide sequence 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, without considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed according to procedures known in the art, for example, as described in EP 1241179 (in particular, pages 9, lines 35 to 10, lines 40, and the definitions used therein, as well as Table 1 regarding possible conservative substitutions, which are incorporated herein by reference). For example, one skilled in the art can use publicly available computer software. Computer program methods for determining sequence identity include, but are not limited to, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. According to one embodiment, the software alignment program used can be BLAST. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. According to one embodiment, percent identity values ​​can be generated using the WU-BLAST-2 computer program (Altschul et al., 1996, Methods in Enzymology, 266:460-480, incorporated herein by reference). According to one embodiment, the following parameters are used when running the WU-BLAST-2 computer program, with most of the WU-BLAST-2 search parameters set to default values. Adjustable parameters were set to the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11, and score matrix=BLOSUM62.The HSP S and HSP S2 parameters used by BLAST-2 are dynamic values ​​established by the program itself depending on the composition of the sequence of interest and the structure of the database being searched. However, the values ​​can be adjusted to increase sensitivity. The percent sequence identity can be determined by dividing (a) the number of identical amino acid residues between the specific amino acid sequence described herein (e.g., a specific polypeptide sequence identified by a sequence identifier in the Sequence Listing) and the candidate amino acid sequence being compared, e.g., the number of identical amino acid residues as determined by WU-BLAST-2, by (b) the total number of amino acid residues in the polypeptide sequence described herein (e.g., a specific polypeptide sequence identified by a SEQ ID NO: in the Sequence Listing).

[0092] "Percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences described herein is defined as the percentage of nucleotides in a candidate sequence being compared that are identical to nucleic acids in a specific nucleic acid sequence described herein (e.g., a particular 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.

[0093] As used herein, the term "homology" or "identity" refers to the identity of subunit sequences between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in two molecules is occupied by the same monomer subunit, e.g., if each position in 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 two sequences (e.g., position 5 in a polymer 10 subunits long) are homologous, then the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

[0094] In the context of two or more nucleic acid or polypeptide sequences, percent identity refers to two or more sequences that are the same. Two sequences are "substantially the same" if, when compared and aligned for maximum correspondence within a comparison window or designated region, they have a certain percentage of identical amino acid residues or nucleotides (e.g., 60% identity, 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 over a designated region, or, if not specified, over the entire sequence), as measured by using one of the following sequence comparison algorithms or by manual alignment and visual inspection. In some cases, identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length (or 20, 50, 200 or more amino acids). For sequence comparison, a sequence typically serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, and subsequence coordinates and sequence algorithm program parameters are designated, if necessary. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Methods of sequence alignment for comparison are well known in the art, as disclosed above.

[0095] A "coding sequence," or a sequence "encoding" 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, such as 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' (carboxy) terminus. A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid but is considered to be part of the coding region; any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region.

[0096] A coding sequence can include, but is 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 usually located 3' to the coding sequence.

[0097] As used herein, the term "recombinant DNA / RNA technology" refers to the manipulation of nucleic acid sequences outside of an organism. This technology includes, but is not limited to, combining nucleic acid sequences (e.g., coding sequences, regulatory elements (e.g., promoters, enhancers, silencers, termination sequences), linkers (e.g., spacers, internal ribosome entry sites, cleavage sites) from various sources, inserting nucleic acid sequences from various sources into appropriate vectors (e.g., delivery vectors, expression vectors, integration vectors), altering or modifying nucleotide sequences (e.g., by mutagenesis, insertion of modified nucleotides, 5' capping, polyadenylation), and synthesizing artificial nucleotide sequences. Nucleic acid sequences can be manipulated outside of an organism using a variety of techniques well known in the art (e.g., molecular cloning, polymerase chain reaction (PCR), restriction enzyme digestion, 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. (See, e.g., Green & Sambrook, "Molecular Cloning: A Laboratory Manual," volumes 1-3, 4th edition).

[0098] As used herein, the term "recombinant" refers to any nucleic acid (e.g., DNA or RNA), peptide (e.g., oligopeptide, polypeptide, or protein), cell, or organism that is produced by combining genetic material from two or more different sources. In some embodiments, a recombinant nucleic acid, peptide, cell, or organism contains a portion of genetic material from at least one source. In some embodiments, a "recombinant DNA" molecule can comprise a DNA molecule derived from one organism and inserted into a host organism to generate a new genetic combination. In some embodiments, a "recombinant RNA" molecule (e.g., a recombinant mRNA molecule) can comprise an RNA molecule derived from one organism and inserted into a host organism to result in expression of a desired gene product in the host organism. In some embodiments, a "recombinant peptide" molecule can comprise an amino acid molecule derived from an organism or cell that is expressed from a recombinant nucleic acid molecule.

[0099] As used herein, the term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as a host cell.

[0100] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0101] As used herein, the term "primary tumor" refers to the original or first tumor that formed in a subject's body.

[0102] As used herein, the terms "metastasis," "metastatic," "secondary tumor," or "metastatic tumor" refer to a cancer (e.g., tumor) formed by cancer cells derived from a primary cancer (e.g., tumor) that spreads to an additional location or area of ​​the body.

[0103] As used herein, the term "specifically binds" refers to an antigen-binding molecule that recognizes and binds to a binding partner protein (such as a tumor antigen) present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0104] As used herein, the term "tumor heterogeneity" refers to the molecular biological or genetic changes that occur in tumor daughter cells after multiple divisions and proliferation during tumor growth, resulting in differences in tumor growth rate, invasive ability, drug sensitivity, prognosis, and other aspects. This is one of the characteristics of malignant tumors.

[0105] As used herein, the term "cancer" refers to a broad group of 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 that can infiltrate adjacent tissues through local spread and metastasize to distant parts of the body via the lymphatic system or bloodstream. In some embodiments, the methods of the present disclosure can be used to reduce the size of or treat primary or metastatic tumors. Conditions that can be treated or prevented by the methods of the present disclosure include various neoplasms, including, for example, benign or malignant tumors, various hyperplasias, and the like. The methods of the present disclosure can achieve inhibition and / or reversal of the unwanted hyperproliferative cell growth involved in such conditions. In some embodiments, the cancer can be ovarian cancer.

[0106] As used herein, "ovarian cancer" refers to cancer that originates in or involves the ovaries, e.g., originates in the ovarian epithelium. As used herein, the terms "cancer" or "tumor" refer to the uncontrolled growth of cells that interfere with the normal function of bodily organs and systems. A subject with cancer or tumor is one who has objectively measurable cancer cells present in the subject's body. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer that migrates from its original location and disseminates to vital organs can ultimately lead to the subject's death through functional deterioration of the affected organ. Ovarian cancer is typically treated by cytoreductive surgery (also referred to herein as "debulking") 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 subject's health, and complex factors that can be assessed by one skilled in the art. In some embodiments, cytoreductive surgery can remove at least 10% of the tumor tissue, e.g., 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.

[0107] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. A cell includes the primary subject cell and its progeny.

[0108] 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. A refractory cancer is also referred to as a resistant cancer. In some aspects, refractory or recurrent malignant tumors can be treated using the methods disclosed herein.

[0109] As used herein, "relapsed" as used herein refers to the return of signs and symptoms of a disease (e.g., cancer) during a period of improvement after therapy, such as a previous treatment of cancer therapy, or the recurrence of a disease, such as cancer.

[0110] As used herein, the term "combination therapy" refers to a therapy that includes at least a gene therapy, an anti-cancer agent, and an antibody with binding specificity for VEGF, which can be administered together or separately. In some embodiments, the compositions of the combination therapy are formulated together as a single composition or as separate compositions.

[0111] As used herein, the terms "treat," "treated," and "treating" refer to both therapeutic and prophylactic treatments or preventative measures, where the objective is to reverse, alleviate, ameliorate, reduce, inhibit undesirable symptoms, complications, conditions, biochemical manifestations of a disorder, or disease; delay the progression, onset, severity, or recurrence of the condition; or achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder, or disease; a stabilized (i.e., non-worsening) state of the condition, disorder, or disease; a delayed onset or delayed progression of the condition, disorder, or disease; an improvement or remission of the condition, disorder, or disease state, whether detectable or undetectable (whether partial or total); an improvement in at least one measurable physical parameter, not necessarily discernible by the patient; or an improvement or amelioration of the condition, disorder, or disease. In some embodiments, treatment involves eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment involves prolonging survival compared to the expected survival if not receiving 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 "prevent" or "prevention" refers to delaying or preventing the onset, development, or progression of a condition or disease over a period of time, including weeks, months, or years. As used herein, the term "prophylactic" (e.g., "prophylactic agent," "prophylactic treatment," "prophylactically effective amount") refers to the complete or partial prevention of a disease or its symptoms, and / or may be therapeutic with respect to a partial or complete cure of the disease and / or adverse effects and / or symptoms resulting from the disease.

[0112] As used herein, the terms "individual" and "subject" have the same meaning herein and may be humans and other species of animals. As used herein, the terms "subject" and "patient" are used interchangeably. A subject may be an animal. In some embodiments, a 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 embodiments, a subject is a human. In some embodiments, a patient is a subject who has a disease, disorder, or condition, or is at risk of suffering from a disease, disorder, or condition, or who is otherwise in need of the compositions and methods provided herein.

[0113] 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, preparation, substance, or composition effective to achieve a particular biological result described herein, such as, but not limited to, treating or reducing the growth of cancer or tumors. When referring to an "immunologically effective amount," an "anti-tumor effective amount," a "tumor suppression effective amount," or a "therapeutically effective amount," the exact number 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, extent 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, a number of immune effector cells that 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; or promote tumor regression, tumor shrinkage, and / or tumor necrosis.

[0114] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, formulations and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0115] The term "excipient" refers to any substance that is not itself a therapeutic agent, which can be used in a composition for delivering an active therapeutic agent to a subject, or can be combined with an active therapeutic agent to improve its handling or storage characteristics, or to enable or facilitate the formation of a dosage unit of the composition (e.g., to create a pharmaceutical composition). Excipients include, but are not limited to, solvents, penetration enhancers, humectants, antioxidants, lubricants, emollients, substances added to improve the appearance or texture of the composition, and substances used to form hydrogels. Any such excipient can be used in any dosage form according to the present disclosure. The foregoing classes of excipients are not meant to be exhaustive but merely exemplary, as one of ordinary skill in the art will recognize that additional types and combinations of excipients can be used to achieve the desired goal of drug delivery. Excipients can be inert, inactive, and / or pharmaceutically non-active substances. Excipients can serve a variety of purposes.

[0116] Those skilled in the art can select one or more excipients for specific desired properties without undue burden through routine experimentation. The amount of each excipient used can vary within a range conventional in the art. The techniques and excipients that can be used to formulate dosage forms are described in "Handbook of Pharmaceutical Excipients" 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009) and "Remington: The Science and Practice of Pharmacy" 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0117] As used herein, the term "immune response" refers to a biological response within an organism to foreign substances or abnormal cells (e.g., tumor cells), which response protects the organism from such agents / cells and the diseases they cause. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the organism of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. In some embodiments, the immune response includes, for example, the activation or inhibition of T cells, e.g., effector T cells or Th cells, e.g., CD4+ or CD8+ T cells, or the inhibition of regulatory T cells (Treg cells).

[0118] As used herein, the term "autologous" refers to any material originating from an individual that is later reintroduced into the same individual.

[0119] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0120] Papain digestion of an intact antibody produces two identical antigen-binding fragments called "Fab" fragments, each containing the heavy and light chain variable domains (VH and VL, respectively) and the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Thus, the term "Fab fragment" refers to an antibody fragment containing a light chain containing the VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains.

[0121] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, as determined, for example, by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0122] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. 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.

[0123] 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 native antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) that contain complementarity-determining regions (CDRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).

[0124] As used interchangeably herein, the terms "paratope" and "antigen-binding site" refer to the portion of an antibody that recognizes and binds to an antigen. The antigen-binding site is formed by several individual amino acid residues from the heavy and light chain variable domains of an antibody arranged in close spatial proximity in the tertiary structure of the Fv region. In one embodiment, the antigen-binding site is defined as the set of six CDRs contained in a cognate VH / VL pair.

[0125] As used herein, the term "complementarity-determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and contains antigen-contacting residues. Generally, antibodies contain 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, CDR residues and other residues in the variable domains (e.g., FR residues) 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).

[0126] For purposes of this specification, an "acceptor human framework" 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.

[0127] As used herein, "framework" or "FR" refers to variable domain amino acid residues other than CDR residues. The framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR amino acid sequences generally appear in the following order: (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.

[0128] Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine-knot family of growth factors. Typically, VEGF refers to any native VEGF from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed VEGF, as well as any form of VEGF resulting from intracellular processing. The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants. A VEGF dimer refers to a homodimer of two identical VEGF molecules. A complex formed by two identical antibody molecules bound to a VEGF dimer is referred to herein as a VEGF dimer-antibody complex.

[0129] The "first and second antigen-binding sites" contained in a VEGF dimeric antibody complex refer to the antigen-binding sites contained in each VH / VL pair of the two antibodies contained in the VEGF dimeric antibody complex. For example, the antigen-binding site of one of the two anti-VEGF antibodies in the VEGF dimeric antibody complex is the "first antigen-binding site," and the antigen-binding site of the other of the two anti-VEGF antibodies is automatically the "second antigen-binding site."

[0130] VEGF stimulates cellular responses by binding to cell surface tyrosine kinase receptors (VEGF receptors, or "VEGFRs"), causing them to dimerize and become activated by transphosphorylation, albeit 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) and interacts with domain 2 of VEGF-R1, while VEGFR-2 (KDR / Flk-1) interacts with domains 2 and 3 of VEGF-R2.

[0131] As used herein, the "VEGF-R1 binding region" and "VEGF-R2 binding region" of a VEGF molecule or VEGF dimer refer to the amino acids on VEGF that interact with domain 2 of VEGF-R1 or domain 2 or 3 of VEGF-R2, respectively.

[0132] As used herein, the terms "anti-VEGF antibody" and "antibody that binds to VEGF" refer to an antibody or antigen-binding fragment thereof that can bind to VEGF with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting VEGF. In one embodiment, the extent of binding of an anti-VEGF antibody to unrelated, non-VEGF proteins is less than about 10% of the antibody binding to VEGF, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to VEGF has a dissociation constant (KD) of less than 1 nM or less than 0.15 nM. An antibody is said to "specifically bind" to VEGF if it has a KD of 1 μM or less.

[0133] "VEGF blocking selectivity" is used herein as an abbreviation to refer to the property of an anti-VEGF antibody that, when bound to a VEGF dimer, preferentially inhibits VEGF binding to VEGF-R2 but not to VEGF-R1. An anti-VEGF antibody that can completely block VEGF binding to VEGF-R2, but not to VEGF-R1, is considered to selectively block VEGF signaling through VEGF-R2, but not through VEGF-R1, i.e., exhibits "VEGF blocking selectivity."

[0134] As used herein, "affinity" refers to the strength of the sum total of 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 expressed by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific exemplary and representative embodiments for measuring binding affinity are described herein.

[0135] As used herein, the term "epitope" refers to a site on either a proteinaceous or non-proteinaceous antigen to which an anti-VEGF antibody binds. An epitope can be formed from a contiguous stretch of amino acids (linear epitope) or can include discontinuous amino acids (conformational epitope), which are spatially adjacent due to, for example, antigen folding, i.e., tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by anti-VEGF antibodies after exposure of the proteinaceous antigen to a denaturing agent, whereas conformational epitopes are typically destroyed by treatment with a denaturing agent. 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.

[0136] Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods routine 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)).

[0137] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows for the identification of a large number of monoclonal antibodies that specifically bind to VEGF based on the binding profile of each antibody to a large number of chemically or enzymatically modified antigen surfaces (see, e.g., U.S. Patent Application Publication No. 2004 / 0101920). Antibodies in each bin bind to the same epitope, which may be a unique epitope distinct from or partially overlapping with an epitope represented by another bin. Furthermore, competitive binding can be used to easily 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 refers to an antibody that blocks the binding of the reference anti-VEGF antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Also, for example, to determine whether an antibody binds to the same epitope as a reference anti-VEGF antibody, the reference antibody is bound to VEGF under saturating conditions. After removing excess reference anti-VEGF antibody, the ability of the anti-VEGF antibody in question to bind to VEGF is evaluated. If the anti-VEGF antibody can 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 cannot bind to VEGF after saturation binding of the reference anti-VEGF antibody, it may bind to the same epitope as the epitope bound by the reference anti-VEGF antibody. To determine whether the antibody in question binds to the same epitope or whether binding is simply hindered by steric reasons, routine experiments can be used (e.g., peptide mutation and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two settings, i.e., in a setting where both antibodies are saturating antibodies.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 binding to VEGF.

[0138] Sometimes, two antibodies are considered to have the same epitope if essentially every amino acid mutation in the antigen that reduces or eliminates binding of one antibody also reduces or eliminates binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0139] 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, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, and a reduction in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent or reduce the frequency of tumor formation.

[0140] As used herein, the terms "chemotherapy" or "chemotherapeutic agent" refer to a wide variety of chemotherapeutic agents that may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to allude to a compound or composition that is administered in the treatment of cancer.

[0141] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. The composition may be sterile.

[0142] 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.

[0143] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art, and such techniques are fully explained in the literature.

[0144] II.GEN-1 (IL-12 DNA nanoparticles) In animal models, recombinant IL-12 has been demonstrated to induce significant T cell-mediated antitumor effects, resulting in the regression of established tumors and subsequent systemic immune memory. (See The Oncologist, 1996, Vol. 1, 88.) However, systemic administration of recombinant IL-12 has resulted in dose-limiting toxicity in several experimental studies and in the first human trials. (See Lab Invest., 1994, Vol. 71, 862; Science, 1995, Vol. 270, 908; J. Interferon Cytokine Res., 1995, Vol. 14, 335.) Dose-limiting toxicity was also observed with intraperitoneal administration of recombinant IL-12 in a recent human clinical trial. (Clin. Cancer Res., 2002, Vol. 8, 3686.) Gene delivery techniques capable of delivering therapeutic levels of IL-12 locally to tumor sites would have the advantage of producing anticancer responses without causing systemic toxicity.

[0145] Both viral and nonviral gene delivery systems have been used for IL-12 gene delivery in animal models of cancer. Viral approaches have serious practical limitations due to toxicity concerns, primarily due to increased cancer incidence and strong immune responses to viral antigens by the host system. Considerable interest has been shown in developing nonviral gene delivery systems due to lower toxicity. The use of polyvinylpyrrolidone (PVP), a nonviral gene delivery system, has been demonstrated to deliver IL-12 to treat renal carcinoma (Renca) and colon cell carcinoma (CT26). See Gene Ther., 1999, vol. 6, 833. When tumors were subjected to this gene therapy, they displayed all the hallmarks of IL-12 protein therapy, including increased infiltration of NK cells, CD4 and CD8 T cells, coupled with increased expression of major histocompatibility complex (MHC) class I molecules. IL-12 gene delivery was well tolerated and highly effective in both Renca and CT26 tumor-bearing animals. Tumor-rejecting mice were also protected from subsequent rechallenge, suggesting the presence of long-lasting systemic immunity. Functionalized, low-toxicity, water-soluble lipopolymers (WSLPs) have been tested for IL-12 gene delivery to CT26 colon cancer tumors. See Mahato et al., Mol. Ther., 2001, vol. 4, 130. IL-12 plasmid (pIL-12) and WSLP (pIL-12 / WSLP) treatment resulted in higher levels of intratumoral gene expression than naked DNA.

[0146] Interleukin-12 (IL-12) is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16:495-521 (1998). IL-12 functions primarily as a 70 kDa heterodimeric protein consisting of two disulfide-linked p35 and p40 subunits. IL-12 p40 homodimers exist but do not appear to mediate biological responses other than functioning as antagonists that bind to the IL-12 receptor. Ibid. The precursor form of the IL-12 p40 subunit (NM_002187; P29460; also known as IL-12B, natural killer cell-stimulating factor 2, and 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-stimulating factor 1, and cytotoxic lymphocyte maturation factor 1) is 219 amino acids long, and the mature form is 197 amino acids long. Id. 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 upon antigen stimulation. The active IL-12 heterodimer is formed after protein synthesis. Id.

[0147] Due to its ability to activate both NK cells and cytotoxic T cells, IL-12 protein has been investigated as a promising anticancer therapeutic agent since 1994 (see Nastala, C. L. et al., J Immunol 153:1697-1706 (1994)). However, despite high expectations, early clinical trials did not produce satisfactory results (see Lasek, W. et al., Cancer Immunol Immunother 63:419-435, 424 (2014)). Repeated administration of IL-12 in most patients resulted in an adaptive response and a gradual decline in IL-12-induced interferon-gamma (IFN-γ) levels in the blood (see Ibid.). Furthermore, although it is recognized that IL-12-induced anticancer activity is primarily mediated by secondary IFN-γ secretion, simultaneous induction of IFN-γ by IL-12 with other cytokines (e.g., TNF-α) or chemokines (IP-10 or MIG) has been associated with severe toxicity. Same as above.

[0148] In addition to negative feedback and toxicity, the limited effectiveness of IL-12 therapy in clinical settings may be caused by the strongly immunosuppressive environment in humans.

[0149] Furthermore, secondary effects of cytokine IL-12 production, namely IFN-γ and nitric oxide (NO) levels, were also higher in WSLP-treated tumors compared with naked DNA. A single injection of pIL-12 / WSLP complexes resulted in suboptimal effects on tumor growth and animal survival, whereas repeated delivery resulted in better efficacy, indicating insufficient delivery by the system. J. Control Release 2003, vol. 87, 177. Similarly, intratumoral injection of IL-12 plasmid in another polymeric carrier, PAGA, resulted in only partial inhibition of CT26 tumors. Gene Ther. 2002, vol. 9, 1075. These results underscore the need for more efficient delivery systems. Despite previous limited preclinical studies, the excellent molecular flexibility of polymeric gene carriers allows for complex modifications and novel functionalization, which are essential for the development of more efficient gene delivery systems.

[0150] To achieve the desired results from combination approaches involving gene therapy, it is important to select an appropriate gene delivery system. The gene delivery system used in the aforementioned combination experiment (Molecular Therapy, 2004, vol. 9, 829) was the water-soluble lipopolymer PEI-cholesterol (WSLP).

[0151] In some embodiments, the DNA plasmid further encodes a synthetic polymer that facilitates plasmid delivery that is a lipopolymer.

[0152] In some embodiments, the lipopolymer further comprises polyethyleneimine (PEI) independently covalently attached to cholesterol and polyethylene glycol (PEG) groups.

[0153] The present disclosure provides a polymer system, PEG-PEI-cholesterol (PPC), which differs from WSLP (PEI-cholesterol) in that it contains a PEG moiety, resulting in significantly higher transfection efficiency in tumors. The addition of PEG is designed to enhance the stability of the nucleic acid / polymer complex in biological environments, circumventing this deficiency in the prior art (WSLP). Furthermore, the addition of PEG chains allows for the incorporation of ligands onto the PPC chains to improve tissue selectivity of delivery. For example, the cholesterol moiety, which is directly linked to the PEI backbone in the prior art (WSLP), can be extended farther from the PEI backbone to create a more flexible geometry for cell receptor interaction. Controlling the number of PEG molecules per unit of the PEI backbone is important to achieve optimal enhancement of transfection activity. The preferred composition range was a PEG:PEI molar ratio of 2 to 4 at a constant cholesterol content. The optimal PEI to cholesterol ratio was 1:0.5 to 1:1.

[0154] 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 in a lipopolymer (e.g., a nanoparticle), and (ii) an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0155] Certain aspects of the present disclosure relate to methods of treating a subject suffering from cancer, 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 in a lipopolymer (e.g., a nanoparticle); and (ii) an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0156] In some embodiments, the polynucleotide encodes human IL-12. In some embodiments, the polynucleotide encodes the p35 and p40 IL-12 subunits.

[0157] In some embodiments, human IL-12 p35 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 3. In some embodiments, human IL-12 p40 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 4.

[0158] In some embodiments, the human IL-12 p35 comprises the following sequence: [ka]

[0159] In some embodiments, the human IL-12 p40 comprises the following sequence: [ka]

[0160] In some embodiments, the polynucleotide encoding human IL-12 p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 5. In some embodiments, the polynucleotide encoding human IL-12 p35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to SEQ ID NO: 6.

[0161] In some embodiments, the polynucleotide encoding human IL-12 p35 comprises the following sequence: [ka]

[0162] In some embodiments, the polynucleotide encoding human IL-12 p35 comprises the following sequence: [ka]

[0163] In some embodiments, the nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL12.

[0164] In some embodiments, the nucleic acid vector (eg, a plasmid) comprises an intron, a 3'UTR (eg, an hGH 3'UTR), an antibiotic resistance gene, or any combination thereof (eg, elements of Figure 4).

[0165] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to cholesterol and polyethylene glycol (PEG) groups (eg, the lipopolymer of Figure 5).

[0166] In some embodiments, the nanoparticles disclosed herein comprise a DNA plasmid encoding human IL-12.

[0167] In some embodiments, the nanoparticles comprise a synthetic polymer that facilitates plasmid delivery that is a lipopolymer.

[0168] In some embodiments, the lipopolymer further comprises polyethyleneimine (PEI) independently covalently attached to cholesterol and polyethylene glycol (PEG) groups.

[0169] In some embodiments, 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 (PEI), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-β-cyclodextrin, spermine, and spermidine. One 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, where the lipid is either directly attached to the polyethyleneimine backbone or covalently attached to a polyethylene glycol spacer, which is attached to the PEI via a biocompatible bond.

[0170] The cationic gene delivery polymers of the present disclosure are suitable for use in the delivery of antibodies or antibody fragments, cell receptors, growth factor receptors, cytokine receptors, folate, transferrin, epidermal growth factor (EGF), insulin, asialoorosomucoid, mannose-6-phosphate (monocytes), mannose (macrophages, some B cells), Lewis X and Cialil Lewis XThe gene delivery polymer may further comprise a targeting moiety including a targeting agent such as a nucleotide sequence (e.g., nucleotide sequence), a targeting moiety ...

[0171] 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-β-cyclodextrin, spermine, and spermidine. One example of a cationic gene delivery polymer suitable for the present invention is a polyethyleneimine derivative comprising a polyethyleneimine (PEI) backbone, a lipid, and a polyethylene glycol spacer, wherein the lipid is either directly attached to the polyethyleneimine backbone or covalently attached to the polyethylene glycol spacer, which is in turn attached to the PEI via a biocompatible bond.

[0172] In some embodiments, nanoparticles comprising a DNA plasmid encoding interleukin-12 (IL-12) and a synthetic polymer that facilitates plasmid delivery are delivered intraperitoneally.

[0173] In some embodiments, the nanoparticles have a concentration of about 35 mg / m 2 ~about 80mg / m 2In some embodiments, the nanoparticles are administered at a dose of about 50 mg / m 2 ~about 100mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 80 mg / m 2 is administered at a dose of

[0174] III. Anti-VEGF antibody In another embodiment of the above combination therapy, the pharmaceutical agent is an anti-VEGF antibody.

[0175] In some embodiments, 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).

[0176] In some embodiments, the antibody with binding specificity for vascular endothelial growth factor (VEGF) is bevacizumab or a biosimilar thereof.

[0177] In some embodiments, the anti-VEGF antibody comprises a first amino acid sequence having a sequence having at least about 85% identity to SEQ ID NO:1 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO:1) and a second amino acid sequence having a sequence having at least about 85% identity to SEQ ID NO:2 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO:2).

[0178] In some embodiments, the heavy chain of the anti-VEGF antibody comprises the following sequence: [ka]

[0179] In some embodiments, the light chain of the anti-VEGF antibody comprises the following sequence: [ka]

[0180] In some embodiments, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0181] In some embodiments, the anti-VEGF antibody is administered intravenously.

[0182] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

[0183] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered prior to, concurrently with, or after the anti-cancer agent.

[0184] In some embodiments, an anti-cancer agent is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third).

[0185] In some embodiments, an anti-cancer drug is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third), followed by surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) (e.g., fourth).

[0186] In some embodiments, an anti-cancer drug is administered, followed by a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0187] In some embodiments, the anti-VEGF antibody is administered prior to interval cytoreductive surgery, eg, at least about 22 days after the first administration of the anti-cancer agent, eg, weekly for at least about 12 weeks up to about 18 weeks.

[0188] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 12 weeks and up to about 18 weeks, beginning at least about 22 days after the first administration of the anti-cancer agent, prior to interval cytoreductive surgery.

[0189] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 9 weeks, beginning at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0190] In some embodiments, 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 embodiments, the anti-VEGF antibody is administered at a dose of about 15 mg / kg IV.

[0191] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-cancer agent. In some embodiments, the interval cytoreductive surgery (ICS) is performed about 28 days after administration of the anti-cancer agent.

[0192] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid. In some embodiments, the interval cytoreductive surgery (ICS) is performed about 7 days after administration of the DNA plasmid.

[0193] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid. In some embodiments, the interval cytoreductive surgery (ICS) is performed about 7 days after administration of the DNA plasmid.

[0194] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days prior to administration of the anti-VEGF antibody. In some embodiments, the interval cytoreductive surgery (ICS) is performed about 28 days prior to administration of the anti-VEGF antibody.

[0195] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-VEGF antibody. In some embodiments, the interval cytoreductive surgery (ICS) is performed about 28 days after administration of the anti-VEGF antibody.

[0196] IV. Anticancer drugs In one embodiment of the foregoing treatment, the anti-cancer agent is a chemotherapeutic agent selected from the group consisting of taxanes, platinum, adriamycin, cyclofosfamide, topotecan, carmustine (BCNU), or combinations thereof. In some embodiments, the anti-cancer therapy is selected from the group consisting of paclitaxel, carboplatin, docetaxel, nab-paclitaxel, doxorubicin, and any combination thereof.

[0197] In some embodiments, the anti-cancer agent is doxorubicin.

[0198] In some embodiments, the anti-cancer agent comprises paclitaxel.

[0199] In some embodiments, the anti-cancer agent comprises carboplatin.

[0200] In some embodiments, the anti-cancer agent comprises docetaxel.

[0201] In some embodiments, the anti-cancer agent comprises nab-paclitaxel.

[0202] In some embodiments, the anti-cancer agent comprises olaparib.

[0203] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0204] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0205] In some embodiments, the anti-cancer agent is administered, for example, every 1-2 weeks (eg, every 3 weeks) for about 8-10 weeks (eg, 9 weeks) starting at least about 28 days after interval cytoreductive surgery.

[0206] In some embodiments, the anti-cancer agent is administered every three weeks for about nine weeks, at least about 28 days after interval cytoreductive surgery.

[0207] In some embodiments, the anti-cancer agent is selected from the group consisting of paclitaxel, carboplatin, docetaxel, nab-paclitaxel, and any combination thereof.

[0208] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered prior to, concurrently with, or after the anti-cancer agent.

[0209] In some embodiments, an anti-cancer agent is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third).

[0210] In some embodiments, an anti-cancer drug is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third), followed by surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) (e.g., fourth).

[0211] In some embodiments, an anti-cancer drug is administered, followed by a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0212] In some embodiments, the anti-cancer agent is administered prior to interval cytoreductive surgery, hi some embodiments, the anti-cancer agent is administered every three weeks for about 12 weeks to about 18 weeks prior to interval cytoreductive surgery.

[0213] In some embodiments, the anti-cancer agent is administered at least about 28 days after interval cytoreductive surgery (eg, every 3 weeks for about 9 weeks).

[0214] In some embodiments, the administration of the anticancer agent comprises paclitaxel at a dose of about 100 to 200 mg / m 2 (about 175mg / m 2 ) optionally followed by carboplatin at a dose of about AUC 5-6 IV.

[0215] In some embodiments, the administration of the anticancer drug is docetaxel at a dose of about 50 to 100 mg / m 2 (e.g., about 75 mg / m 2 ) optionally followed by carboplatin at a dose of about AUC 5-6 IV.

[0216] In some embodiments, the administration of the anticancer drug is nab-paclitaxel at a dose of about 200-300 mg / m 2 (e.g., about 260 mg / m 2 ) optionally followed by carboplatin at a dose of about AUC 5-6 IV.

[0217] In some embodiments, administration of nanoparticles prior to interval cytoreductive surgery begins 15 days after the first administration of anticancer agent and occurs weekly for at least about 12 weeks to about 18 weeks.

[0218] In some embodiments, the administration of the anti-cancer agent comprises paclitaxel at a dose of about 150 mg / m 2 ~about 200mg / m 2 (e.g., about 175 mg / m 2 ) followed by carboplatin at a dose of about AUC 4-8 IV (e.g., about AUC 5-6 IV).

[0219] In some embodiments, the administration of the anti-cancer agent comprises paclitaxel at a dose of about 175 mg / m 2 followed by carboplatin at a dose of about AUC 5-6 IV.

[0220] In some embodiments, the administration of the anticancer agent comprises docetaxel at a dose of about 50 mg / m 2 ~about 100mg / m 2 (e.g., about 75 mg / m 2 ) followed by carboplatin at a dose of about AUC 4-8 IV (e.g., about AUC 5-6 IV).

[0221] In some embodiments, the administration of the anticancer agent comprises docetaxel at a dose of about 75 mg / m 2 followed by carboplatin at a dose of about AUC 5-6 IV.

[0222] In some embodiments, the administration of the anticancer agent comprises nab-paclitaxel at a dose of about 240 mg / m 2 ~about 300mg / m 2 (e.g., about 260 mg / m 2 ) followed by carboplatin at a dose of about AUC 4-8 IV (e.g., about AUC 5-6 IV).

[0223] In some embodiments, the administration of the anticancer agent comprises nab-paclitaxel at a dose of about 260 mg / m 2 followed by carboplatin at a dose of about AUC 5-6 IV.

[0224] V. Treatment method Certain aspects of the present disclosure relate to a method of treating a subject suffering from cancer, the method comprising administering to the subject a combination of (i) nanoparticles and (ii) an antibody having binding specificity for vascular endothelial growth factor (VEGF).

[0225] The present invention also provides a method for treating mammalian cancer or hyperproliferative disorders by intratumoral, intraperitoneal, intravenous, intravesical, intratracheal, intracranial, or systemic administration of a pharmaceutical composition comprising a plasmid-based gene expression system and a gene delivery polymer without the use of chemotherapy. 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.

[0226] Treatment of tumors with pharmaceutical compositions (nucleic acid and gene delivery polymers and one or more chemotherapeutic agents) results in tumor shrinkage and extended lifespan. Combining gene therapy (nucleic acid and gene delivery polymers) with chemotherapy (chemotherapeutic agents) according to the methods of the present disclosure results in additive and / or synergistic efficacy. Efficacy of the methods herein is defined as, but not limited to, a reduction in tumor size or tumor density, an increase in lymphocyte or neutrophil counts, or improved survival, or all of the above. Furthermore, combining gene therapy (nucleic acid and gene delivery polymers) with chemotherapy (chemotherapeutic agents) according to the methods of the present invention reduces the toxicity of the chemotherapeutic agents and reverses tumor resistance to chemotherapy. Toxicity, as used herein, is defined as any treatment-related adverse effect on clinical findings, including, but not limited to, abnormal hematology or serum chemistry or organ toxicity. Furthermore, combining gene therapy (nucleic acid and gene delivery polymers) with suboptimal doses of chemotherapy (chemotherapeutic agents) according to the methods of the present invention enhances anti-cancer efficacy above that achieved with optimal doses of the chemotherapeutic agents, but with lower toxicity.

[0227] New cancer treatment strategies focus on delivering polymers carrying genetic information rather than therapeutic proteins themselves, allowing exogenously delivered genes to be expressed in the tumor environment. Although nonviral gene delivery systems are considered safer than viral delivery systems, the practical application of current polymer systems has been unsatisfactory due to their low efficiency. A strategy to enhance the gene transfection efficiency of low-molecular-weight PEI by covalently attaching cholesterol to form water-soluble lipopolymers (WSLPs) has recently been described. See Mol. Ther., 2001, 4, 130. IL-12 gene transfer into solid tumors using WSLPs was significantly better than unmodified PEI, resulting in more significant tumor inhibition.

[0228] It is widely recognized that single treatment strategies for cancer are generally ineffective due to the multifactorial nature of this disease. The benefits of combining two or more drugs to maximize anticancer responses are increasingly recognized. In this disclosure, the inventors combined a chemotherapeutic agent with gene delivery of an anticancer gene administered locally to the tumor site to improve the safety and efficacy of the treatment. Combining safe and efficient local delivery of an anticancer gene with standard chemotherapeutic agents enhances anticancer responses and patient survival without increasing toxicity. This combination therapy reduces chemotherapy doses and increases tumor sensitivity to chemotherapy. This disclosure demonstrates that a pharmaceutical composition comprising an anticancer gene complexed with a gene delivery polymer and at least one adjunctive chemotherapeutic agent is more effective than gene therapy or chemotherapy administered alone. Furthermore, when administered by different routes, the combination therapy is effective against a wide variety of tumors and does not increase toxicity compared to the individual therapies.

[0229] 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 that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0230] Certain aspects of the present disclosure relate to methods of treating a subject suffering from cancer, 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 that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0231] In some embodiments, the polynucleotide encodes human IL-12.

[0232] In some embodiments, the nucleic acid vector (e.g., a plasmid) comprises a promoter operably linked to a nucleic acid encoding the p35 subunit of IL-12 and a promoter operably linked to a nucleic acid encoding the p40 subunit of IL12.

[0233] In some embodiments, the nucleic acid vector (eg, a plasmid) comprises an intron, a 3'UTR (eg, an hGH 3'UTR), an antibiotic resistance gene, or any combination thereof (eg, elements of Figure 4).

[0234] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to cholesterol and polyethylene glycol (PEG) groups (eg, the lipopolymer of Figure 5).

[0235] In some embodiments, the combination further comprises an anti-cancer agent.

[0236] In some embodiments, the anti-cancer agent is a chemotherapeutic agent.

[0237] In some embodiments, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0238] In some embodiments, the anti-cancer agent is doxorubicin.

[0239] In some embodiments, the anti-cancer agent is paclitaxel.

[0240] In some embodiments, the anti-cancer agent is carboplatin.

[0241] In some embodiments, the anti-cancer agent is docetaxel.

[0242] In some embodiments, the anti-cancer agent is nab-paclitaxel.

[0243] In some embodiments, the anti-cancer agent is olaparib.

[0244] In some embodiments, 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).

[0245] In some embodiments, the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO:1 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO:1), and a variable light chain (VL) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO:2 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO:2).

[0246] In some embodiments, the method further comprises surgery to remove all or part of the tissue or tumor in the subject (eg, interval cytoreductive surgery).

[0247] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered intratumorally or intraperitoneally.

[0248] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered intravenously.

[0249] In some embodiments, the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravesically, or any combination thereof.

[0250] In some embodiments, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0251] In some embodiments, the anti-VEGF antibody is administered intravenously.

[0252] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

[0253] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered prior to, concurrently with, or after the anti-cancer agent.

[0254] In some embodiments, an anti-cancer agent is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third).

[0255] In some embodiments, an anti-cancer drug is administered (e.g., first), followed by administration of a lipopolymer-formulated nucleic acid vector (e.g., second), followed by administration of an anti-VEGF antibody (e.g., third), followed by surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) (e.g., fourth).

[0256] In some embodiments, an anti-cancer drug is administered, followed by a DNA plasmid, followed by an anti-VEGF antibody, followed by interval cytoreductive surgery.

[0257] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0258] In some embodiments, the anti-cancer agent is administered at least about 28 days after interval cytoreductive surgery (eg, every 3 weeks for about 9 weeks).

[0259] In some embodiments, the administration of the anticancer agent comprises paclitaxel at a dose of about 100 to 200 mg / m 2 (about 175mg / m 2 ) optionally followed by carboplatin at a dose of about AUC 5-6 IV.

[0260] In some embodiments, the administration of the anticancer drug is docetaxel at a dose of about 50 to 100 mg / m 2 (e.g., about 75 mg / m 2 ) optionally followed by carboplatin at a dose of about AUC 5-6 IV.

[0261] In some embodiments, the administration of the anticancer drug is nab-paclitaxel at a dose of about 200-300 mg / m 2 (e.g., about 260 mg / m 2 ) optionally followed by carboplatin at a dose of about AUC 5-6 IV.

[0262] In some embodiments, administration of the anticancer agent comprises oral administration of olaparib twice at a dose of about 250 mg to about 350 mg (eg, about 300 mg).

[0263] In some embodiments, administration of nanoparticles prior to interval cytoreductive surgery begins 15 days after the first administration of anticancer agent and occurs weekly for at least about 12 weeks to about 18 weeks.

[0264] In some embodiments, the nanoparticles are administered at least about 28 days after interval cytoreductive surgery, and administration begins 15 days after the first administration of the anti-cancer agent and occurs weekly for at least about 9 weeks.

[0265] In some embodiments, the interleukin-12 (IL-12) formulated with lipopolymers (e.g., nanoparticles) is administered at a concentration of about 35 mg / m 2 ~about 80mg / m 2 In some embodiments, interleukin-12 (IL-12) formulated with lipopolymers (e.g., nanoparticles) is administered at a dose of about 50 mg / m 2 ~about 100mg / m 2 In some embodiments, interleukin-12 (IL-12) formulated with lipopolymers (e.g., nanoparticles) is administered at a dose of about 80 mg / m 2 is administered at a dose of

[0266] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 12 weeks and up to about 18 weeks, beginning at least about 22 days after the first administration of the anti-cancer agent, prior to interval cytoreductive surgery.

[0267] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 9 weeks, beginning at least about 28 days after interval cytoreductive surgery and at least about 22 days after the first administration of the anti-cancer agent.

[0268] In some embodiments, 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 embodiments, the anti-VEGF antibody is administered at a dose of about 15 mg / kg IV.

[0269] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-cancer agent.

[0270] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid.

[0271] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 7 days after administration of the DNA plasmid.

[0272] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days prior to administration of the anti-VEGF antibody.

[0273] In some embodiments, the interval cytoreductive surgery (ICS) is performed at least about 28 days after administration of the anti-VEGF antibody.

[0274] In some embodiments, 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, as well as metastasis of any cancer.

[0275] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0276] In some embodiments, the subject is a human.

[0277] In some embodiments, 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-look laparoscopy (SLL), BEV + Olaparive, and BEV + GEN-1.

[0278] In some embodiments, the subject is (a) administered neoadjuvant chemotherapy (NACT), bevacizumab (BEV), and GEN-1. In some embodiments, in (a), the subject is treated with NACT for 4 to 6 cycles. In some embodiments, in (a), the subject is administered BEV in cycles 2, 3, 6, and 7. In some embodiments, in (a), BEV is included in each cycle except for the following cycles: (1) cycle 1, (2) the last cycle of neoadjuvant therapy immediately prior to ICS (which may be C4, C4+1, or C4+2), and (3) the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). In some embodiments, in (a), the subject is administered GEN-1 weekly starting on C1D15. In some embodiments, each cycle is a 21-day cycle.

[0279] In some embodiments, the NACT is carboplatin and paclitaxel. In some embodiments, the NACT is administered once per cycle (e.g., every 3 weeks). In some embodiments, paclitaxel is administered at a dose of about 175 mg / m 2 In some embodiments, BEV is administered IV at a dose of about 15 mg / kg IV on day 1 of the included cycle. In some embodiments, GEN-1 is administered IV at a dose of about 80 mg / m 2 It is administered in IP doses.

[0280] In some embodiments, (a) is followed by (b) interval cytoreductive surgery. In some embodiments, the ICS is administered at least 4 weeks after the last administration of NACT from (a).

[0281] In some embodiments, the subject is administered (c) neoadjuvant chemotherapy (NACT), bevacizumab (BEV), and GEN-1. In some embodiments, in (c), the subject is treated with NACT for three cycles. In some embodiments, in (c), the subject is administered BEV in cycles 2, 3, 6, and 7. In some embodiments, in (c), BEV is included in each cycle except for the following cycles: (1) cycle 1, (2) the last cycle of neoadjuvant therapy immediately before ICS (which may be C4, C4+1, or C4+2), and (3) the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). In some embodiments, in (c), the subject is administered GEN-1 weekly. In some embodiments, each cycle is a 21-day cycle.

[0282] In some embodiments, the NACT is carboplatin and paclitaxel. In some embodiments, the NACT is administered once per cycle (e.g., every 3 weeks). In some embodiments, paclitaxel is administered at a dose of about 175 mg / m 2 In some embodiments, BEV is administered IV at a dose of about 15 mg / kg IV on day 1 of the included cycle. In some embodiments, GEN-1 is administered IV at a dose of about 80 mg / m 2 It is administered in IP doses.

[0283] In some embodiments, (b) is followed by (c).

[0284] In some embodiments, (c) is followed by (d) minimal residual disease (MRD) detection by second-look laparoscopy (SLL).

[0285] In some embodiments, during the maintenance phase, the subject is administered (e) BEV and olaparib. In some embodiments, during the maintenance phase, if the subject is BRCA+ / homologous recombination deficiency positive (HRD+), the subject is administered (e). In some embodiments, in (e), BEV is administered at about 15mg / kg IV every day for 3 weeks, for up to 18 cycles. In some embodiments, in (e), olaparib is orally administered twice at a dose of about 300mg. In some embodiments, (d) is followed by (e).

[0286] In some embodiments, during the maintenance phase, the subject is administered (f) BEV and GEN-1. In some embodiments, during the maintenance phase, if the subject is BRCA- / homologous recombination competent (HRP), the subject is administered (f). In some embodiments, in (f), BEV is administered at about 15 mg / kg IV every 3 weeks for up to 18 cycles. In some embodiments, in (f), GEN-1 is administered at about 80 mg / m 2 IP every 21 days for up to 18 additional cycles. In some embodiments, (d) is followed by (f).

[0287] In some embodiments, the subject is treated with one, two, three, four or more of (a), (b), (c), (d), and (e). In some embodiments, the subject is treated with all of (a), (b), (c), (d), and (e).

[0288] In some embodiments, the subject is treated with one, two, three, four or more of (a), (b), (c), (d), and (f).

[0289] In some embodiments, the subject is treated with all of (a), (b), (c), (d), and (f).

[0290] The following examples are illustrative and do not limit the scope of the claimed embodiments.

[0291] example Example 1. GEN-1 enhances the activity of anti-VEGF antibodies in a mouse model. Figure 1 shows the potential synergistic effectiveness of lowering VEGF levels and inhibiting production by administering an anti-VEGF antibody (e.g., bevacizumab) simultaneously with GEN-1. Results show that SKOV-3 (human ovarian epithelial adenocarcinoma) cells (7 x 10 6 Cell) nude-foxn1 nu Anti-VEGF antibodies were administered intravenously to mice at different concentrations: 5 mg / kg (low), 10 mg / kg (medium), and 20 mg / kg (high).

[0292] Anti-VEGF antibodies (e.g., bevacizumab) were administered 9 days after the initial tumor implantation and once a week for 6 weeks thereafter, followed by intraperitoneal administration of mGEN-1 (100 μg DNA) starting 14 days after the initial tumor implantation and once a week for 4 weeks thereafter.

[0293] 59 days after the initial tumor implantation, the mice were euthanized, and the tumors were then removed and weighed. Improved efficacy of low-dose anti-VEGF antibodies (e.g., bevacizumab) was demonstrated when administered in combination with GEN-1, which improved the therapeutic index and cost. See Figure 1.

[0294] Example 2. GEN-1 enhances the activity of anti-VEGF antibodies in combination with anti-cancer drugs in a mouse model. Figure 2 shows the potential synergistic effectiveness of lowering VEGF levels and inhibiting production by co-administering an anti-VEGF antibody (e.g., bevacizumab) with an anti-cancer drug, GEN-1. Results show that SKOV-3-Luc (human ovarian epithelial adenocarcinoma) cells (7 x 10 6 Cell) nude-foxn1 nu The tumors were obtained by intraperitoneal injection into mice. Doxil was administered intraperitoneally at a dose of 7.5 mg / kg every other week starting two weeks after tumor implantation. Anti-VEGF antibodies (e.g., bevacizumab) were administered intravenously at 10 mg / kg every week starting 10 days after tumor implantation.

[0295] mGEN-1 was then administered intraperitoneally (100 μg of DNA) weekly starting two weeks after the initial tumor implantation. Tumor burden was then quantified using IVIS imaging, as shown in Figure 2. Whole-body images of mice taken with IVIS imaging are shown in Figure 3.

[0296] Example 3. Clinical combination of GEN-1 + NACT + anti-VEGF antibody This is a 1:1 randomized, open-label, multicenter Phase II study with a safety lead to evaluate the safety, dosing, efficacy, and biological activity of adding GEN-1 to neoadjuvant chemotherapy (NACT) plus an anti-VEGF antibody (e.g., bevacizumab) (BEV) compared with NACT plus BEV alone. NACT is a standard regimen of carboplatin plus paclitaxel administered every 3 weeks for 7–9 cycles. The protocol requires at least 4 cycles of neoadjuvant chemotherapy and allows up to 2 additional cycles (C4+1, C4+2) prior to ICS at the investigator's discretion based on response and other clinical considerations. ICS will be administered after a 3–4 week break from the last dose of NACT. After at least 4 weeks of recovery from ICS, 3 further adjuvant cycles of study treatment will be administered.

[0297] In addition, BEV was included in each cycle except for the following cycles: cycle 1, the last cycle of neoadjuvant therapy immediately prior to ICS, and the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). 2 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 cannot be administered within 30 days before or after surgery. In the experimental group, GEN-1 will be added weekly to each cycle of NACT + BEV starting on cycle 1, day 15. FDA-approved BEV biosimilars may also be used.

[0298] The safety run-in will evaluate the safety of adding weekly GEN-1 to the NACT+BEV regimen in up to 12 subjects. This is a standard 3+3 design with a Data Safety Monitoring Board (DSMB) evaluating a cohort of three subjects who received at least two cycles of NACT+BEV+GEN-1 before initiating the main phase of the study. Run-in patients will also be randomized. To be evaluable for safety, subjects must have received at least two cycles of chemotherapy+BEV+GEN-1. At least six subjects in the GEN-1 group must be evaluable for safety prior to the Phase II dose of GEN-1.

[0299] Phase II of the study may begin once the DSMB determines a recommended safe dose from the safety phase. Approximately 50 subjects will be randomized in this study. Upon completion of NACT, all subjects will undergo second-look laparoscopy (SLL) to determine whether they are minimal residual disease (MRD) positive. SLL will be performed according to a standardized surgical technique by a gynecologic oncologist.

[0300] Maintenance treatment will be determined by BRCA+ / HRD status. All subjects will receive BEV, with only BRCA+ / HRD subjects receiving olaparib in addition to BEV. Experimental subjects who are BRCA- / HRP will receive GEN-1 along with BEV. All subjects will be followed for disease progression and survival.

[0301] Testing phase Run-in To ensure the NACT+BEV+GEN-1 combination is safe, the study will enroll at least six subjects in the experimental arm before initiating the main phase of the protocol. Prior to initiating the main phase of the study, no more than two of six subjects treated in the experimental arm may exhibit dose-limiting toxicities. An independent DSMB will review safety data from subjects receiving at least two cycles of NACT+BEV+GEN-1 and provide recommendations regarding dose modifications, safety monitoring, and dosing for the main phase of the study. The DSMB authorization letter will define the committee's responsibilities, which will include defining dose-limiting toxicities (DLTs) for the safety phase and recommending the phase II dose of GEN-1.

[0302] Phase II The primary phase of the study may begin once the DSMB determines a recommended safe dose from the safety phase. The study will randomize approximately 50 subjects (25 per group) across Phase II and III. 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 two cycles of treatment after surgery. At the end of the final cycle of chemotherapy, all subjects will undergo SLL before starting the maintenance phase.

[0303] maintain After completion of the SLL subject, maintenance treatment begins. This phase begins no earlier than 4 weeks (ideally 5-7 weeks) from the date of SLL.

[0304] All subjects will receive BEV (or an FDA-approved biosimilar) every 21 days until disease progression or unacceptable toxicity, or for a maximum of 15 months.

[0305] BRCA+ / HRD subjects only: For all BRCA+ / HRD subjects, administer olaparib 300 mg orally twice daily starting in the maintenance phase until disease progression or unacceptable toxicity, or for a maximum of 24 months, plus BEV 15 mg / kg as a single agent every 3 weeks until disease progression or unacceptable toxicity for up to 18 additional cycles.

[0306] BRCA- / HRP subjects only: Subjects who are BRCA- / HRP will receive GEN-1 with BEV 15 mg / kg every 21 days until disease progression or unacceptable toxicity for up to 18 additional cycles.

[0307] Study population Approximately 50 subjects (including up to 12 safety run-ins) with newly diagnosed advanced ovarian cancer will be randomized.

[0308] Inclusion criteria 1. Subjects must have a suspected diagnosis of high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer with pretreatment biopsy by laparoscopy or histologic confirmation by interventional radiology or CT or ultrasound-guided core biopsy. Histologic documentation of the original primary tumor is required via a pathology report. 2. Subjects must be International Federation of Gynecology and Obstetrics (FIGO) stage III or IV, where clinical considerations have determined that they would benefit from neoadjuvant therapy based on standard of care. 3. Subjects with only epithelial cell type histology of high-grade serous adenocarcinoma are eligible. 4. Subjects must meet the following criteria: I. Bone marrow function: Absolute neutrophil count (ANC) ≥ 1,500 / mcl. This ANC is not induced or supported by granulocyte colony-stimulating factor. Platelets ≥ 100,000 / mcl. II. Renal function: Cockcroft-Gold estimated GFR ≥ 50 ml / min. Urine dipstick showing proteinuria ≤ 1+ (patients with a dipstick reading ≥ 2+ must have a 24-hour urine collection with < 2g protein / 24 hours). III. Liver function: bilirubin ≦1.5×ULN. SGOT (AST) and SGPT (ALT) ≦3.0×ULN and alkaline phosphatase ≦2.5×ULN. IV. Neurological function: Neurological impairment (sensory and motor) grade 1 or less. 5. Subjects must be free of active infection requiring isolation, parenteral antibiotics, or uncontrolled serious medical illness or disorder within 4 weeks of study entry. 6. Hormone therapy for malignant tumors must be discontinued at least 1 week before the first treatment. Continuation of hormone replacement therapy is permitted. 7. Subjects must have an Eastern Cooperative Group (ECOG) performance status score of 0-1. 8. Subjects of childbearing potential must have a negative serum pregnancy test within 14 days prior to the start of protocol therapy and must be practicing an effective form of contraception. If applicable, subjects must discontinue breastfeeding prior to study entry. 9. Subject must have satisfactory results on baseline laboratory analyses and diagnostic procedures as specified in the protocol. 10. Subjects must have signed an IRB / EC approved informed consent and authorization allowing release of personal health information. 11. Subjects must be 18 years of age or older.

[0309] 1.Subjects who have been previously treated with GEN-1. 2. History of allergic reaction caused by compounds of similar chemical or biological composition to GEN-1 or other drugs used in this study. 3. Subjects who have received oral or parenteral corticosteroids within 2 weeks of study entry or have a clinical requirement for ongoing systemic immunosuppressive therapy, such as chronic steroid use (greater than 10 mg / day prednisone equivalent) unrelated to chemotherapy administration. Steroid prophylaxis for intravenous contrast allergy is permitted. 4. Subjects with an autoimmune disease requiring immunosuppressive therapy within the past two years. Examples of autoimmune diseases include systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. 5. Subjects with known human immunodeficiency virus (HIV) or human T-lymphotropic virus (HTLV) infection will be excluded. 6. Subjects with other invasive malignancies will be excluded if there is evidence of the presence of an invasive malignancy within the past 3 years. Subjects will also be excluded if previous cancer treatment contraindicates this protocol therapy. Subjects with non-invasive malignancies, such as non-melanoma skin cancer and in-situ melanoma, are eligible. 7. Subjects who have received prior radiation therapy to any part of the abdominal cavity or pelvis are excluded. Prior radiation for localized cancer of the breast, head and neck, or skin is acceptable provided it was completed more than 3 years prior to enrollment and the patient remains free of recurrent or metastatic disease. 8. Subjects who have received prior chemotherapy for abdominal or pelvic tumors are excluded. Subjects may have received prior adjuvant chemotherapy for localized breast cancer, provided it was completed more than 3 years prior to enrollment and the patient remains free of recurrent or metastatic disease. 9. Subjects with known active hepatitis. 10. Subjects with known renal syndrome (proteinuria grade 2 or higher). 11. Subject has a concurrent severe medical problem unrelated to malignancy that significantly limits full compliance with the study or places the subject at extreme risk or reduced life expectancy. 12. Subjects with clinically significant cardiovascular disease. This includes: a) Uncontrolled hypertension, defined as systolic blood pressure (BP) >150 mmHg or diastolic BP >90 mmHg on at least two separate days. (Subjects with uncontrolled hypertension may become eligible once their hypertension is controlled.) b) Myocardial infarction or unstable angina within 6 months prior to enrollment. c) severe ventricular arrhythmia (i.e., ventricular tachycardia or ventricular fibrillation) requiring antiarrhythmic medication or a history of cardiac arrhythmia (excluding atrial fibrillation well controlled with antiarrhythmic medication). d) QTc interval ≥ 450 ms on baseline ECG (electrocardiogram). e) Baseline ejection fraction ≤ 50% as assessed by echocardiogram or MUGA. f) New York Heart Association (NYHA) class II or higher congestive heart failure. 13. Subjects of childbearing potential without adequate contraception, pregnant subjects, or breastfeeding subjects are not eligible for this study. 14. Subject has a history or evidence on physical examination of CNS disease, including a primary brain tumor, seizures uncontrolled with standard medical therapy, any brain metastases, or a history of cerebrovascular accident (CVA, stroke), transient ischemic attack (TIA), or subarachnoid hemorrhage within 6 months of the first treatment date on this study. 15. Subjects with a history of diverticulitis. Diverticulosis is not excluded. 16. Subjects who have coughed up blood within the last month. 17. Subject with any condition / abnormality that would prevent proper placement of an IP catheter for study drug administration, including abdominal surgery (for reasons other than IP port placement) within 4 weeks of study entry, bowel dysfunction, fistula, or extensive adhesions suspected from previous medical history or laparoscopic findings.

[0310] Registration and Cancellation Procedures Subjects with newly diagnosed advanced ovarian cancer will be randomized to receive either NACT+BEV+GEN-1 or NACT+BEV.

[0311] Study treatment and medication Treatment Allocation Eligible subjects will be randomized in a 1:1 ratio to either NACT+BEV+GEN-1 or NACT+BEV alone. This is an open-label trial, so site staff, subjects, and sponsors will be aware of each subject's treatment.

[0312] Neoadjuvant chemotherapy (NACT) Control group NACT: Paclitaxel 175 mg / m² intravenously (IV) followed by carboplatin AUC 5-6 IV on C1D1. This is repeated every 21 days on day 1 for 4 cycles pre-ICS and 3 cycles post-ICS. Up to 2 additional cycles may be added pre-ICS at the physician's discretion (C4+1, C4+2). If there is a paclitaxel response, 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.

[0313] BEV will be included in every cycle except for the following: cycle 1, the last cycle of neoadjuvant therapy immediately prior to ICS, and the first cycle of adjuvant chemotherapy (i.e., the first cycle after ICS). FDA-approved BEV biosimilars may be used in this study.

[0314] In the experimental group, GEN-1 will begin on C1D15 and continue weekly until the last cycle of adjuvant therapy.

[0315] Interval cytoreductive surgery (ICS) is performed at least 28 days after the last cycle of neoadjuvant chemotherapy, and protocol therapy is resumed as adjuvant chemotherapy after recovery from ICS (at least 28 days) for three more cycles. BEV cannot be administered within 28 days before or after surgery.

[0316] Prophylactic dexamethasone to prevent hypersensitivity reactions is permitted only with the initial dose of NACT. BEV 15 mg / kg IV is administered on days 1 of cycles 2, 3, 6, and 7. During the maintenance phase, BEV 15 mg / kg is administered as a single agent every 3 weeks until disease progression or unacceptable toxicity, for up to 18 additional cycles. BEV may be administered for up to 24 total cycles.

[0317] Prevention of hypersensitivity On Day 1 of Cycle 1, all patients treated with paclitaxel should be pretreated with corticosteroids such as dexamethasone. Subsequent administration of corticosteroids (e.g., C2D1) during chemotherapy is prohibited to avoid blunting the efficacy of GEN-1. Patients who experience a hypersensitivity reaction may be switched to docetaxel or Abraxane.

[0318] Dose modification Investigators should follow institutional standards for dose modifications due to adverse reactions from chemotherapy, BEV, or olaparib. Any dose modifications or skipped doses of GEN-1 should be approved by the study committee or study monitor. GEN-1 doses can be skipped for up to 2 weeks at any time if necessary due to chemotherapy-related adverse events to maintain the schedule. However, it is preferable that delays in GEN-1 administration do not delay or shift the timing of chemotherapy administration. In the event of grade 3 or grade 4 toxicity due to GEN-1, subsequent doses are skipped until recovery to grade 1 or less. In general, GEN-1 can be administered as long as the ANC is >500 and the PLT is >50K; otherwise, a dose modification may be considered.

[0319] If a subject develops grade 3 abdominal pain after the analgesic regimen, the dose of GEN-1 will be reduced to 60 mg / m2 for all subsequent doses.

[0320] For complete prescribing information, refer to carboplatin, paclitaxel, Abraxane, bevacizumab (or biosimilar), and docetaxel labeling and local institutional guidelines.

[0321] GEN-1 (investigational drug) Human IL-12 plasmid (phIL-12-005) is formulated with the lipopolymer PEG-PEI-cholesterol (PPC) in 10% lactose.

[0322] Human IL-12 Plasmid The phIL-12-005 plasmid was obtained from Kan r The gene-containing plasmid contains an hIL-12 gene expression cassette. The hIL-12 gene expression cassette in phIL-12-005 contains a cytomegalovirus (CMV)-derived immediate-early enhancer and promoter, a 5' untranslated region (UTR), a synthetic intron, a p35 gene, a human growth hormone (hGH) 3' UTR and polyadenylation signal sequence, a CMV promoter, a 5' UTR, a synthetic intron, a p40 gene, an hGH 3' UTR and polyadenylation signal sequence. The two hIL-12 subunits are individually under the control of two separate CMV promoters (see Figure 4).

[0323] PEG-PEI-cholesterol PEG-PEI-cholesterol (PPC) consists of a PEI backbone to which polyethylene glycol and cholesterol are independently covalently attached. The molecular weights of PEI, PEG, and cholesterol carbonyl are 1800, 550, and 414, respectively (see Figure 5).

[0324] Route and administration of GEN-1 GEN-1 is administered after peritoneal lavage and translational specimen collection using an IP port.

[0325] To check catheter patency, flush 25 mL of 0.9% saline through the IP port. Heparin should not be used to flush the catheter during sample collection or drug infusion.

[0326] Reconstituted GEN-1 (in a 50 mL glass vial or IV bag) is stable at room temperature for up to 24 hours. Once catheter patency is confirmed, the IV bag containing GEN-1 is administered through the patient's IP catheter. GEN-1 is infused from the IV bag via gravity through the catheter, with the valve fully open and free flowing. A typical administration can take approximately 1 hour.

[0327] After GEN-1 infusion, a second flush of at least 25 ml of 0.9% saline for injection is administered to ensure that the study drug is removed from the catheter.

[0328] To date, toxicities associated with systemic administration of recombinant IL-12 therapy have not been detected with GEN-1 administration. Phase 1 studies of GEN-1 indicate that there is little to no systemic uptake of GEN-1 or its downstream cytokines.

[0329] GEN-1 may be administered into the peritoneal cavity using a subcutaneously implanted IP silicone catheter. GEN-1 has been demonstrated to be compatible with silicone catheters in previous preclinical compatibility studies and in a previous Phase I study. The Port-A-Cath catheter (Deltec, Inc., St. Paul, MN) has been successfully used for IP delivery of GEN-1, with few to no catheter-related serious complications. While the Port-A-Cath catheter is preferred, any other approved catheter may be used with a subcutaneous port for IP delivery if suitable for aspiration of biological samples for translational studies. For venous access, a Bard 9.6 Fr silicone single-lumen catheter, or an equivalent with or without a cuff manufactured by Bard Access Systems (West Amelia Earhart Drive, Salt Lake City, Utah), can be used. Catheter compatibility studies performed with the Port-A-Cath catheter demonstrated that catheter exposure to GEN-1 did not significantly affect the physicochemical properties or transfection activity of GEN-1. Similar compatibility studies were performed with a Bard catheter. Subjects may be treated through an existing IP catheter, provided it is of similar nature and functionality to the Port-A-Cath catheter device. If there are concerns regarding catheter patency or function, a cathetergram can be obtained to verify intraperitoneal infusion.

[0330] Catheter Insertion: IP catheters will be implanted according to the institution's standard process. The procedure and risks associated with IP catheter placement must be explained to the subject, and the subject will sign a procedure consent form prior to catheter placement. Subjects will undergo IP catheter insertion at least 7 days prior to the scheduled 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 an equivalent device according to current institutional clinical practice, will be used. The port will be located in the lower chest.

[0331] Study medication will be infused through this port during the course of the study. Prior to each infusion of study medication, at least 25 mL of saline will be flushed through the catheter to check catheter patency. Heparin should not be used to flush the catheter during sample collection or drug infusion. Peritoneal / ascites washes for translational studies should be obtained prior to GEN-1 infusion. For subjects enrolled in the NACT+BEV+GEN-1 group, the catheter may be removed upon completion of GEN-1 administration at the clinician's discretion.

[0332] Test Procedure Screening Overview (Day -21 to Day 1 of Cycle 1) Written informed consent must be obtained prior to the start of treatment and within 21 days before any specific procedure for inclusion in the protocol is performed. Regardless of the group to which they are randomized, all eligible subjects will undergo a baseline assessment prior to dosing.

[0333] Screening assessments will be conducted after obtaining informed consent and within 21 days prior to treatment initiation. For both treatment groups, screening procedures will include medical history, laparoscopy / biopsy, physical examination, vital signs, Eastern Cooperative Group (ECOG) performance status, electrocardiogram (ECG), laboratory tests including serum pregnancy test and CA-125, and radiological imaging scans. Radiological imaging scans can be completed within 21 days prior to treatment initiation. Screening procedures may be repeated to assess eligibility parameters during the screening period.

[0334] Screening assessments must be collected, reviewed, and deemed acceptable by the investigator prior to randomization, allowing sufficient time for the subject to have an IP catheter inserted. Any changes in health status assessed by physical examination or vital signs should be acceptable to the investigator before study treatment begins.

[0335] Eligible subjects will be randomized 1:1 to receive either NACT+BEV+GEN-1 or NACT+BEV.

[0336] NACT and interval cytoreductive surgery All subjects receive seven to nine cycles of standard NACT every 21 days (see Figure 6). BEV is administered on C2D1, C3D1, and ICS followed by C6D1, C7D1 (and additional neoadjuvant cycles as needed, except for the cycle immediately preceding ICS). GEN-1 is added to the regimen for subjects in the experimental group every week starting on C1D15. For subjects randomized to the experimental group, an IP port must be placed at least seven days before GEN-1 administration to allow for healing. ICS is administered four weeks after the last dose of the neoadjuvant chemotherapy cycle (based on response as judged by the investigator). The remaining three cycles of the adjuvant treatment regimen are initiated after a recovery period of at least four weeks from ICS.

[0337] maintain All subjects will receive BEV every 21 days until resistance or unacceptable toxicity occurs, for up to 18 additional cycles.

[0338] 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 arm in a 3+3 design. To be evaluable for safety, subjects must have received at least two cycles of NACT+BEV+GEN-1. At least six subjects in the GEN-1 arm must be evaluable for safety before the DSMB can recommend a Phase II dose of GEN-1. Generally, fewer than two of the six subjects may progress to Phase II with dose-limiting toxicities. The DSMB will review the safety data from evaluable subjects and make recommendations to the sponsor and trial committee.

[0339] Subjects will be monitored for safety (including physical examination and assessment of AEs) at every treatment visit from the time they sign informed consent until at least 30 days after the last dose of chemotherapy or GEN-1. Suspected drug-related AEs may be reported at any time during follow-up until they resolve to Grade 2 or less (CTCAE v5.0).

[0340] Subjects will be monitored for antitumor activity clinically (CA-125) and by CT or MRI scan at screening, prior to ICS, and approximately 4 weeks after completion of all carboplatin + paclitaxel chemotherapy.Subjects will then be monitored clinically by CA-125 and CT / MRI every 3 months until progression.Radiological progression (per RECIST v.1.1) as determined by the investigator will be observed and documented before initiating alternative treatment.

[0341] SLL is performed approximately 6-8 weeks (±1 week) after day 1 of the last cycle of adjuvant chemotherapy. SLL is performed at least 4 weeks after the last dose of BEV.

[0342] evaluation The presence of histopathologically confirmed MRD at SLL will be used as the primary endpoint for evaluating efficacy. For purposes of the primary endpoint, the presence of minimal residual disease (MRD) at SLL will be defined as any histopathological or cytological evidence of viable residual cancer in peritoneal biopsies or washings from the SLL.

[0343] The rate of MRD in SLL is defined as the proportion of subjects who are MRD+ (microscopic only or gross disease) at the time of SLL.

[0344] For exploratory analyses, residual disease at SLL will be further classified according to the following three categories: a) complete response (no evidence of disease in any biopsy or peritoneal washings); b) macroscopic MRD (suspected disease visible at surgery or confirmed by histopathological diagnosis); c) microscopic MRD (no definitive visible residual disease at SLL, but microscopically positive biopsy or positive cytology in peritoneal washings). Fisher's exact test will be used to compare the difference in MRD rates in SLL-positive patients (including partial response and persistent disease) between the two groups. 80% and 95% Clopper Pearson exact confidence intervals will be derived for individual treatment rates, with continuity correction for the difference in rates.

[0345] Secondary endpoints include progression-free survival (PFS) and overall survival (OS). PFS is defined as the time 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 the absence of confirmed death or for subjects still alive as of the OS cutoff date, survival will be censored at the date of last study follow-up or the cutoff date, whichever occurs first.

[0346] This protocol allows for the enrollment of subjects with both measurable and non-measurable disease and therefore deviates slightly from standard RECIST 1.1 wording regarding the use of CA-125 to define biochemical response and progression. Note: "Response" below refers to clinical response, not "biochemical response," unless otherwise specified.

Claims

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

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

3. 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 to 3, wherein the nucleic acid vector comprises an intron, a 3'UTR, an antibiotic resistance gene, or any combination thereof (eg, an element of Figure 4).

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

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

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

8. 7. 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 anti-cancer 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 anti-cancer agent is nab-paclitaxel.

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

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

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

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

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

2.

20. 20. The combination therapy of claim 19, wherein the anti-VEGF antibody comprises a variable heavy chain (VH) comprising an amino acid sequence having 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% identity to SEQ ID NO: 1, and a variable light chain (VL) comprising an amino acid sequence having 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% identity to SEQ ID NO:

2.

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

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

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

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

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

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

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

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

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

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

31. The method of any one of claims 23 to 30, comprising administering an anti-cancer agent, followed by administering the nucleic acid vector formulated in the lipopolymer, followed by administering the anti-VEGF antibody, followed by surgery to remove all or part of a tissue or tumor.

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

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

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

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

36. 35. The method of any one of claims 23 to 34, wherein the anti-cancer agent is administered every three weeks for about nine weeks after the interval cytoreductive surgery.

37. The administration of the anticancer drug comprises administering paclitaxel at a dose of about 100 to 200 mg / m ２ 36. The method of any one of claims 23 to 35, comprising administering lorcaserin at a dose of about AUC 5-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 5-6 IV.

38. The administration of the anticancer drug is paclitaxel at about 175 mg / m ２ 40. The method of claim 37, comprising administering lorcaserin at a dose of about AUC 5-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 5-6 IV.

39. The administration of the anticancer drug is docetaxel at about 50 to 100 mg / m ２ 40. The method of any one of claims 23 to 38, comprising administering lorcaserin at a dose of about AUC 5-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 5-6 IV.

40. The administration of the anticancer drug comprises administering docetaxel at about 75 mg / m ２ 40. The method of claim 39, comprising administering lorcaserin at a dose of about AUC 5-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 5-6 IV.

41. The administration of the anticancer drug comprises administering nab-paclitaxel at a dose of about 200 to 300 mg / m ２ 41. The method of any one of claims 23 to 40, comprising administering lorcaserin at a dose of about AUC 5-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 5-6 IV.

42. The administration of the anticancer drug is nab-paclitaxel at about 260 mg / m ２ 42. The method of claim 41, comprising administering lorcaserin at a dose of about AUC 5-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 5-6 IV.

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

44. 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 wherein administration begins 15 days after the first administration of the anticancer agent and occurs weekly for at least about 9 weeks.

45. The lipopolymer-formulated interleukin-12 (IL-12) plasmid is administered at a concentration of about 35 mg / m ２ ~about 80mg / m ２ The method of any one of claims 23 to 44, wherein the dose is

46. The lipopolymer-formulated interleukin-12 (IL-12) plasmid is administered at a concentration of about 50 mg / m ２ ~Approx. 100mg / m ２ The method of any one of claims 23 to 44, wherein the dose is

47. The interleukin-12 (IL-12) plasmid formulated in lipopolymer is administered at a concentration of about 80 mg / m ２ The method of any one of claims 23 to 44, wherein the dose is

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

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

50. 50. The method of any one of claims 23-49, wherein the anti-VEGF antibody is administered weekly for at least about 9 weeks, beginning 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. 51. The method of any one of claims 23 to 50, wherein the anti-VEGF antibody is administered at a dose of about 10 to 20 mg / kg IV.

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

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

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

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

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

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

58. 58. The method of any one of claims 23 to 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 metastasis of any of the foregoing cancers.

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

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

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