IL-12 gene therapy for treating BRCA-negative / homologous repair-proficient cancers

Abstract

The present disclosure relates to methods of treating cancer in a subject that is BRCA-negative and homologous repair proficient (HRP), comprising administering to the subject a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12), formulated in a lipopolymer (e.g., a nanoparticle). In some embodiments, the method further comprises administering to the subject an anti-cancer agent (e.g., a chemotherapeutic agent), an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody), an immune checkpoint inhibitor, or any combination thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 375,535, filed September 13, 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_083PC01_SequenceListing_ST26; Size: 113,069 bytes; and Creation Date: August 31, 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] BRCA1 and BRCA2 mutations increase an individual's risk of developing certain malignancies, particularly ovarian and breast cancer. If an individual has a BRCA1 mutation, they have a 39-46% chance of developing ovarian cancer. Women with a BRCA1 mutation have a 57-65% chance of developing breast cancer over their lifetime. If they have a BRCA2 mutation, they have a 45-49% chance of developing breast cancer and an 11-18% chance of developing ovarian cancer.

[0005] Homologous recombination and DNA repair are important for genome maintenance. Genetic mutations in essential homologous recombination genes, including BRCA1 and BRCA2, result in homologous recombination deficiency (HRD) and can be targeted by therapeutic strategies, including poly(ADP-ribose) polymerase inhibitors (PARPi). However, responses are limited in patients who are not HRD (i.e., homologous recombination proficient (HRP)), highlighting the need for improved treatment options for these patients. See, e.g., Creeden et al., BMC Cancer 1154 (2021).

[0006] 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 potential for cytokine expression specifically within the tumor microenvironment, with the goal of achieving increased efficacy while minimizing potential systemic toxicity.

[0007] While traditional chemotherapy regimens are designed to inhibit tumor growth through cytotoxic mechanisms, immunocytokine therapy is designed to induce tumor killing 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. Summary of the Invention

[0008] Certain aspects of the present disclosure relate to methods of treating a subject suffering from cancer, comprising administering to the subject a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12), formulated in a lipopolymer (e.g., a nanoparticle), wherein the cancer comprises a BRCA-negative genotype (having wild-type BRCA1 and wild-type BRCA2) and is homologous recombination proficient (HRP).

[0009] Certain embodiments of the present disclosure relate to methods of treating a subject, comprising: (i) determining whether the subject is (a) positive or negative for BRCA negation (having wild-type BRCA1 or wild-type BRCA2) and (b) whether the subject is Homologous Repair Proficient (HRP) or Homologous Repair Deficient (HRD) (e.g., by performing a genotyping assay); and (ii) if the subject is BRCA negative and HRP (BRCA- / HRP), administering to the subject a therapy comprising a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12), formulated in a lipopolymer (e.g., nanoparticle). In some embodiments, the therapy is a combination therapy further comprising administering an anti-cancer agent (e.g., a chemotherapeutic agent), an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody), an immune checkpoint inhibitor, or any combination thereof.

[0010] In some embodiments, the cancer and / or subject is BRCA1 negative. In some embodiments, the cancer and / or subject is BRCA2 negative. In some embodiments, the BRCA negative genotype comprises wild-type BRCA1 and / or BRCA2 genes.

[0011] In some embodiments, genotyping assays are performed to determine whether a subject is BRCA positive or BRCA negative, homologous repair proficient and / or homologous repair deficient.

[0012] In some embodiments, the administering further comprises administering an anti-cancer agent (e.g., a chemotherapeutic agent). In some embodiments, the method further comprises administering an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody). In some embodiments, the method further comprises administering an immune checkpoint inhibitor. In some embodiments, the method further comprises administering a maintenance dose of a polynucleotide encoding IL-12 formulated in a lipopolymer (e.g., nanoparticle).

[0013] In some embodiments, the polynucleotide encodes human IL-12.

[0014] 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 IL-12.

[0015] 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, an element of Figure 1).

[0016] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group (eg, the lipopolymer of Figure 2).

[0017] In some embodiments, the method further comprises administering an anti-cancer agent (eg, a chemotherapeutic agent).

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

[0019] In some embodiments, the anti-cancer agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0020] In some embodiments, the anti-cancer agent is paclitaxel.

[0021] In some embodiments, the anti-cancer agent is carboplatin.

[0022] In some embodiments, the anti-cancer agent is docetaxel.

[0023] In some embodiments, the anti-cancer agent is nab-paclitaxel.

[0024] In some embodiments, the anti-cancer agent is olaparib.

[0025] In some embodiments, the method further comprises administering to the subject an antibody or antigen-binding fragment thereof that specifically binds vascular endothelial growth factor (VEGF) (an anti-VEGF antibody).

[0026] 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).

[0027] 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: 85 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 85), and a variable light chain (VL) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO: 86 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 86).

[0028] In some embodiments, the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravenously, intravesicularly, or any combination thereof.

[0029] In some embodiments, the anti-VEGF antibody is administered intravenously.

[0030] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

[0031] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

[0032] In some embodiments, an anti-cancer agent is administered (e.g., first), then a lipopolymer-formulated nucleic acid vector is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third).

[0033] In some embodiments, an anti-cancer drug is administered (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., fourth).

[0034] In some aspects, the methods involve administration of a maintenance dose of a nucleic acid vector formulated in a lipopolymer.

[0035] 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.

[0036] In some embodiments, the interleukin-12 (IL-12) plasmid formulated in lipopolymer (e.g., nanoparticles) is administered at a concentration of about 35 mg / m 2 ~about 100mg / m 2 is administered at a dose of

[0037] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 12 weeks and up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anti-cancer agent.

[0038] 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.

[0039] In some embodiments, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV.

[0040] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the anti-VEGF antibody.

[0041] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody.

[0042] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0043] 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).

[0044] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0045] In some embodiments, the administration of the anticancer agent is between 25 and 250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0046] In some embodiments, the administration of the anticancer agent is from 25 to 350 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0047] In some embodiments, administration of lipopolymer (e.g., nanoparticles) prior to interval cytoreductive surgery begins 15 days after the first administration of the anticancer agent weekly for at least about 12 weeks to about 18 weeks.

[0048] In some embodiments, the lipopolymer (e.g., nanoparticles) is administered at least about 28 days after interval cytoreductive surgery, with administration beginning 15 days after the first administration of the anticancer agent weekly for at least about 9 weeks.

[0049] In some embodiments, the interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-cancer agent.

[0050] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0051] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0052] In some embodiments, the method comprises administering an immune checkpoint inhibitor to the subject. In some embodiments, the immune checkpoint inhibitor comprises two or more (e.g., two, three, or four) immune checkpoint inhibitors.

[0053] In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0054] In some embodiments, the immune checkpoint inhibitor is an antibody.

[0055] In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor.

[0056] In some embodiments, the immune checkpoint inhibitor comprises a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostallimab, cemiplimab, and any combination thereof.

[0057] In some embodiments, the PD-1 antagonist comprises nivolumab.

[0058] In some embodiments, the immune checkpoint inhibitor comprises a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, avelumab, and any combination thereof.

[0059] In some embodiments, the immune checkpoint inhibitor comprises a CTLA-4 antagonist that is ipilimumab.

[0060] In some embodiments, the immune checkpoint inhibitor comprises a LAG-3 antagonist, and the LAG-3 antagonist is leratolimab.

[0061] In some embodiments, the method further comprises administering a second immune checkpoint inhibitor.

[0062] In some embodiments, the second immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0063] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered intratumorally or intraperitoneally.

[0064] In some aspects, the lipopolymer-formulated nucleic acid vector is administered intravenously.

[0065] In some aspects, the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, intravenously, intravesically, or any combination thereof.

[0066] In some aspects, the immune checkpoint inhibitor is administered intravenously.

[0067] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

[0068] In some aspects, the lipopolymer-formulated nucleic acid vector is administered prior to, concurrently with, or after the immune checkpoint inhibitor.

[0069] In some embodiments, an anti-cancer agent is administered (e.g., first), then a lipopolymer-formulated nucleic acid vector is administered (e.g., second), then an immune checkpoint inhibitor is administered (e.g., third).

[0070] In some embodiments, an anti-cancer drug is administered (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), then an immune checkpoint inhibitor is administered (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., fourth).

[0071] In some embodiments, an anti-cancer drug is administered, followed by a DNA plasmid, followed by an immune checkpoint inhibitor, followed by interval cytoreductive surgery.

[0072] In some embodiments, the interleukin-12 (IL-12) plasmid formulated in lipopolymer (e.g., nanoparticles) is administered at a concentration of about 35 mg / m 2 ~about 80mg / m 2 is administered at a dose of

[0073] In some embodiments, the nanoparticles have a concentration of about 60 mg / m 2 is administered at a dose of

[0074] In some embodiments, the immune checkpoint inhibitor comprises nivolumab, and optionally, the nivolumab is administered at about 240 mg.

[0075] In some embodiments, the nivolumab and nanoparticles are administered every 1 to 4 weeks (eg, every 2 weeks) during treatment.

[0076] In some embodiments, the second inhibitor comprises ipilimumab, and optionally, the ipilimumab is administered at about 1 mg / kg.

[0077] In some embodiments, ipilimumab is administered every 2-8 weeks (eg, every 6 weeks) during treatment.

[0078] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer (e.g., glioblastoma), lung cancer, and any combination thereof, and metastasis of any of the cancers.

[0079] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0080] In some embodiments, the cancer is ovarian cancer.

[0081] In some embodiments, the subject is a human.

[0082] In some embodiments, interleukin-12 (IL-12) plasmid formulated in lipopolymers (eg, nanoparticles) is administered at a dose of about 35 mg / m 2 to about 80 mg / m 2 .

[0083] In some embodiments, interleukin-12 (IL-12) plasmid formulated in lipopolymers (eg, nanoparticles) is administered at about 80 mg / m 2 .

[0084] In some embodiments, interleukin-12 (IL-12) plasmids formulated in lipopolymers (eg, nanoparticles) are administered weekly.

[0085] In some embodiments, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV.

[0086] In some embodiments, the anti-VEGF antibody is administered at a dose of about 15 mg / kg IV.

[0087] In some embodiments, the anti-VEGF antibody is administered every 1 to 6 weeks (eg, every 3 weeks) during treatment.

[0088] In some embodiments, the nucleic acid vector is a plasmid.

[0089] In some embodiments, the lipopolymer is a nanoparticle. [Brief explanation of the drawings]

[0090] [Figure 1] An exemplary hIL-12 expression plasmid is shown.

[0091] [Figure 2] PEG-PEI-cholesterol structure.

[0092] [Figure 3] The difference in tumor weight (mg) compared to the control group after administration of mGEN-1, various dose levels of Bevicuzimab, and mGEN-1 in combination with various dose levels of Bevacizumab is shown.

[0093] [Figure 4] 1 shows various tumor burden levels following tumor implantation in untreated nude Foxn1nu mice, nude Foxn1nu mice treated with Doxil + Bebicuzimab, and nude Foxn1nu mice treated with Doxil + Bebicuzimab + mGEN-1, plotted via IVIS signal quantification.

[0094] [Figure 5] IVIS whole-body imaging shows various tumor burden levels following tumor implantation in untreated nude Foxn1nu mice, nude Foxn1nu mice treated with Doxil + Bebicuzimab, and nude Foxn1nu mice treated with Doxil + Bebicuzimab + mGEN-1.

[0095] [Figure 6] The administration schedules for the NACT + bevacizumab arm and the NACT + bevacizumab + GEN-1 arm of the clinical protocol are shown.

[0096] [Figure 7]The schedules for the NACT + bevacizumab arm and the NACT + bevacizumab + GEN-1 arm of the clinical protocol are outlined.

[0097] [Figure 8A] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8B] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8C] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8D] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8E] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8F] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8G] Survival rates of subjects treated with neoadjuvant chemotherapy (NACT), or NACT plus GEN-1 are shown. [Figure 8H]Figure 8 shows the survival rates of subjects treated with neoadjuvant chemotherapy (NACT) or NACT+GEN-1. Figure 8A shows the progression-free survival for all subjects. Figure 8B shows the progression-free survival for only subjects with known BRCA status. Figure 8C shows the progression-free survival for only subjects with BRCA-positive status. Figure 8D shows the progression-free survival for only subjects with BRCA-negative status. Figure 8E shows the hazard ratio for NACT treatment versus NACT+GEN-1 treatment without stratification for all subjects. Figure 8F shows the hazard ratio for NACT treatment versus NACT+GEN-1 treatment without stratification for subjects with known BRCA status. Figure 8G shows the hazard ratio for NACT treatment versus NACT+GEN-1 treatment by BRCA status. Figure 8H shows the hazard ratio for NACT treatment versus NACT+GEN-1 treatment stratified by BRCA status. DETAILED DESCRIPTION OF THE INVENTION

[0098] 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 plurals 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.

[0099] 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 present disclosure will be apparent from the detailed description and claims.

[0100] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "a" (or "an"), and the terms "one or more" and "at least one" may 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 "plurality."

[0101] 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).

[0102] 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 present disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges and 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.

[0103] 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.

[0104] 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 is 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. Multiple elements of the present disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0105] The term "and / or" 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" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" 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 (single); B (single); and C (single).

[0106] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0107] 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.

[0108] 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 and 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. Additionally, 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.

[0109] 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 by methods taught in this disclosure, which induces a desired biological or pharmacological effect, which may include, but is not limited to, (1) having a prophylactic effect on an organism and preventing 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, reducing, or completely eliminating disease from an organism. The effect may be local, such as providing a local anesthetic effect, or may be systemic.

[0110] 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 living organisms.

[0111] 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.

[0112] 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 stated.

[0113] As used herein, a "derivative" of a carbohydrate includes, 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.

[0114] As used herein, "administering" and similar terms refer to delivering a composition to an individual to be treated so that the composition can circulate systemically, bind to target cells, and be taken up by endocytosis.Therefore, the composition is preferably administered systemically to an individual, typically by subcutaneous, intramuscular, transdermal, intravenous, or intraperitoneal route.Injectables for such use can be prepared in conventional forms, as liquid solutions or suspensions, or as solid forms suitable for preparation as liquid solutions or suspensions before injection, or as emulsions.Suitable excipients that can be used for administration include, for example, water, saline, dextrose, glycerol, ethanol, etc., and, if desired, include minor amounts of auxiliary substances such as wetting agents or emulsifying agents, buffers, etc.

[0115] As used herein, "efficacy" and like terms means tumor disappearance, or reduction in tumor size, or reduction in tumor density, or increase in lymphocyte count, or increase in neutrophil count, or improved survival, or all of the above.

[0116] 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.

[0117] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence operably linked to the promoter / regulatory sequence, which is necessary for expressing a gene product. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. The term "inducible" promoter means that, when operably linked to a polynucleotide encoding a particular gene product, an inducer corresponding to the promoter essentially results in the production of the gene in a cell only when the nucleotide sequence of the product is present in the cell.

[0118] 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 produces a "gene product."

[0119] 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.

[0120] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence 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) incorporating a recombinant polynucleotide.

[0121] 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 when 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.

[0122] 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 into the interior of a cell.Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.The term transfer vector should also be interpreted as including non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc.

[0123] 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.

[0124] "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 of skill in the art can use publicly available computer software. Computer program methods for determining sequence identity include, but are not limited to, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. According to one embodiment, the software alignment program used can be BLAST. 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 are set to the following values: overlap span=1, overlap portion=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 database in which the sequence is being searched. However, the values ​​can be adjusted to increase sensitivity. The percent sequence identity can be determined by dividing (a) the number of matching 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 matching identical amino acid residues 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).

[0125] "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.

[0126] As used herein, the term "homology" or "identity" refers to the identity of subunit sequences between two polymer molecules, for example, 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, for example, if each position in two DNA molecules is occupied by adenine, 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 10-subunit-long polymer) are homologous, the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are identical or homologous, the two sequences are 90% homologous.

[0127] 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" when compared and aligned for maximum correspondence within a comparison window, or designated region, as measured by using one of the following sequence comparison algorithms or by manual alignment and visual inspection; two sequences are "substantially the same" if 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). Optionally, 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.

[0128] 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; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region.

[0129] 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 will usually be located 3' to the coding sequence.

[0130] 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), modifying or altering 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).

[0131] 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 made by combining genetic material from two or more different sources. In some embodiments, a recombinant nucleic acid, recombinant peptide, recombinant cell, or recombinant 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.

[0132] As used herein, the term "isolated" means altered or removed from its 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.

[0133] 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.

[0134] As used herein, the term "primary tumor" refers to the original or first tumor that formed in a subject's body.

[0135] 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.

[0136] 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 substantially bind to other molecules in the sample.

[0137] As used herein, the term "tumor heterogeneity" refers to the fact that after multiple divisions and proliferation during tumor growth, daughter cells of a tumor exhibit molecular biological or genetic changes, resulting in differences in tumor growth rate, invasive ability, drug sensitivity, prognosis, and other aspects, which is one of the characteristics of malignant tumors.

[0138] 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, for example, various neoplasms, including 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.

[0139] As used herein, "ovarian cancer" refers to cancer that originates in or involves the ovaries, e.g., originates in or involves the ovarian epithelium. As used herein, the term "cancer" or "tumor" refers to the uncontrolled growth of cells that interferes 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.

[0140] 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. The cell includes the primary subject cell and its progeny.

[0141] 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, a refractory malignant tumor or a recurrent malignant tumor can be treated using the methods disclosed herein.

[0142] 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.

[0143] As used herein, the term "combination therapy" refers to a therapy that includes multiple treatments (e.g., active agents or procedures). In some embodiments, the combination therapy herein includes at least a gene therapy and one or more of an anti-cancer agent, an antibody having binding specificity for VEGF, and / or an immune checkpoint inhibitor, which may be administered together or separately. In some embodiments, the compositions of the combination therapy are formulated together in a single composition or as separate compositions.

[0144] 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, or slow the progression, onset, severity, or recurrence of undesirable symptoms, complications, conditions, biochemical manifestations of disorders, or diseases, or to achieve beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; reduction in the extent of a condition, disorder, or disease; a stabilized (i.e., non-worsening) state of a condition, disorder, or disease; a delayed onset or delayed progression of a condition, disorder, or disease; an improvement or remission of a 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 a condition, disorder, or disease. In some embodiments, treatment involves eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment includes extending survival compared to expected survival in the absence of 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 a disease and / or adverse effects and / or symptoms resulting from the disease.

[0145] 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.

[0146] 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, an amount that increases, enhances, or prolongs the anti-tumor activity of immune effector cells; increases the number of anti-tumor immune effector cells or activated immune effector cells; or is capable of promoting tumor regression, tumor shrinkage, and / or tumor necrosis.

[0147] As used herein, the term "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.

[0148] The term "excipient" refers to any substance that is not itself a therapeutic agent and that may be used in a composition for delivering an active therapeutic agent to a subject, or that may 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 excipients 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.

[0149] 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 the 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).

[0150] 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 caused by them. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes (T cells), B lymphocytes (B cells), natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by either these cells or the liver, which results in the selective targeting, binding to, damaging, destroying, and / or eliminating from the organism's body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. In some embodiments, the immune response includes, for example, activation or inhibition of T cells, e.g., effector T cells or Th cells, e.g., CD4+ or CD8+ T cells, or inhibition of regulatory T cells (Treg cells).

[0151] As used herein, the term "autologous" refers to any material derived from an individual that is later reintroduced into the same individual.

[0152] 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.

[0153] 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.

[0154] 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 methods (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion-exchange HPLC or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0155] "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.

[0156] 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 (VH and VL, respectively) of native antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR) that contain complementarity-determining regions (CDR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).

[0157] 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 that are arranged in close spatial proximity in the tertiary structure of the Fv region. In one embodiment, the antigen-binding site is defined as a set of six CDRs contained in a cognate VH / VL pair.

[0158] As used herein, the term "complementarity-determining region" or "CDR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and contain 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 within the variable domain (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).

[0159] For purposes herein, an "acceptor human framework" is a framework that comprises 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.

[0160] 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, namely, 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.

[0161] 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.

[0162] As used herein, the term "epitope" refers to a site on either a proteinaceous or non-proteinaceous antigen to which an antibody binds. Epitopes can be formed from a contiguous stretch of amino acids (linear epitopes) or can include discontinuous amino acids that are spatially adjacent, for example, due to antigen folding, i.e., tertiary folding of a proteinaceous antigen (conformational epitopes). Linear epitopes are typically still bound by antibodies after exposing a 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.

[0163] 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, including, 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)).

[0164] Antigen Structure-Based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows for the identification of multiple monoclonal antibodies that specifically bind to an antigen based on the binding profile of each antibody to multiple chemically or enzymatically modified antigen surfaces (see, e.g., U.S. Patent No. 2004 / 0101920). Antibodies in each bin bind to the same epitope, which may be a unique epitope that is distinct from or partially overlaps with an epitope represented by another bin. Competitive binding can also be used to easily determine whether an antibody binds to the same epitope as a reference antibody or competes for binding with the reference antibody. For example, an "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competitive assay; conversely, the reference antibody blocks 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 antibody, the reference antibody is bound to the antigen under saturating conditions.After removing excess reference antibody, the ability of the antibody to bind to the antigen is evaluated.If the antibody can bind to the antigen after saturating binding of the reference antibody, it can be concluded that the antibody binds to a different epitope than the reference antibody.However, if the antibody cannot bind to the antigen after saturating binding of the reference antibody, the antibody may bind to the same epitope as the epitope bound by the reference antibody.To confirm whether the antibody binds to the same epitope or whether binding is simply hindered by steric reasons, routine experiments can be used (for example, 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, that is, in the setting where both antibodies are saturating antibodies.In both settings, if only the first (saturating) antibody is able to bind to the antigen of interest, it can be concluded that the antibody and the reference antibody compete for binding to the same antigen.

[0165] Sometimes, two antibodies are considered to bind to 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 reduce or eliminate binding of the other.

[0166] 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 tumor cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be expressed by the ability of the peptides, polynucleotides, cells, and antibodies of the present disclosure to prevent or reduce the frequency of tumor formation.

[0167] As used herein, the term "chemotherapy" or "chemotherapeutic agent" refers 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.

[0168] 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.

[0169] Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors.Typically, VEGF refers to any natural VEGF derived from any vertebrate source, 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 and any form of VEGF resulting from intracellular processing.The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants.VEGF dimer refers to a homodimer of two identical VEGF molecules.The complex formed by two identical antibody molecules bound to a VEGF dimer is referred to herein as a VEGF dimer-antibody complex.

[0170] 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."

[0171] 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.

[0172] 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 domains 2 or 3 of VEGF-R2, respectively.

[0173] 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 an unrelated, non-VEGF protein 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 <1 nM or <0.15 nM. An antibody is said to "specifically bind" to VEGF if it has a KD of 1 μM or less.

[0174] "VEGFR 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 "VEGFR blocking selectivity."

[0175] As used herein, "BRCA positive" or "BRCA+" refers to a patient or cancer that tests positive for a mutation in one of the breast cancer genes, BRCA1 or BRCA2.

[0176] As used herein, "BRCA negative" or "BRCA-" refers to a patient or cancer that tests negative for a mutation in one of the breast cancer genes, BRCA1 or BRCA2 (i.e., a wild-type BRCA gene).

[0177] 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.

[0178] 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 that are within the skill of those in the art, and such techniques are fully explained in the literature.

[0179] II.GEN-1 (IL-12 DNA nanoparticles) In animal models, recombinant IL-12 has been demonstrated to induce significant T cell-mediated antitumor effects, leading to 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 recent human clinical trials (see 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.

[0180] In animal models of cancer, both viral and nonviral gene delivery systems have been used for IL-12 gene delivery. Viral approaches have serious practical limitations due to toxicity concerns, primarily due to the increased incidence of cancer and the strong immune response to viral antigens by the host system. Due to their low toxicity, there has been considerable interest in developing nonviral gene delivery systems. The use of polyvinylpyrrolidone (PVP), a nonviral gene delivery system, has been demonstrated to deliver IL-12 to treat renal cell carcinoma (Renca) and colon cell carcinoma (CT26). See Gene Ther., 1999, vol. 6, 833. When tumors were subjected to this gene therapy, they exhibited all the characteristics of IL-12 protein therapy, including increased infiltration of NK cells, CD4 T cells, 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.

[0181] 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 binding to the IL-12 receptor. Ibid. The precursor form of the IL-12 p40 subunit (e.g., NM_002187; NP_002178; 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 (e.g., NM_000882; NP_000873; 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.

[0182] IL-12 protein has been tested as a promising anti-cancer therapeutic agent since 1994 due to its ability to activate both NK cells and cytotoxic T cells. See Nastala, C. L. et al., J Immunol 153:1697-1706 (1994). However, despite high expectations, early clinical trials did not produce satisfactory results. Lasek, W. et al., Cancer Immunol Immunother 63:419-435, 424 (2014). Repeated administration of IL-12 resulted in an adaptive response and a gradual decline in circulating IL-12-induced interferon gamma (IFN-γ) levels in most patients. Ibid. Furthermore, it is recognized that IL-12-induced anticancer activity is primarily mediated by secondary secretion of IFN-γ, but the simultaneous induction of IFN-γ by IL-12 along with other cytokines (e.g., TNF-α) or chemokines (IP-10 or MIG) caused severe toxicity.

[0183] In addition to negative feedback and toxicity, the marginal effectiveness of IL-12 therapy in clinical settings may be caused by the strongly immunosuppressive environment in humans.

[0184] Furthermore, secondary effects of cytokine IL-12 production, i.e., 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 even better efficacy, indicating insufficient delivery by this 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. See Gene Ther., 2002, vol. 9, 1075. These results support the need for more efficient delivery systems. Despite previous limitations in 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.

[0185] To achieve the desired results from combination approaches involving gene therapy drugs, 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) is the water-soluble lipopolymer PEI-cholesterol (WSLP).

[0186] IL-12 is primarily secreted by antigen-presenting cells (phagocytes, dendritic cells) in response to pathogens and promotes the differentiation of CD4+ cells into Th1 cells. It has a positive synergistic proliferative effect on preactivated NK cells and T cells, independently and / or synergistically enhancing the cytolytic capacity of both NK cells and CD8+ T cells by upregulating genes encoding cytotoxic granule-associated proteins. It also increases ADCC of antibody-coated tumors at concentrations significantly lower than IL-2. IL-12-stimulated effector cells produce several cytokines, including GMCSF and TNF-α, but primarily IFN-γ. IL-12 consists of p35 and p40 subunits, the latter of which is also shared by IL-23. The IL-12 receptor (IL-12R) consists of two chains, IL-12Rβ1 and IL-12Rβ2, and signals primarily via STAT4. IL-12R is expressed primarily by activated NK cells and T cells, is barely detectable on resting T cells, but is expressed at low levels on NK cells, which likely explains their rapid response to IL-12. Nevertheless, TCR activation and costimulation such as B7, IFN-α, IFN-γ, and IL-12 per se upregulate IL-12R expression (especially IL-12Rβ2).

[0187] Preclinical studies on various tumor cell lines have established the antitumor activity of systemically or locally administered IL-12. Dose- and model-dependent responses, as well as memory antitumor effects, have been observed. Other studies suggest that IL-12 is more effective against early-stage or microscopic tumors than advanced disease. T cell responses are involved in its antitumor activity, and NK / NKT cell activation appears to prevent metastasis. In addition to effector T cell (Teff)-mediated tumor control, IL-12 inhibits tumor-derived regulatory T cells (Tregs) by either suppressing T cell IL-2 production, inducing apoptosis, or IFN-γ-mediated cell arrest, thus enhancing the Teff / Treg ratio both in vitro and in vivo. IL-12 has also been reported to mediate the reprogramming of intratumoral myeloid-derived suppressor cells (MDSCs) in a B16 melanoma model, enhancing CTL activity and reversing their suppressive role in vivo. IL-12Rβ2 has also emerged as a potential tumor suppressor gene, particularly in hematological malignancies. Epigenetic IL-12Rβ2 gene silencing by promoter hypermethylation or gene downregulation has been observed in multiple B-cell myeloma specimens, and IL-12 significantly reduced tumor burden upon receptor restoration. IL-12Rβ2- / - mice consistently develop spontaneous malignancies and lymphoproliferative disorders. IL-12 also has "direct" antitumor activity in IL-12Rβ1-expressing tumors via IFN-γ-mediated upregulation of MHC class I molecules.

[0188] Although early clinical trials were temporarily halted after unexpected toxicity-related deaths were observed in a phase II study in patients with metastatic RCC and melanoma, a prior phase I study had established a safe MTD. This was attributed to a small but biologically significant change in the treatment schedule, which involved eliminating the test dose before the 5-day treatment course. Evaluation suggested that the test dose attenuated the subsequent effect of IL-12 on IFN-γ production through an unknown mechanism. Various IL-12 regimens were subsequently tested, but clinical results were modest in patients with solid tumors, with more promising results seen in hematologic malignancies. Because IL-12 is a key mediator of Th1 responses, efforts focused on using IL-12 as an adjunct to vaccine therapy against tumor-associated antigens for both resectable and metastatic disease. In general, IL-12 improved immune responses, but this did not translate into clinically significant antitumor effects.

[0189] Demonstration in preclinical models that locally delivered IL-12 is effective without compromising systemic administration has prompted research into the development of methods for intratumoral (IT) expression of IL-12. Studies of IT injection of IL-12 into primary tumors in patients with head and neck cancer have demonstrated induction of B cell activation, which correlates with increased survival. Enabling tumor cells to produce IL-12 has attracted interest, and several small studies using viral vectors or plasmid DNA have tested this approach, but results have been limited. Antibody formation against viral vectors and the unproven efficiency of gene delivery have been limiting factors. A novel method for locally administering IL-12 involves injection of IL-12-encoding plasmid DNA followed by electroporation of superficial tumors to promote cell entry. Results from a phase I study demonstrated antitumor activity at both the local injection site and non-systemic non-injection sites. Two trials in patients with gynecological malignancies evaluated IL-12 plasmids formulated in PEG-PEI-cholesterol lipopolymer delivered intraperitoneally, but the clinical benefit was modest. Current trials are also testing IL-12 as a gene therapy, including electroporation-mediated plasmid transfer, adenoviral vectors combined with orally administered activating ligands, or mesenchymal engineered cells for IL-12 expression. NHS-IL-12 is a novel immunokine consisting of two IL-12 molecules fused to a tumor necrosis-targeting human IgG1 with an even longer half-life. NHS-IL-12 is currently in phase II clinical trials.

[0190] Carson et al. conducted an NCI-sponsored phase I trial to determine the safety and optimal biological dose of IL-12 when administered in combination with trastuzumab. Patients with metastatic HER2-positive malignancies received trastuzumab (initially 4 mg / kg, then 2 mg / kg) on ​​day 1 of each weekly cycle. Patients also received intravenous (iv) injections of IL-12 on days 2 and 5 of the weekly cycle, starting in week 3. Within cohorts of three patients, the dose of the IL-12 component was escalated (30, 100, 300, or 500 ng / kg). Fifteen patients were treated. The regimen was well tolerated, with no drug-related grade 3 or 4 toxicities. Evaluation of dose-limiting toxicities and biological endpoints suggested that the 300 ng / kg dose was the maximum tolerated and optimal biological dose of IL-12 for use in combination with trastuzumab. Correlation assays demonstrated sustained IFN-γ production by NK cells in only three patients who responded favorably to the regimen. Enhanced circulating levels of NK cell-derived chemokines (MIP-1α, IL-8, and RANTES) were also detected in these three patients. These factors exert potent chemotactic effects on naive and activated T cells, and their presence correlated with tumor tissue infiltration by CD8+ T cells. Importantly, NK cell-mediated ADCC against tumor targets in vitro did not appear to correlate with clinical response or dose of IL-12. Thus, as expected, clinical response to IL-12 / trastuzumab therapy was associated with immune cell production of IFN-γ and chemokines.

[0191] Carson et al. conducted a follow-up study using IL-12 in combination with trastuzumab and paclitaxel. 175 mg / m every 3 weeks 2Paclitaxel was administered intravenously (iv) in 100 patients. Starting with cycle 2, trastuzumab was administered on day 1 of each week (initially 4 mg / kg, then 2 mg / kg) in combination with IL-12 injections on days 2 and 5. This study enrolled 21 patients with metastatic 2+ and 3+ HER2-positive tumors, 16 of whom had previously received chemotherapy. The dose of the IL-12 component was escalated (100 ng / kg, then 300 ng / kg) in a cohort of three patients; however, due to dose-limiting grade 3 fatigue at the 300 ng / kg dose level, the IL-12 component was reduced to 200 ng / kg subcutaneously. Overall, a 52% rate of clinical benefit was observed. Clinical benefit was accompanied by a significant increase in ERK activation in peripheral blood mononuclear cells and an increase in plasma levels of IFN-γ (complete response, partial response, or stable disease), but not in patients with progressive disease. Patients with progressive disease did not have measurable levels of IFN-γ in their circulation. Within any one cycle, IFN-γ levels typically peaked after IL-12 injection (range 124–1612 pg / ml) and then declined to baseline. Analysis of patient time-to-progression data using the nonparametric Mann-Whitney U test showed that IFN-γ induction was associated with a statistically significant increase in progression-free survival (p=0.004). Intracellular flow cytometry analysis of cryopreserved PBMCs from day 5 of the treatment cycle revealed high levels of IFN-γ within CD56+ NK cells only in patients who demonstrated a clinical response or stabilization of disease. Circulating levels of MIP-1α also correlated with clinical response, as did levels of IP-10 and MIG, two antiangiogenic factors induced by IFN-γ.

[0192] The GOG trial evaluated GEN-1 in 16 patients with persistent or recurrent platinum-resistant EOC. The trial consisted of intravenous pegylated liposomal doxorubicin (PLD) 40 mg / m every 28 days. 2 (dose levels 1 and 2) or 50 mg / m 2 (dose level 3), and 24 mg / m on days 1, 8, 15, and 22 of a 28-day cycle2 (dose level 1) or 36 mg / m 2 High-dose GEN-1 was evaluated in combination with intraperitoneal GEN-1 (at dose levels 2 and 3). Cycles were repeated every 28 days until disease progression. In 14 patients with measurable disease, a clinical benefit of 57.1% (PR=21.4%; SD=35.7%) was observed. The greatest number of partial responses (28.6%) and stable disease (57.1%) was observed at dose level 3. The maximum tolerated dose was not reached. Increased levels of IL-12, IFN-γ, and TNF-α were observed in the ascites after GEN-1 treatment.

[0193] Ovarian cancer is the fifth most common form of cancer affecting women, the most lethal gynecological malignancy, and the fourth leading cause of cancer deaths in women. First-line chemotherapy regimens for the treatment of ovarian cancer are typically platinum-based combination therapies administered intravenously (IV) every 21–28 days for four to six treatments. While nearly 90% of women initially respond to platinum-based therapy, 55–75% of women develop recurrent ovarian cancer within two years. Second- and third-line therapies are generally ineffective and toxic, including immune checkpoint inhibitors, which have response rates of 11–15% in patients with platinum-resistant recurrent disease. Safer and more effective therapies for the treatment of ovarian cancer are urgently needed.

[0194] Cancers can evade detection and destruction by the immune system, despite the fact that many tumors elicit strong immune responses evident in lymphocytic infiltration of primary lesions. Tumor immune evasion can be categorized into induction of immune tolerance and resistance to killing by activated immune effector cells. The "immunoediting" hypothesis suggests that tumors manipulate their microenvironment by creating complex local and regional immunosuppressive networks involving various tumor-derived cytokines and other soluble factors. Thus, by the time tumors become clinically detectable, they have already evolved mechanisms to evade host-initiated immune responses against them. Such resistance mechanisms must be overcome to generate effective and sustained antitumor immunity. One of the most promising strategies for enhancing patients' antitumor responses appears to be the use of antibodies that block immune regulatory mechanisms that can suppress host responses to tumor-associated antigens, with remarkable results demonstrated so far by CTLA4- and PD1 / PDL1-blocking monoclonal antibodies. However, a significant number of patients with solid tumors do not respond to these agents. In these cases, the tumor microenvironment (TME) is thought to be less immunogenic, and pro-inflammatory cytokines such as IL-12 may play an important role in transforming the TME, acting synergistically with CTLA4 blockade and PD1 blockade.

[0195] GEN-1 nanoparticles contain a DNA plasmid encoding the IL-12 gene and a synthetic polymer that facilitates plasmid delivery. GEN-1 is designed to be delivered locally (e.g., intraperitoneally), offering the potential for cytokine expression specifically within the tumor microenvironment, with the goal of achieving increased efficacy while minimizing potential systemic toxicity. GEN-1 is being tested in subjects with recurrent ovarian cancer as a single agent or in combination with standard chemotherapy. Safety and tolerability are assessed using a 0.6 mg / m2 dose administered weekly every 4 weeks. 2 , 3 mg / m 2 , 12 mg / m 2 and 24 mg / m 2A phase I monotherapy trial evaluating 13 patients with platinum-resistant EOC treated with IP GEN-1 at 12 mg / m IP. The most common adverse events (AEs) were fever and abdominal pain. A significant increase in IFN-γ was observed in the ascites but not in serum. Escalating doses of IP GEN-1: 12 mg / m IP. 2 , 18 mg / m 2 or 24 mg / m 2 ×4 doses or 24 mg / m 2 Another phase I study was conducted in patients with platinum-sensitive EOC treated with intravenous (IV) carboplatin and docetaxel at 6 to 8 doses each. Similar AEs and a dose-dependent increase in IFN-γ concentrations were observed. No dose-limiting toxicities (DLTs) were observed, and the MTD was not reached. The addition of GEN-1 to chemotherapy did not reduce efficacy or worsen side effects.

[0196] The GOG trial evaluated GEN-1 in 16 patients with persistent or recurrent platinum-resistant EOC. The trial consisted of intravenous pegylated liposomal doxorubicin (PLD) 40 mg / m every 28 days. 2 (dose levels 1 and 2) or 50 mg / m 2 (dose level 3), and 24 mg / m on days 1, 8, 15, and 22 of a 28-day cycle 2 (dose level 1) or 36 mg / m 2 High-dose GEN-1 was evaluated in combination with intraperitoneal GEN-1 (at dose levels 2 and 3). Cycles were repeated every 28 days until disease progression. In 14 patients with measurable disease, a clinical benefit of 57.1% (PR=21.4%; SD=35.7%) was observed. The greatest number of partial responses (28.6%) and stable disease (57.1%) was observed at dose level 3. The maximum tolerated dose was not reached. Increased levels of IL-12, IFN-γ, and TNF-α were observed in the ascites after GEN-1 treatment.

[0197] Translational research findings demonstrate that GEN-1 is biologically active in ovarian cancer patients, promoting the dynamics of pro-immune T cell populations within the tumor microenvironment and the conversion of tumor-naive T cells into cytotoxic effector T cells. To date, over 100 ovarian cancer patients have been administered GEN-1. The most common adverse events attributed to high-dose GEN-1 are abdominal pain, nausea, fatigue, fever, vomiting, and diarrhea.

[0198] In some embodiments, the GEN-1 DNA plasmid is delivered using a lipopolymer, a synthetic polymer that facilitates plasmid delivery.

[0199] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group.

[0200] The present disclosure provides a polymer system, PEG-PEI-cholesterol (PPC), which differs from WSLP (PEI-cholesterol) in that it contains PEG moieties, resulting in significantly higher transfection efficiency within 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 directly linked to the PEI backbone of the prior art (WSLP) can be extended further 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.

[0201] 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 immune checkpoint inhibitor.

[0202] 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), the nucleic acid vector (e.g., a plasmid) formulated in a lipopolymer (e.g., a nanoparticle), and (ii) an immune checkpoint inhibitor.

[0203] In some embodiments, the polynucleotide encodes human IL- 12. In some embodiments, the polynucleotide encodes the p35 subunit of IL-12 and the p40 subunit of IL-12.

[0204] 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.

[0205] 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: 94. 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: 95.

[0206] In some embodiments, the human IL-12 p35 comprises the following sequence:

number

[0207] In some embodiments, the human IL-12 p40 comprises the following sequence:

number

[0208] 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: 96. 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:97.

[0209] In some embodiments, the polynucleotide encoding human IL-12 p35 comprises the following sequence:

number

[0210] In some embodiments, the polynucleotide encoding human IL-12 p35 comprises the following sequence:

number

[0211] 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, an element of Figure 1).

[0212] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group (eg, the lipopolymer of Figure 2).

[0213] In some embodiments, the nanoparticles disclosed herein comprise a DNA plasmid encoding human IL-12.

[0214] In some embodiments, the nanoparticles comprise a synthetic polymer that facilitates plasmid delivery, which is a lipopolymer.

[0215] In some embodiments, the lipopolymer further comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group.

[0216] 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 the polyethylene glycol spacer, and the polyethylene glycol spacer is attached to the PEI via a biocompatible bond.

[0217] 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 phosphodiesterase (e.g., phosphodiesterase B) (endothelial cells), N-acetyllactosamine (T cells), galactose (colon cancer cells), and thrombomodulin (mouse pulmonary endothelial cells), a fusogenic agent such as polymyxin B and hemagglutinin HA2, a lysosomotrophic agent, a nuclear localization signal (NLS), such as a T antigen. Another gene delivery polymer is a non-condensing polymer selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, poly(lactide-co-glycolide) (PLGA), and a triblock copolymer of PLGA and PEG. The gene delivery polymer may also be a non-condensing polymer. Examples of such non-condensing polymers include polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, polyglutamate, gelatin, polyphosphoester, silk-elastin-like hydrogel, agarose hydrogel, lipid microtubules, poly(lactide-co-glycolide), and polyethylene glycol-conjugated poly(lactide-co-glycolide).

[0218] 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 directly bound to the polyethyleneimine backbone or covalently bound to the polyethylene glycol spacer, and the polyethylene glycol spacer is bound to the PEI via a biocompatible bond.

[0219] In some embodiments, the gene delivery polymer comprises a lipopolyamine having the following formula:

[0220] [ka] (Staramine).

[0221] In some embodiments, the gene delivery polymer comprises a mixture of lipopolyamine and an alkylated derivative of lipopolyamine. In some embodiments, the alkylated derivative of lipopolyamine is polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinyl ether maleic anhydride. In some embodiments, the alkylated derivative of lipopolyamine has the following formula:

[0222] [ka] (Methoxypolyethylene glycol (mPEG) modified stalamine),

[0223] wherein n represents an integer of 10 to 100 repeating units each containing 2 to 5 carbon atoms. In some embodiments, the alkylated derivatives of lipopolyamines have the formula:

[0224] [ka]

[0225] wherein n=11 (stalamine-mPEG515). In some embodiments, the alkylated derivative of lipopolyamine has the formula:

[0226] [ka] (Stalamine-mPEG11).

[0227] In some embodiments, the ratio of lipopolyamine to alkylated derivative of lipopolyamine in the mixture is 1:1 to 10:1. In some embodiments, the lipopolyamine is present in an amount sufficient to provide a ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector of about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1). In some embodiments, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector is about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some embodiments, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector is about 1:10 to about 10:1.

[0228] In some embodiments, the gene delivery polymer comprises a lipopolyamine having the following formula:

[0229] [ka] (Crossamine).

[0230] In some embodiments, the gene delivery polymer comprises a mixture of lipopolyamine and an alkylated derivative of lipopolyamine. In some embodiments, the alkylated derivative of lipopolyamine is polyoxyalkylene, polyvinylpyrrolidone, polyacrylamide, polydimethylacrylamide, polyvinyl alcohol, dextran, poly(L-glutamic acid), styrene maleic anhydride, poly-N-(2-hydroxypropyl) methacrylamide, or polydivinyl ether maleic anhydride. In some embodiments, the ratio of lipopolyamine to alkylated derivative of lipopolyamine in the mixture is 1:1 to 10:1. In some embodiments, the lipopolyamine is present in an amount sufficient to provide a ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector of about 0.01:1 to about 50:1 (e.g., about 0.01:1 to about 40:1; about 0.01:1 to about 30:1; about 0.01:1 to about 20:1; about 0.01:1 to about 10:1, or about 0.01:1 to about 5:1). In some embodiments, the ratio of amine nitrogen in the lipopolyamine to phosphate in the nucleic acid vector is about 0.1:1 to about 50:1 (e.g., about 0.1:1 to about 40:1; about 0.1:1 to about 30:1; about 0.1:1 to about 20:1; about 0.1:1 to about 10:1, or about 0.1:1 to about 5:1). In some embodiments, the ratio of amine nitrogens in the lipopolyamine to phosphate in the nucleic acid vector is about 1:10 to about 10:1.

[0231] In some embodiments, the gene delivery polymer comprises a poloxamer backbone having a metal chelator covalently attached to at least one end of the poloxamer backbone. In some embodiments, the metal chelator is attached to at least two ends of the poloxamer backbone. In some embodiments, the poloxamer backbone is a poloxamer backbone disclosed in U.S. Patent Publication No. 2010 / 0004313, the entire contents of which are incorporated herein by reference. In some embodiments, the metal chelator is a metal chelator disclosed in U.S. Patent Publication No. 2010 / 0004313. In some embodiments, the gene delivery polymer is a polymer having the formula:

[0232] [ka]

[0233] and pharmaceutically acceptable salts thereof, wherein:

[0234] A represents an integer from 2 to 141,

[0235] B represents an integer from 16 to 67,

[0236] C represents an integer of 2 to 141;

[0237] R A and R C are the same or different and are R′-L-, or H, and at least one of R A and R C is R′-L-;

[0238] L is a bond, —CO—, —CH—O—, or —O—CO—;

[0239] R' is a metal chelator.

[0240] In some embodiments, the metal chelator is RNNH—, RN2N—, or (R″—(N(R″—CH2CH2)x)2-N—CH2CO—, where each x is independently 0 to 2, and R″ is HO2C—CH2—. In some embodiments, the metal chelator is a crown ether selected from the group consisting of 12-crown-4, 15-crown-5, 18-crown-6, 20-crown-6, 21-crown-7, and 24-crown-8. In some embodiments, the crown ether is a substituted crown ether, wherein the substituted crown ether has the following:

[0241] (1) one or more crown ether oxygens independently substituted by NH or S;

[0242] (2) one or more crown ether -CH-CH- moieties substituted by -C6H4-, -C10H6-, or -C6H10-;

[0243] (3) one or more crown ether -CH2-O-CH2- moieties substituted by -C4H2O-, or -C5H3N-; or

[0244] (4) Any combination thereof.

[0245] In some embodiments, the metal chelator is a cryptand, wherein the cryptand is selected from the group consisting of a (1,2,2) cryptand, a (2,2,2) cryptand, a (2,2,3) cryptand, and a (2,3,3) cryptand. In some embodiments, the cryptand is a substituted cryptand, wherein the substituted cryptand has:

[0246] (1) one or more of the cryptand ether oxygens independently substituted by NH or S;

[0247] (2) one or more crown ether -CH-CH- moieties substituted by -C6H4-, -C10H6-, or -C6H10-;

[0248] (3) one or more crown ether -CH2-O-CH2- moieties substituted by -C4H2O-, or -C5H3N-; or

[0249] (4) Any combination thereof.

[0250] In some embodiments, the gene delivery polymer is a Crown Poloxamer (aza-crown linked poloxamer), a crown poloxamer is a polymer having the following formula:

[0251] [ka]

[0252] or a pharmaceutically acceptable salt thereof, wherein:

[0253] a represents an integer of approximately 10 units,

[0254] b represents an integer of approximately 21 units;

[0255] The total molecular weight of the polymer is about 2,000 Da to about 2,200 Da.

[0256] In some embodiments, the gene delivery polymer is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0257] In some embodiments, the gene delivery polymer is a β-amino ester. In some embodiments, the polymer is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0258] In some embodiments, the gene delivery polymer is polyinosinic-polycytidylic acid, which is present in a solution containing the nucleic acid vector at about 0.1% to about 5%, or about 0.5% to about 5%.

[0259] In some embodiments, the gene delivery polymer further comprises benzalkonium chloride.

[0260] In some embodiments, the gene delivery polymer comprises BD15-12. In some embodiments, the ratio of nucleotide to BD15-12 polymer (N:P) is 5:1.

[0261] In some embodiments, the gene delivery polymer comprises Omnifect. In some embodiments, the ratio of nucleotides to Omnifect polymer (N:P) is 10:1.

[0262] In some embodiments, the gene delivery polymer comprises a crown poloxamer (aza-crown-linked poloxamer). In some embodiments, the ratio of nucleotide to crown poloxamer (N:P) is 5:1. In some embodiments, the gene delivery polymer comprises a crown poloxamer and a PEG-PEI-cholesterol (PPC) lipopolymer. In some embodiments, the gene delivery polymer comprises a crown poloxamer and benzalkonium chloride. In some embodiments, the gene delivery polymer comprises a crown poloxamer and Omnifect. In some embodiments, the gene delivery polymer comprises a crown poloxamer and linear polyethyleneimine (LPEI). In some embodiments, the gene delivery polymer comprises a crown poloxamer and BD15-12.

[0263] In some embodiments, the gene delivery polymer comprises stalamine and mPEG-modified stalamine. In some embodiments, the mPEG-modified stalamine is stalamine-mPEG515. In some embodiments, the mPEG-modified stalamine is stalamine-mPEG11. In some embodiments, the ratio of stalamine to mPEG-modified stalamine is 10:1. In some embodiments, the nucleotide to polymer (N:P) ratio is 5:1. In some embodiments, the gene delivery polymer comprises stalamine, mPEG-modified stalamine, and crown poloxamer. In some embodiments, the gene delivery polymer comprises stalamine, stalamine-mPEG515, and crown poloxamer. In some embodiments, the gene delivery polymer comprises stalamine, stalamine-mPEG11, and crown poloxamer.

[0264] In some embodiments, the gene delivery polymer comprises a poloxamer backbone, as disclosed in WO 2022 / 072910, which is incorporated by reference in its entirety.

[0265] In some embodiments, nanoparticles comprising a DNA plasmid encoding interleukin-12 (IL-12) and a synthetic polymer that facilitates plasmid delivery are delivered intraperitoneally.

[0266] In some embodiments, the nanoparticles have a concentration of about 35 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 40 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 45 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 50 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 55 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 60 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 65 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 70 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 75 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 75mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 70mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 65mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 60mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 55mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2~about 50mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 45mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 40mg / m 2 is administered at a dose of

[0267] In some embodiments, the nanoparticles have a concentration of about 35 mg / m 2 , about 40mg / m 2 , about 45mg / m 2 , about 50mg / m 2 , about 55mg / m 2 , about 60mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , about 75mg / m 2 or approximately 80 mg / m 2 is administered at a dose of

[0268] In some embodiments, the nanoparticles have a concentration of about 60 mg / m 2 is administered at a dose of

[0269] III. Immune Checkpoint Inhibitors Immune checkpoint proteins interact with specific ligands that send signals to T cells that inhibit T cell function. Cancer cells exploit this by promoting high levels of checkpoint proteins on their surface, thereby suppressing anti-cancer immune responses.

[0270] Immune checkpoint inhibitors include any compound capable of inhibiting the function of an immune checkpoint protein. Inhibition includes reduction and complete blockage of function. In some embodiments, the immune checkpoint protein is a human checkpoint protein.

[0271] In some aspects, the immune checkpoint inhibitor is an antagonist of an immune checkpoint protein. In some aspects, the immune checkpoint inhibitor is an agonist of an immune checkpoint protein.

[0272] In some embodiments, the immune checkpoint protein is selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG-3, VISTA, KIR, BTLA, TIGIT, IDO-1, CEA, PVRIG, GARP, STING, Siglec-15, CD20, CD27, CD38, CD39, CD47, CD66a (CEACAM1), CD73, CD80, CD86, CD93, CD96, and / or CD161.

[0273] In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0274] In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor.

[0275] In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that specifically binds to LAG-3.

[0276] In some embodiments, the immune checkpoint inhibitor is an antibody. In some embodiments, the immune checkpoint inhibitor comprises an antibody or fragment thereof that specifically binds to an immune checkpoint protein. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody, fully human antibody, chimeric antibody, humanized antibody, or fragment thereof that can at least partially antagonize an immune checkpoint protein.

[0277] In some embodiments, the immune checkpoint inhibitor comprises a heavy chain variable region (VH) amino acid sequence and a light chain variable region (VL) amino acid sequence disclosed in Table 1. In some embodiments, the immune checkpoint inhibitor comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence disclosed in Table 2. In some embodiments, the immune checkpoint inhibitor comprises a VH complementarity-determining region (CDR)1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 disclosed in Table 3.

[0278] [Table 1-1] [Table 1-2]

[0279] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0280] [Table 3-1] [Table 3-2] [Table 3-3]

[0281] In some embodiments, the immune checkpoint inhibitor is ipilimumab. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 1 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 1.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 2 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 2.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 3 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 3.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 4 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 4 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 3 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 2.5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 2 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 1.5 mg / kg.

[0282] In some embodiments, ipilimumab is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, or about 5 mg / kg.

[0283] In some embodiments, ipilimumab is administered at a dose of about 1 mg / kg.

[0284] In some embodiments, ipilimumab is administered every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0285] In some embodiments, ipilimumab is administered every 2-8 weeks (eg, every 6 weeks) during treatment.

[0286] In some embodiments, ipilimumab is administered intravenously.

[0287] In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 140 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 160 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 180 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 200 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 220 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 240 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 340 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 320 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 300 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 280 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 260 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 220 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 200 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 180 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 160 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 140 mg. In some embodiments, nivolumab is administered at a dose of about 240 mg to about 360 mg.In some embodiments, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 140 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 160 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 180 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 200 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 220 mg to about 240 mg.

[0288] In some embodiments, nivolumab is administered at a dose of about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, or about 360 mg.

[0289] In some embodiments, nivolumab is administered at a dose of about 240 mg.

[0290] In some embodiments, nivolumab is administered intravenously.

[0291] In some embodiments, nivolumab is administered every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0292] In some embodiments, nivolumab is administered every 1 to 4 weeks (e.g., every 2 weeks) during treatment.

[0293] PD-1 Human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity are disclosed in U.S. Patent Nos. 8,008,449 and 8,779,105. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication No. WO 2012 / 145493. Each of the anti-PD-1 HuMAbs disclosed in U.S. Patent No. 8,008,449 has been demonstrated to exhibit one or more of the following characteristics: (a) a specific binding affinity of 1×10 to PD-1 as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D (b) binds to human PD-1 in a mixed lymphocyte reaction (MLR) assay; (b) does not substantially bind to human CD28, CTLA-4, or ICOS; (c) increases T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and cynomolgus PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates an antigen-specific memory response; (i) stimulates an Ab response; and (j) inhibits tumor cell growth in vivo. In some embodiments, the anti-PD-1 antibodies of the combination therapy disclosed herein comprise a mAb that specifically binds human PD-1 and exhibits at least one of the foregoing characteristics.

[0294] In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab ("OPDIVO®"; also known as BMS-936558; formerly known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In some embodiments, the anti-PD-1 antibody or a fragment thereof binds to the same epitope as nivolumab. In some embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0295] Anti-PD-1 antibodies useful in the disclosed compositions also include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105; International Publication No. WO 2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that they bind to the same epitope region of the antigen and sterically interfere with the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of nivolumab due to their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., International Publication No. WO 2013 / 173223).

[0296] In certain embodiments, the antibody or antigen-binding fragment thereof that cross-competes with nivolumab for binding to human PD-1 or binds to the same epitope region of human PD-1 as nivolumab is a mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, humanized antibodies, or human antibodies. Such chimeric mAbs, humanized mAbs, or human mAbs can be prepared and isolated by methods well known in the art.

[0297] In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®," lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 (S228P) antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587. Pembrolizumab has been approved by the FDA for the treatment of recurrent or refractory melanoma.

[0298] In some embodiments, the anti-PD-1 antibody is REGN2810. In some embodiments, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011). In some embodiments, the anti-PD-1 antibody is the monoclonal antibody MEDI0608 (formerly AMP-514). MEDI0608 is described, for example, in U.S. Patent No. 8,609,089. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is the humanized monoclonal antibody BGB-A317. BGB-A317 is described in U.S. Patent Publication No. 2015 / 0079109. Antibodies or antigen-binding fragments thereof that bind to the same epitope or have the same CDRs as any of these antibodies can be used.

[0299] Anti-human PD-1 antibodies (or VH and / or VL domains derived therefrom) suitable for use as disclosed herein can be generated using methods well known in the art. Alternatively, art-recognized anti-PD-1 antibodies can be used.

[0300] In embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO 2008 / 156712), PDR001 (Novartis; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al. al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; see WO 2015 / 35606 and U.S. Patent No. 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO 2015 / 085847; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al., J. Hematol. Oncol.10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), and IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540), which references are incorporated herein by reference.

[0301] Other anti-PD-1 monoclonal antibodies are described in, for example, U.S. Patent No. 6,808,710, U.S. Patent No. 7,488,802, U.S. Patent No. 8,168,757, and U.S. Patent No. 8,354,509, U.S. Patent Publication No. 2016 / 0272708, and PCT Publication No. WO 2012 / 145493, WO 2012 / 145496, each of which is incorporated herein by reference. 008 / 156712, WO 2015 / 112900, WO 2012 / 145493, WO 2015 / 112800, WO 2014 / 206107, WO 2015 / 35606, WO 2015 / 085847, WO 2014 / 179664, WO 2017 / 020291, WO 201 7 / 020858, WO 2016 / 197367, WO 2017 / 024515, WO 2017 / 025051, WO 2017 / 123557, WO 2016 / 106159, WO 2014 / 194302, WO 2017 / 040790, WO 2017 / 133540, WO 201 and WO 2017 / 132827, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 106061, WO 2017 / 19846, WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540.

[0302] Anti-PD-1 antibodies useful in the combination therapy of the disclosed invention also include antigen-binding portions of the antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domain and CH1 domain; and (iv) a Fv fragment consisting of the VL domain and VH domain of a single antibody arm.

[0303] Anti-PD-1 antibodies suitable for use in the disclosed combination therapies are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In certain embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1. In some aspects, the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric, humanized, or human monoclonal antibody, or portion thereof. In some aspects, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype can be used.

[0304] In some aspects, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of the human IgG1 or human IgG4 isotype. In some aspects, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding fragment thereof contains a S228P mutation, which replaces a serine residue in the hinge region with a proline residue normally found at the corresponding position in IgG1 isotype antibodies. This mutation, present in nivolumab, prevents Fab arm exchange with endogenous IgG4 antibodies while retaining the low affinity for activating Fc receptors associated with wild-type IgG4 antibodies (Wang et al., 2014). In some aspects, the antibody comprises a light chain constant region that is a human kappa constant region or a human lambda constant region. In other embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is a mAb or antigen-binding portion thereof. In certain embodiments of any of the therapeutic methods described herein that include administration of an anti-PD-1 antibody, the anti-PD-1 antibody is nivolumab.

[0305] In some embodiments, the PD-1 antagonist is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, and dostarlimab.

[0306] PD-L1 Anti-human PD-L1 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the combination therapies disclosed herein can be generated using methods known in the art. Examples of anti-PD-L1 antibodies useful in the methods of the present disclosure include those disclosed in U.S. Patent No. 9,580,507, which is incorporated herein by reference. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has been demonstrated to exhibit one or more of the following characteristics: (a) a specific activity of 1×10 as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D(b) binds to human PD-L1 in a mixed lymphocyte reaction (MLR) assay; (c) increases interferon-γ production in an MLR assay; (d) increases IL-2 secretion in an MLR assay; (e) stimulates antibody responses; and (f) reverses the effects of T regulatory cells on T cell effector cells and / or dendritic cells. Anti-PD-L1 antibodies useful in the methods disclosed herein include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one of the foregoing characteristics.

[0307] Recognized anti-PD-L1 antibodies can be used. For example, the human anti-PD-L1 antibodies disclosed in U.S. Patent No. 7,943,743, the contents of which are incorporated herein by reference, can be used. Such anti-PD-L1 antibodies include 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Other art-recognized anti-PD-L1 antibodies that can be used include, for example, those described in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Patent Publication No. 2009 / 0317368, and PCT Publication Nos. WO 2011 / 066389 and WO 2012 / 145493, the teachings of which are also incorporated herein by reference. Other examples of anti-PD-L1 antibodies include atezolizumab (TECENTRIQ; RG7446), or durvalumab (IMFINZI; MEDI4736), or avelumab (Bavencio). Antibodies or antigen-binding fragments thereof that compete with any of these art-recognized antibodies or inhibitors for binding to PD-L1 can also be used.

[0308] In some embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO 2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al. 2013 J Clin Oncol 31(suppl):3000; U.S. Patent No. 8,217,149), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802), or MSB0010718C (also known as avelumab; see U.S. Patent No. 2014 / 0341917). In some aspects, the antibody that cross-competes with the above-mentioned PD-L1 antibodies for binding to human PD-L1, or binds to the same epitope region of human PD-L1 as the above-mentioned PD-L1 antibodies, is a mAb. For administration to human subjects, these cross-competing antibodies can be chimeric, or humanized or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (12A4, also known as MDX-1105; see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche; TECENTRIQ®; MPDL3280A, also known as RG7446; see U.S. Patent No. 8,217,149; Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; IMFINZI™, also known as MEDI-4736; see WO 2011 / 066389), avelumab (Pfizer; BAVENCIO®, also known as MSB-0010718C; see WO 2013 / 079174), STI-1014 (Sorrento; see WO 2013 / 181634), CX-072 (Cytomx; see WO 2016 / 149201), KN035 (3D Med / Alphamab; see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g., WO 2017 / 034916) and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (Apr 2016)).

[0309] In some embodiments, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0310] In some embodiments, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0311] In some embodiments, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0312] In some embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, and any combination thereof.

[0313] In some aspects, the anti-PD-L1 antibodies of the disclosed methods include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein, e.g., atezolizumab, durvalumab, and / or avelumab, for binding to human PD-L1. In some embodiments, the anti-PD-L1 antibody binds to the same epitope as any of the anti-PD-L1 antibodies described herein, e.g., atezolizumab, durvalumab, and / or avelumab. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0314] In some embodiments, the antibodies that cross-compete with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1 or that bind to the same epitope region of human PD-L1 antibodies as atezolizumab, durvalumab, and / or avelumab are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric, engineered, humanized, or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0315] Anti-PD-L1 antibodies for use in the methods disclosed herein may comprise antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0316] CTLA-4 Monoclonal antibodies that specifically bind to CTLA-4 include, but are not limited to, ipilimumab (Yervoy®; BMS) and tremelimumab (AstraZeneca / MedImmune), as well as antibodies disclosed in U.S. Patent Application Publication Nos. 2005 / 0201994, 2002 / 0039581, and 2002 / 0086014, the contents of each of which are incorporated herein by reference. Nos. 5,811,097, 5,855,887, 6,051,227, 6,984,720, 6,682,736, 6,207,156, 5,977,318, 6,682,736, 7,109,003, 7,132,281, and 8,491,895, or antibodies comprising the heavy chain variable region and light chain variable region of any of these antibodies.

[0317] A human monoclonal antibody that specifically binds to CTLA-4 with high affinity is disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described, for example, in U.S. Patent No. 7,034,121 and International Publication Nos. WO 2012 / 122444, WO 2007 / 113648, WO 2016 / 196237 and WO 2000 / 037504. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is selected from the group consisting of ipilimumab and tremelimumab. In some embodiments, the CTLA-4 antagonist is ipilimumab.

[0318] In some aspects, the anti-CTLA-4 antibodies of the disclosed methods include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any of the anti-CTLA-4 antibodies disclosed herein, e.g., ipilimumab, for binding to human CTLA-4. In some embodiments, the anti-CTLA-4 antibody binds to the same epitope as any of the anti-CTLA-4 antibodies described herein, e.g., ipilimumab. Cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab in standard CTLA-4 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0319] In some embodiments, the antibody cross-competes with ipilimumab for binding to human CTLA-4 or binds to the same epitope region of CTLA-4 as ipilimumab. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric monoclonal antibodies, engineered monoclonal antibodies, humanized monoclonal antibodies, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0320] Anti-CTLA-4 antibodies for use in the methods disclosed herein can include antigen-binding portions of such antibodies. It is well established that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0321] LAG-3 The pathway involving LAG-3, BTLA, B7-H3, B7-H4, TIM-3, and KIR constitutes an immune checkpoint pathway similar to the CTLA-4-dependent and PD-1-dependent pathways (see, e.g., Pardoll, 2012, Nature Rev Cancer 12:252-264; Mellman et al., 2011, Nature 480:480-489).

[0322] Anti-human LAG-3 antibodies (or VH / VL domains derived therefrom) suitable for use in the combination therapies disclosed herein can be generated using methods well known in the art, or art-recognized anti-LAG-3 antibodies can be used.

[0323] For example, an anti-human LAG-3 antibody called monoclonal antibody 25F7 (also known as "25F7" and "LAG3.1"), described in U.S. Patent No. 2011 / 0150892, the teachings of which are incorporated herein by reference, can be used. Other art-recognized anti-LAG-3 antibodies that may be used include IMP731 (H5L7BW), described in U.S. Patent No. 2011 / 007023, MK-4280 (28G-10), described in WO 2016028672, REGN3767, described in Journal for ImmunoTherapy of Cancer, (2016) Vol. 4, Supp. Supplement 1 Abstract Number: P195, BAP050, IMP-701 (LAG-525), IMP321 (eftiragimodo alfa), Sym022, TSR-033, MGD013, BI754111, FS118, AVA-017, and GSK2831781, described in WO 2017 / 019894. These and other anti-LAG-3 antibodies useful in the claimed invention are described in, for example, WO 2016 / 028672, WO 2017 / 106129, WO 2017 / 062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 2205 ...8 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 069500, WO 2018 / 0 2017 / 220569, WO 2018 / 071500, WO 2017 / 015560, WO 2017 / 025498, WO 2017 / 087589, WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, U.S. Patent No. 2017 / 0260271, WO 2017 / 086367, WO 2017 / 086419, WO 2018 / 034227 and WO 2014 / 140180.The contents of each of these references are incorporated herein by reference.

[0324] Antibodies that compete with any of the above antibodies for binding to LAG-3 can also be used.

[0325] An exemplary anti-LAG-3 antibody is BMS-986016, which is described in U.S. Patent No. 9,505,839, incorporated herein by reference. In some embodiments, the anti-LAG-3 antibody is BMS-986016.

[0326] In some embodiments, the antibody has the heavy and light chain CDRs or variable regions of BMS-986016. In some embodiments, the antibody competes for binding with the above antibodies and / or binds to the same epitope on LAG-3 as the above antibodies. In some embodiments, the antibody binds to an epitope on human LAG-3 comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 82). In some embodiments, the antibody binds to an epitope on human LAG-3 comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 83) or PAAPSSWG (SEQ ID NO: 84).

[0327] IV. Anti-VEGF antibody In another embodiment of the above combination therapy, the pharmaceutical agent is an anti-VEGF antibody.

[0328] 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).

[0329] In some embodiments, the antibody with binding specificity for vascular endothelial growth factor (VEGF) is bevacizumab or a biosimilar thereof.

[0330] 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: 85 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 85) and a second amino acid sequence having a sequence having at least about 85% identity to SEQ ID NO: 86 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 86).

[0331] In some embodiments, the heavy chain of the anti-VEGF antibody comprises the following sequence:

number

[0332] In some embodiments, the light chain of the anti-VEGF antibody comprises the following sequence:

number

[0333] In some embodiments, the anti-VEGF antibody comprises any of the sequences in Table 4.

[0334] [Table 4]

[0335] In some embodiments, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0336] In some embodiments, the anti-VEGF antibody is administered intravenously.

[0337] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

[0338] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

[0339] In some embodiments, an anti-cancer agent is administered (e.g., first), then a lipopolymer-formulated nucleic acid vector is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third).

[0340] In some embodiments, an anti-cancer drug is administered (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., fourth).

[0341] 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.

[0342] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 12 weeks and up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anti-cancer agent.

[0343] 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.

[0344] 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.

[0345] In some embodiments, the interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-cancer agent.

[0346] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0347] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0348] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the anti-VEGF antibody.

[0349] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody.

[0350] V. Anticancer drugs In one embodiment of the aforementioned therapy, the anti-cancer agent is a chemotherapeutic agent selected from the group consisting of taxanes, platinum, adriamycin, cylcophosphamide, topotecan, carmustine (BCNU), or combinations thereof.

[0351] In some embodiments, the chemotherapeutic agent is selected from the group consisting of topoisomerase inhibitors (e.g., irinotecan, topotecan, doxorubicin, epirubicin, idarubicin), anti-microtubule agents (e.g., paclitaxel, docetaxel), alkylating agents (e.g., cyclophosphamide, dacarbidine), platinum-based drugs (cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., gemcitabine, methotrexate, 5-fluorouracil), or combinations thereof.

[0352] In some embodiments, the anti-cancer therapy is selected from the group consisting of paclitaxel, carboplatin, docetaxel, nab-paclitaxel, doxorubicin, and any combination thereof.

[0353] In some embodiments, the anti-cancer agent is doxorubicin.

[0354] In some embodiments, the anti-cancer agent comprises paclitaxel.

[0355] In some embodiments, the anti-cancer agent comprises carboplatin.

[0356] In some embodiments, the anti-cancer agent comprises docetaxel.

[0357] In some embodiments, the anti-cancer agent comprises nab-paclitaxel.

[0358] In some embodiments, an anti-cancer drug is included.

[0359] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0360] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0361] In some embodiments, the anti-cancer agent is administered every three weeks for about nine weeks, beginning at least about 28 days after interval cytoreductive surgery.

[0362] In some embodiments, the anti-cancer agent is selected from the group consisting of paclitaxel, carboplatin, docetaxel, nab-paclitaxel, and any combination thereof.

[0363] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

[0364] In some embodiments, an anti-cancer agent is administered (e.g., first), then a lipopolymer-formulated nucleic acid vector is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third).

[0365] In some embodiments, an anti-cancer drug is administered (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., fourth).

[0366] 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.

[0367] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0368] 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).

[0369] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0370] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 , about 50~250mg / m 2 , about 75~250mg / m 2 , about 100~250mg / m 2 , about 125~250mg / m 2 , about 150~250mg / m 2 , about 175~250mg / m 2 , about 200~250mg / m 2 , about 225~250mg / m 2 , about 25~225mg / m 2 , about 25~200mg / m 2 , about 25~175mg / m 2 , about 25~150mg / m 2, about 25~125mg / m 2 , about 25~250mg / m 2 , about 25~100mg / m 2 , about 25~75mg / m 2 , or approximately 25-50 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0371] In some embodiments, the administration of the anti-cancer agent is about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 or approximately 250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0372] In some embodiments, the administration of the anticancer agent is between 25 and 250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0373] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 , about 50~250mg / m 2 , about 75~250mg / m 2 , about 100~250mg / m 2 , about 125~250mg / m 2 , about 150~250mg / m 2 , about 175~250mg / m 2 , about 200~250mg / m 2 , about 225~250mg / m 2 , about 25~225mg / m 2 , about 25~200mg / m 2, about 25~175mg / m 2 , about 25~150mg / m 2 , about 25~125mg / m 2 , about 25~250mg / m 2 , about 25~100mg / m 2 , about 25~75mg / m 2 , or approximately 25-50 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0374] In some embodiments, the administration of the anti-cancer agent is about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 or approximately 250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0375] In some embodiments, the administration of the anticancer agent is from 25 to 350 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0376] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 , about 50~250mg / m 2 , about 75~250mg / m 2 , about 100~250mg / m 2 , about 125~250mg / m 2 , about 150~250mg / m 2 , about 175~250mg / m 2 , about 200~250mg / m 2 , about 225~250mg / m 2, about 25~225mg / m 2 , about 25~200mg / m 2 , about 25~175mg / m 2 , about 25~150mg / m 2 , about 25~125mg / m 2 , about 25~250mg / m 2 , about 25~100mg / m 2 , about 25~75mg / m 2 , or approximately 25-50 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0377] In some embodiments, the administration of the anti-cancer agent is about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 or approximately 250 mg / m 2 nab-paclitaxel at a dose of about AUC 4-6 IV, optionally followed by carboplatin at a dose of about AUC 4-6 IV. In some embodiments, administration of the nanoparticles prior to interval cytoreductive surgery begins 15 days after the first administration of the anticancer agent, and is weekly for at least about 12 weeks to about 18 weeks.

[0378] In some embodiments, the administration of the anti-cancer agent is about 175 mg / m 2 followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0379] In some embodiments, the administration of the anti-cancer agent is about 75 mg / m 2 followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0380] In some embodiments, the administration of the anti-cancer agent is about 260 mg / m 2 followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0381] VI. Treatment method Certain aspects of the present disclosure relate to methods for treating cancer in a subject that is BRCA-negative and homologous repair proficient (HRP), comprising administering to the subject a nucleic acid vector (e.g., a plasmid) comprising a polynucleotide encoding interleukin-12 (IL-12), formulated in a lipopolymer (e.g., nanoparticle). In some aspects, the method further comprises administering to the subject an immune checkpoint inhibitor. In some aspects, the method further comprises administering to the subject an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

[0382] The present invention also provides a method for treating mammalian cancer or hyperproliferative disorders without chemotherapy by intratumoral, intraperitoneal, intravenous, intravesicular, intratracheal, intracranial, or systemic administration of a pharmaceutical composition comprising a plasmid-based gene expression system and a gene delivery polymer. The mammalian cancer is selected from the group consisting of primary or metastatic ovarian tumors. Preferably, the nucleic acid is a plasmid-based gene expression system containing a DNA sequence encoding interleukin-12.

[0383] Treatment of tumors with the pharmaceutical composition (nucleic acid + gene delivery polymer and one or more chemotherapeutic agents) results in tumor shrinkage and extended lifespan. Combining gene therapy (nucleic acid and gene delivery polymer) with chemotherapy (chemotherapeutic agent) according to the disclosed methods results in additive and / or synergistic efficacy. Efficacy of the methods of the present invention is defined as, but not limited to, a reduction in tumor size or tumor density, an increase in lymphocyte count or neutrophil count, or improved survival, or all of the above. Additionally, combining gene therapy (nucleic acid and gene delivery polymer) with chemotherapy (chemotherapeutic agent) according to the methods of the present invention reduces the toxicity of the chemotherapeutic agent 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 polymer) with suboptimal doses of chemotherapy (chemotherapeutic agent) according to the methods of the present invention enhances anti-cancer efficacy to levels above those achieved by optimal doses of the chemotherapeutic agent, but with reduced toxicity.

[0384] New cancer treatment strategies focus on delivering polymers carrying genetic information rather than therapeutic proteins themselves, allowing exogenously delivered genes to be expressed within the tumor environment. While nonviral gene delivery systems are considered safer than viral delivery systems, the practical application of current polymer systems has been hampered by poor efficiency. Recently, a strategy was disclosed to enhance the gene transfection efficiency of low-molecular-weight PEI by covalently attaching cholesterol to form water-soluble lipopolymers (WSLPs). See Mol. Ther., 2001, 4, 130. IL-12 gene transfer into solid tumors using WSLPs was significantly better than unmodified PEI and resulted in even more pronounced tumor inhibition.

[0385] 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 multiple drugs to maximize anti-cancer responses are increasingly recognized. In this disclosure, the inventors combined a chemotherapeutic agent with gene delivery of an anti-cancer gene administered locally to the tumor site to improve the safety and efficacy of the treatment. Combining safe and efficient local delivery of the anti-cancer gene with standard chemotherapeutic agents enhances anti-cancer 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 anti-cancer gene complexed with a gene delivery polymer and at least one adjuvant chemotherapeutic agent is more effective than gene therapy or chemotherapy treatment administered alone. Furthermore, the combination therapy is effective against a wide variety of tumors when administered via various routes of administration and does not increase toxicity compared to the individual therapies.

[0386] 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 immune checkpoint inhibitor.

[0387] Certain aspects of the present disclosure relate to methods of treating cancer in a subject that is BRCA-negative and homologous repair proficient (HRP), 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), the nucleic acid vector (e.g., a plasmid) formulated in a lipopolymer (e.g., a nanoparticle), and (ii) an immune checkpoint inhibitor.

[0388] 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).

[0389] Certain aspects of the present disclosure relate to methods of treating cancer in a subject that is BRCA-negative and homologous repair proficient (HRP), 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).

[0390] In some embodiments, the polynucleotide encodes human IL-12.

[0391] 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.

[0392] 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, an element of Figure 1).

[0393] In some embodiments, the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group (eg, the lipopolymer of Figure 2).

[0394] In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0395] In some embodiments, the immune checkpoint inhibitor is an antibody.

[0396] In some embodiments, the immune checkpoint inhibitor is a small molecule inhibitor.

[0397] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostallimab, and cemiplimab.

[0398] In some embodiments, the immune checkpoint inhibitor is a PD-1 antagonist, and the PD-1 antagonist is nivolumab.

[0399] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0400] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 antagonist that is ipilimumab.

[0401] In some embodiments, the immune checkpoint inhibitor is a LAG-3 antagonist, and the LAG-3 antagonist is leratolimab.

[0402] In some embodiments, the combination further comprises an anti-cancer agent.

[0403] In some embodiments, the anti-cancer agent is a chemotherapeutic agent.

[0404] In some embodiments, the chemotherapeutic agent is selected from the group consisting of topoisomerase inhibitors (e.g., irinotecan, topotecan, doxorubicin, epirubicin, idarubicin), anti-microtubule agents (e.g., paclitaxel, docetaxel), alkylating agents (e.g., cyclophosphamide, dacarbidine), platinum-based drugs (cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., gemcitabine, methotrexate, 5-fluorouracil), or combinations thereof.

[0405] In some embodiments, the chemotherapeutic agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

[0406] In some embodiments, the anti-cancer agent is doxorubicin.

[0407] In some embodiments, the anti-cancer agent is paclitaxel.

[0408] In some embodiments, the anti-cancer agent is carboplatin.

[0409] In some embodiments, the anti-cancer agent is docetaxel.

[0410] In some embodiments, the anti-cancer agent is nab-paclitaxel.

[0411] In some embodiments, the anti-cancer agent is olaparib.

[0412] 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).

[0413] 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: 85 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 85), and a variable light chain (VL) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO: 86 (e.g., 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 86).

[0414] 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).

[0415] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered intratumorally or intraperitoneally.

[0416] In some aspects, the lipopolymer-formulated nucleic acid vector is administered intravenously.

[0417] In some aspects, the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, intravesically, or any combination thereof.

[0418] In some embodiments, the immune checkpoint inhibitor is administered intratumorally or intraperitoneally.

[0419] In some aspects, the immune checkpoint inhibitor is administered intravenously.

[0420] In some aspects, the lipopolymer-formulated nucleic acid vector is administered prior to, concurrently with, or after the immune checkpoint inhibitor.

[0421] In some embodiments, the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravesically, or any combination thereof.

[0422] In some embodiments, the anti-VEGF antibody is administered intratumorally or intraperitoneally.

[0423] In some embodiments, the anti-VEGF antibody is administered intravenously.

[0424] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

[0425] In some embodiments, the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

[0426] In some embodiments, surgery to remove all or part of the tissue or tumor is performed (e.g., cytoreductive surgery) (e.g., first), followed by administration of an anti-cancer agent and an anti-VEGF antibody, followed by administration of a nucleic acid vector formulated in a lipopolymer. In some embodiments, the anti-VEGF antibody is administered at least 4 weeks after the surgery. In some embodiments, the anti-VEGF antibody is not administered within 4 weeks after the surgery. In some embodiments, the anti-VEGF antibody is not administered within 4 weeks before the surgery. In some embodiments, the anti-VEGF antibody is administered simultaneously with the anti-cancer agent, except for the first administration. In some embodiments, the nucleic acid vector is administered 8 days after the anti-cancer agent is administered. In some embodiments, the first dose of the anti-cancer agent is administered with a steroid.

[0427] In some embodiments, surgery to remove all or part of the tissue or tumor is performed (e.g., cytoreductive surgery) (e.g., first), followed by administration of an anti-cancer drug at 3-week intervals for 6 cycles, starting at least 4 weeks after surgery, and an anti-VEGF antibody for each cycle of chemotherapy, followed by administration of a nucleic acid vector formulated in a lipopolymer for weekly administration, starting 8 days after the first administration of the anti-cancer drug.

[0428] In some embodiments, an anti-cancer agent is administered (e.g., first), then a lipopolymer-formulated nucleic acid vector is administered (e.g., second), then an immune checkpoint inhibitor is administered (e.g., third).

[0429] In some embodiments, an anti-cancer drug is administered (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), then an immune checkpoint inhibitor is administered (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., fourth).

[0430] In some embodiments, an anti-cancer drug is administered, followed by a DNA plasmid, followed by an immune checkpoint inhibitor, followed by interval cytoreductive surgery.

[0431] In some embodiments, an anti-cancer agent is administered (e.g., first), then a lipopolymer-formulated nucleic acid vector is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third).

[0432] In some embodiments, an anti-cancer drug is administered (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), then an anti-VEGF antibody is administered (e.g., third), and then surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., fourth).

[0433] 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.

[0434] In some embodiments, surgery to remove all or part of the tissue or tumor (e.g., interval cytoreductive surgery) is performed (e.g., first), then a nucleic acid vector formulated in a lipopolymer is administered (e.g., second), and then an immune checkpoint inhibitor is administered (e.g., third).

[0435] In some embodiments, the anti-cancer agent is administered every three weeks for about 12 to about 18 weeks prior to interval cytoreductive surgery.

[0436] 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).

[0437] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0438] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 , about 50~250mg / m 2 , about 75~250mg / m 2 , about 100~250mg / m 2 , about 125~250mg / m 2 , about 150~250mg / m 2 , about 175~250mg / m 2 , about 200~250mg / m 2 , about 225~250mg / m 2 , about 25~225mg / m 2 , about 25~200mg / m 2 , about 25~175mg / m 2 , about 25~150mg / m 2 , about 25~125mg / m 2 , about 25~250mg / m 2 , about 25~100mg / m 2 , about 25~75mg / m 2 , or approximately 25-50 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0439] In some embodiments, the administration of the anti-cancer agent is about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 or approximately 250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0440] In some embodiments, the administration of the anticancer agent is between 25 and 250 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0441] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 , about 50~250mg / m 2 , about 75~250mg / m 2 , about 100~250mg / m 2 , about 125~250mg / m 2 , about 150~250mg / m 2 , about 175~250mg / m 2 , about 200~250mg / m 2 , about 225~250mg / m 2 , about 25~225mg / m 2 , about 25~200mg / m 2 , about 25~175mg / m 2 , about 25~150mg / m 2 , about 25~125mg / m 2 , about 25~250mg / m 2 , about 25~100mg / m 2 , about 25~75mg / m 2 , or approximately 25-50 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0442] In some embodiments, the administration of the anti-cancer agent is about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 or approximately 250 mg / m 2optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0443] In some embodiments, the administration of the anticancer agent is from 25 to 350 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0444] In some embodiments, the administration of the anticancer agent is about 25-250 mg / m 2 , about 50~250mg / m 2 , about 75~250mg / m 2 , about 100~250mg / m 2 , about 125~250mg / m 2 , about 150~250mg / m 2 , about 175~250mg / m 2 , about 200~250mg / m 2 , about 225~250mg / m 2 , about 25~225mg / m 2 , about 25~200mg / m 2 , about 25~175mg / m 2 , about 25~150mg / m 2 , about 25~125mg / m 2 , about 25~250mg / m 2 , about 25~100mg / m 2 , about 25~75mg / m 2 , or approximately 25-50 mg / m 2 optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

[0445] In some embodiments, the administration of the anti-cancer agent is about 25 mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m2 , about 225mg / m 2 or approximately 250 mg / m 2 nab-paclitaxel at a dose of about AUC 4-6 IV, optionally followed by carboplatin at a dose of about AUC 4-6 IV. In some embodiments, administration of the nanoparticles prior to interval cytoreductive surgery begins 15 days after the first administration of the anticancer agent, and is weekly for at least about 12 weeks to about 18 weeks.

[0446] In some embodiments, the nanoparticles are administered at least about 28 days after interval cytoreductive surgery, with administration beginning 15 days after the first administration of the anti-cancer agent weekly for at least about 9 weeks.

[0447] 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

[0448] In some embodiments, the nanoparticles have a concentration of about 35 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 40 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 45 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 50 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 55 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 60 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 65 mg / m 2 ~about 80mg / m 2In some embodiments, the nanoparticles are administered at a dose of about 70 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 75 mg / m 2 ~about 80mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 75mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 70mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 65mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 60mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 55mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 50mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 45mg / m 2 In some embodiments, the nanoparticles are administered at a dose of about 35 mg / m 2 ~about 40mg / m 2 is administered at a dose of

[0449] In some embodiments, the nanoparticles have a concentration of about 35 mg / m 2 , about 40mg / m 2 , about 45mg / m 2 , about 50mg / m 2 , about 55mg / m 2 , about 60mg / m 2 , about 65mg / m 2 , about 70mg / m 2 , about 75mg / m 2 or approximately 80 mg / m 2 is administered at a dose of

[0450] In some embodiments, the nanoparticles have a concentration of about 60 mg / m 2 is administered at a dose of

[0451] In some embodiments, the immune checkpoint inhibitor is ipilimumab. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 1 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 1.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 2 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 2.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 3 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 3.5 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 4 to about 5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 4 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 3 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 2.5 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 2 mg / kg. In some embodiments, ipilimumab is administered at a dose of about 0.5 to about 1.5 mg / kg.

[0452] In some embodiments, ipilimumab is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, or about 5 mg / kg.

[0453] In some embodiments, ipilimumab is administered at a dose of about 1 mg / kg.

[0454] In some embodiments, ipilimumab is administered every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0455] In some embodiments, ipilimumab is administered every six weeks.

[0456] In some embodiments, ipilimumab is administered intravenously.

[0457] In some embodiments, the immune checkpoint inhibitor is nivolumab. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 140 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 160 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 180 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 200 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 220 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 240 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 340 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 320 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 300 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 280 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 260 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 220 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 200 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 180 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 160 mg. In some embodiments, nivolumab is administered at a dose of about 120 mg to about 140 mg. In some embodiments, nivolumab is administered at a dose of about 240 mg to about 360 mg.In some embodiments, nivolumab is administered at a dose of about 260 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 280 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 300 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 320 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 340 mg to about 360 mg. In some embodiments, nivolumab is administered at a dose of about 140 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 160 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 180 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 200 mg to about 240 mg. In some embodiments, nivolumab is administered at a dose of about 220 mg to about 240 mg.

[0458] In some embodiments, nivolumab is administered at a dose of about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, or about 360 mg.

[0459] In some embodiments, nivolumab is administered at a dose of about 240 mg.

[0460] In some embodiments, nivolumab is administered intravenously.

[0461] In some embodiments, nivolumab is administered every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0462] In some embodiments, nivolumab is administered every two weeks.

[0463] In some embodiments, the method further comprises administering a second immune checkpoint inhibitor.

[0464] In some embodiments, the second immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

[0465] In some embodiments, the second immune checkpoint inhibitor is an antibody.

[0466] In some embodiments, the second immune checkpoint inhibitor is a small molecule inhibitor.

[0467] In some embodiments, the second immune checkpoint inhibitor is a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostallimab, and cemiplimab.

[0468] In some embodiments, the second immune checkpoint inhibitor is a PD-1 antagonist, and the PD-1 antagonist is nivolumab.

[0469] In some embodiments, the second immune checkpoint inhibitor is a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

[0470] In some embodiments, the second immune checkpoint inhibitor is a CTLA-4 antagonist that is ipilimumab.

[0471] In some embodiments, the second immune checkpoint inhibitor is a LAG-3 antagonist, and the LAG-3 antagonist is leratolimab.

[0472] In some embodiments, the first immune checkpoint inhibitor is nivolumab and the second immune checkpoint inhibitor is ipilimumab.

[0473] In some embodiments, nivolumab is administered at about 240 mg every two weeks and ipilimumab is administered at about 1 mg / kg every six weeks.

[0474] In some embodiments, the immune checkpoint inhibitor is administered weekly for at least about 12 weeks and up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anti-cancer agent.

[0475] In some embodiments, the immune checkpoint inhibitor 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.

[0476] In some embodiments, the anti-VEGF antibody is administered weekly for at least about 12 weeks and up to about 18 weeks, prior to interval cytoreductive surgery, at least about 22 days after the first administration of the anti-cancer agent.

[0477] 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.

[0478] 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).

[0479] In some embodiments, the interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-cancer agent.

[0480] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0481] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 7 days after administration of the DNA plasmid.

[0482] In some embodiments, the interval cytoreductive surgery (ICS) is administered at least about 28 days prior to administration of the immune checkpoint inhibitor.

[0483] In some embodiments, the interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the immune checkpoint inhibitor.

[0484] In some embodiments, interval cytoreductive surgery (ICS) is administered at least about 28 days after administration of the anti-VEGF antibody.

[0485] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer (e.g., glioblastoma), lung cancer, and any combination thereof, and metastasis of any of the cancers.

[0486] In some embodiments, the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

[0487] Certain aspects of the present disclosure relate to methods of treating a subject, including: (i) obtaining a biological sample from a subject; (ii) performing a genotyping assay on the biological sample to determine whether the subject is BRCA+ or BRCA−; (iii) performing a genotyping assay on the biological sample to determine whether the subject is homologous repair proficient (HRP) or homologous repair deficient (HRD); and, if the subject is BRCA− / HRP, 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); and, if the subject is BRCA+ / HRD, administering to the subject a PARP inhibitor.

[0488] In some embodiments, the interleukin-12 (IL-12) plasmid formulated in lipopolymer (e.g., nanoparticles) is administered at a concentration of about 35 mg / m 2 ~about 80mg / m 2 is administered at a dose of

[0489] In some embodiments, the interleukin-12 (IL-12) plasmid formulated in lipopolymer (e.g., nanoparticles) is administered at a concentration of about 80 mg / m 2 It is administered at .

[0490] In some embodiments, interleukin-12 (IL-12) plasmids formulated in lipopolymers (eg, nanoparticles) are administered weekly.

[0491] In some embodiments, the anti-VEGF antibody is administered at a dose of about 10-20 mg / kg IV.

[0492] In some embodiments, the anti-VEGF antibody is administered at a dose of about 15 mg / kg IV.

[0493] In some embodiments, the anti-VEGF antibody is administered every three weeks.

[0494] In some embodiments, the PARP inhibitor is olaparib.

[0495] In some embodiments, olaparib is administered at a dose of about 300 mg.

[0496] In some embodiments, olaparib is administered daily.

[0497] In some embodiments, the subject is a human.

[0498] The following examples are illustrative and do not limit the scope of the claimed embodiments. example Example 1. GEN-1 enhances the activity of anti-VEGF antibodies in a mouse model.

[0499] Figure 3 shows the potential synergistic effectiveness of lowering VEGF levels and inhibiting production by administering GEN-1 and an anti-VEGF antibody (e.g., bevacizumab) simultaneously. Results show that SKOV-3 (human ovarian epithelial adenocarcinoma) cells (7 x 10 6 Cell) nude-foxn1 nu Mice were intraperitoneally injected with anti-VEGF antibodies at different levels: 5 mg / kg (low), 10 mg / kg (medium), and 20 mg / kg (high).

[0500] Anti-VEGF antibodies (e.g., bevacizumab) were administered 9 days after the initial tumor implantation and then weekly for 6 weeks. mGEN-1 (100 μg DNA) was then administered intraperitoneally starting 14 days after the initial tumor implantation and then weekly for 4 weeks.

[0501] 59 days after the initial tumor implantation, the mice were euthanized, and then the tumors were removed and weighed.The efficacy of low-dose anti-VEGF antibodies (e.g., bevacizumab) administered in combination with GEN-1 was shown to improve, which improves the therapeutic index and cost.See Figure 3. Example 2. GEN-1 enhances the activity of anti-VEGF antibodies in combination with anti-cancer agents in a mouse model.

[0502] Figure 4 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 along with an anti-cancer drug. Results show that SKOV-3-Luc (human ovarian epithelial adenocarcinoma) cells (7 x 10 6 Cell) nude-foxn1 nu Mice were injected intraperitoneally with Doxil at a dose of 7.5 mg / kg every other week, starting two weeks after tumor implantation. Ten days after tumor implantation, anti-VEGF antibodies (e.g., bevacizumab) were administered intravenously at 10 mg / kg every week.

[0503] Then, starting 2 weeks after the initial tumor implantation, mGEN-1 was administered intraperitoneally weekly (100 μg of DNA). Tumor burden in animals was then quantified using IVIS imaging, as shown in Figure 4. Whole-body images of mice taken with IVIS imaging are shown in Figure 5.

[0504] Example 3. Clinical combination of GEN-1 + NACT + anti-VEGF antibody (predicted) This is a 1:1 randomized, open-label, multicenter Phase II study with a safety lead-in 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 four cycles of neoadjuvant chemotherapy and allows up to two additional cycles (C4+1, and 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, three additional adjuvant cycles of study treatment will be administered.

[0505] In addition, BEV will be included in each 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). In the experimental arm, GEN-1 will begin on Day 15 of Cycle 1 (C1D15) and continue weekly until the last cycle of adjuvant therapy. BEV cannot be administered within 30 days before or after surgery. In the experimental arm, GEN-1 will be added weekly to each cycle of NACT + BEV starting on Day 15 of Cycle 1. FDA-approved BEV biosimilars may be used.

[0506] 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 cohorts of three subjects who received at least two cycles of NACT+BEV+GEN-1 before starting the main phase of the study. Run-in patients will also be randomized.

[0507] 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 the 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.

[0508] Maintenance treatment will be determined by breast cancer gene / homologous recombination deficiency (BRCA / HRD) status. All patients will receive BEV, with BRCA+ / HRD patients receiving olaparib in addition to BEV. Non-BRCA+ / HRD patients in the experimental arm will receive GEN-1 (every 3 weeks) along with BEV.

[0509] All subjects will be followed for disease progression and overall survival (OS).

[0510] Trial Phase

[0511] 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 primary phase of the protocol. Prior to initiating the primary phase of the study, no more than two of the six subjects treated in the experimental arm may exhibit dose-limiting toxicities. An independent DSMB will review safety data from subjects who have received at least two cycles of NACT+BEV+GEN-1 and provide recommendations regarding dose modifications, safety monitoring, and dosing for the primary phase of the study. The DSMB approval letter will define the definition of DLTs for the safety phase and the committee's responsibilities, including dose modifications and recommendations for the Phase II dose of GEN-1.

[0512] 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 arm) for Phase II. All subjects will be randomized to either NACT + BEV + GEN-1 or NACT + BEV alone. Subjects will receive 4-6 cycles of treatment before interval cytoreductive surgery (ICS), followed by at least 2 cycles of treatment after surgery. At the end of the final cycle of chemotherapy for all subjects, SLL will be administered before the start of the maintenance phase.

[0513] Maintenance: After completion of SLL, subjects begin maintenance treatment. This phase begins no sooner than 4 weeks (ideally 5-7 weeks) from the date of SLL.

[0514] All patients will receive BEV (or an FDA-approved biosimilar) every 21 days until disease progression or unacceptable toxicity, or for a maximum of 15 months.

[0515] BRCA+ / HRD subjects will receive olaparib with BEV until disease progression or unacceptable toxicity, or for up to 24 months.

[0516] Non-BRCA+ / HRD subjects in the experimental arm will receive GEN-1 every 21 days with BEV until disease progression or unacceptable toxicity, or for a maximum of 15 months.

[0517] All subjects will be followed for progression and overall survival.

[0518] Study population

[0519] Approximately 50 subjects with newly diagnosed advanced ovarian cancer (including up to 12 safety run-ins) will be randomized at approximately four U.S. study centers. The sample size may be expanded based on the results of an interim analysis after completion of the initial 50 subjects.

[0520] Recruitment criteria

[0521] 1. Subjects must have a suspected diagnosis of high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer with pre-treatment biopsy by laparoscopy, or histologic confirmation by interventional radiology or CT or ultrasound-guided core biopsy. Histologic demonstration of the original primary tumor via pathology report is required.

[0522] 2. Subjects must have International Federation of Gynecology and Obstetrics (FIGO) stage III or IV disease with clinical considerations determined to benefit from neoadjuvant therapy based on standard of care.

[0523] 3. Subjects with only epithelial cell type histology of high-grade serous adenocarcinoma are eligible.

[0524] 4. Subjects must meet the following criteria:

[0525] 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.

[0526] b. Renal function: Cockcroft-Gault estimated GFR ≥ 50. Urine dipstick showing ≤ 1+ proteinuria (patients with a dipstick reading of ≥ 2+ must have a 24-hour urine collection with < 2g protein / 24 hours).

[0527] Liver function: bilirubin ≤ 1.5 x ULN, SGOT (AST) and SGPT (ALT) ≤ 3.0 x ULN, and alkaline phosphatase ≤ 2.5 x ULN.

[0528] d. Neurological function: Neurological impairment (sensory and motor) grade 1 or less.

[0529] 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.

[0530] 6. Hormone therapy for malignant tumors must be discontinued at least 1 week before the first treatment. Continuation of hormone replacement therapy is permitted.

[0531] 7. Subjects must have an Eastern Cooperative Group (ECOG) performance status score of 0-1.

[0532] 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 cease breastfeeding prior to study entry.

[0533] 9. Subject must have satisfactory results on baseline clinical analyses and diagnostic procedures as specified in the protocol.

[0534] 10. Subjects must have signed an IRB / EC approved informed consent and authorization allowing release of personal health information.

[0535] 11. Subjects must be 18 years of age or older.

[0536] Exclusion criteria

[0537] 1.Subjects who have previously received treatment with GEN-1.

[0538] 2. History of allergic reaction caused by compounds with similar chemical or biological composition to GEN-1 or other drugs used in this study.

[0539] 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) not related to chemotherapy administration. Steroid prophylaxis for intravenous contrast allergy is permitted.

[0540] 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.

[0541] 5. Subjects with known human immunodeficiency virus (HIV) infection or human T-lymphotropic virus (HTLV) infection will be excluded.

[0542] 6. Subjects with other invasive malignancies will be excluded if there is evidence of such malignancies within the past three 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 intraepidermal melanoma, are eligible.

[0543] 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.

[0544] 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.

[0545] 9. Subjects with known active hepatitis.

[0546] 10. Subjects with known nephrotic syndrome (proteinuria grade 2 or higher).

[0547] 11. Subject has concurrent severe medical problems unrelated to malignancy that significantly limit full compliance with the study or that place the subject at extreme risk or reduced life expectancy.

[0548] 12. Subjects with clinically significant cardiovascular disease. This includes:

[0549] Uncontrolled hypertension, defined as systolic blood pressure (BP) >150 mm Hg or diastolic BP >90 mm Hg on at least two separate days. (Subjects with uncontrolled hypertension may become eligible once their hypertension is controlled.)

[0550] b. Myocardial infarction or unstable angina within 6 months prior to enrollment.

[0551] c. Severe ventricular arrhythmia (i.e., ventricular tachycardia or ventricular fibrillation) requiring antiarrhythmic medication or a history of cardiac arrhythmia (excluding atrial fibrillation adequately controlled by antiarrhythmic medication).

[0552] d. QTc interval ≥ 450 ms on baseline ECG (electrocardiogram).

[0553] e. Baseline ejection fraction ≤ 50% as assessed by echocardiogram or MUGA.

[0554] f. New York Heart Association (NYHA) Class II or higher congestive heart failure.

[0555] 13. Subjects of childbearing potential, pregnant, or breastfeeding who are not using adequate contraception are not eligible for this study.

[0556] 14. Subject has a history of CNS disease, or evidence on physical examination of CNS disease, including a primary brain tumor, seizures not controlled by 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 date of first treatment on this study.

[0557] 15. Subjects with a history of diverticulitis. Diverticulosis is not excluded.

[0558] 16. Subjects who have coughed up blood within the last month.

[0559] 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 previous laparoscopy findings.

[0560] Study treatment and administration

[0561] Treatment Allocation

[0562] Eligible subjects will be randomized 1:1 to either NACT+BEV+GEN-1 or NACT+BEV alone. Because this is an open-label trial, site staff, subjects, and sponsors will be aware of each subject's treatment.

[0563] Neoadjuvant chemotherapy (NACT)

[0564] Control arm.

[0565] NACT: Paclitaxel 175 mg / m 2 Administer intravenously (IV) followed by carboplatin AUC 5-6 IV on C1D1. This is repeated every 21 days on day 1 for 4 cycles before ICS and 3 cycles after ICS. Up to 2 additional cycles may be added before ICS at the physician's discretion (C4+1 and C4+2). If there is a paclitaxel response, docetaxel 75 mg / m is administered according to institutional guidelines.2 or nab-paclitaxel (Abraxane) 260 mg / m 2 may be used instead. Body surface area (BSA) is calculated according to local practice.

[0566] 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.

[0567] In the experimental arm, GEN-1 will begin on C1D15 and continue weekly until the last cycle of adjuvant therapy.

[0568] Interval cytoreductive surgery (ICS) will be performed at least 28 days after the last cycle of neoadjuvant chemotherapy, and protocol therapy will be resumed as adjuvant chemotherapy for three more cycles after recovery from ICS (at least 28 days). BEV cannot be administered within 28 days before or after surgery.

[0569] 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 maintenance, BEV 15 mg / kg is administered as a single agent every 3 weeks until disease progression or unacceptable toxicity occurs for up to 18 additional cycles.

[0570] In addition to the control arm regimen, GEN-1 80 mg / m every 7 days starting on C1D15 2 IP is administered. Note that the IP port should be placed at least 7 days before the first dose of GEN-1 (to allow for healing). During the maintenance phase, patients who are BRCA- / HRP receive GEN-1 every 21 days with BEV (using the Myriad MyChoice assay) for up to 18 additional cycles until disease progression or unacceptable toxicity occurs.

[0571] Hypersensitivity prevention

[0572] On Day 1 of Cycle 1, all patients treated with paclitaxel should be conditioned 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 develop a hypersensitivity reaction may be switched to docetaxel or Abraxane.

[0573] BRCA+ / HRD subjects

[0574] All BRCA+ / HRD patients will receive olaparib 300 mg orally twice daily from the maintenance phase until disease progression or unacceptable toxicity, or for a maximum of 24 months.

[0575] Dose modification

[0576] 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 Principal Investigator or study monitor.

[0577] For complete prescribing information, refer to the labeling of carboplatin, paclitaxel, Abraxane, bevacizumab (or biosimilar), and docetaxel and local institutional guidelines.

[0578] GEN-1 (test drug)

[0579] Human IL-12 plasmid (phIL-12-005) is formulated in lipopolymer PEG-PEI-cholesterol (PPC) in 10% lactose.

[0580] Human IL-12 Plasmid

[0581] 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 1).

[0582] PEG-PEI-cholesterol

[0583] 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 2).

[0584] Route and administration of GEN-1

[0585] To confirm 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.

[0586] Reconstituted GEN-1 (in a 50 mL glass vial or IV bag) is stable at room temperature for up to 24 hours. After confirmation of catheter patency, the IV bag containing GEN-1 is administered through the patient's IP catheter. GEN-1 is infused by gravity from the IV bag through the catheter, with the valve fully open and free flowing. A typical administration can take approximately 1 hour.

[0587] 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.

[0588] Risks Associated with Recombinant IL-12 Therapy

[0589] To date, GEN-1 administration has not detected any toxicity associated with systemic administration of recombinant IL-12 therapy. Phase 1 trials of GEN-1 have shown little to no systemic uptake of GEN-1 or its downstream cytokines.

[0590] Intraperitoneal catheter

[0591] GEN-1 may be administered to 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 previous Phase I studies. Port-A-Cath catheters (Deltec, Inc., St. Paul, MN) have been successfully used for IP delivery of GEN-1, with few to no catheter-related serious complications. While Port-A-Cath catheters are preferred, any other approved catheter may be used with a subcutaneous port for IP delivery if suitable for aspiration of biological samples for bridging studies. For venous access, Bard 9.6 Fr silicone single-lumen catheters, or equivalents with or without a cuff manufactured by Bard Access Systems (West Amelia Earhart Drive, Salt Lake City, Utah), may also be used. Catheter compatibility studies performed with Port-A-Cath catheters 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 Bard catheters. 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 may be obtained to verify intraperitoneal infusion.

[0592] 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.

[0593] 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 confirm catheter patency. Heparin should not be used to flush the catheter during sample collection or drug infusion. Peritoneal lavage for bridging studies should be performed prior to GEN-1 infusion. For subjects enrolled in the NACT+BEV+GEN-1 arm, the catheter may be removed upon completion of GEN-1 administration at the clinician's discretion.

[0594] Test Procedure

[0595] Screening Overview (Day -21 to Day 1 of Cycle 1)

[0596] 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. All eligible subjects, regardless of arm to which they are randomized, will undergo a baseline assessment prior to dosing.

[0597] Screening assessments will be conducted after obtaining informed consent and within 21 days prior to treatment initiation. For both treatment arms, 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.

[0598] 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.

[0599] Eligible subjects will be randomized 1:1 to receive either NACT+BEV+GEN-1 or NACT+BEV.

[0600] NACT and interval cytoreductive surgery

[0601] All subjects will receive seven to nine cycles of standard NACT every 21 days (see Figure 6). BEV will be administered on C2D1 and C3D1, and after ICS on C6D1 and C7D1 (and additional neoadjuvant cycles as needed, except for the cycle immediately prior to ICS). GEN-1 will be added weekly to the regimen for subjects in the experimental arm, starting on C1D15. For subjects randomized to the experimental arm, an IP port must be placed at least seven days before GEN-1 administration to allow for healing. ICS will be 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 will begin after a recovery period of at least four weeks from ICS.

[0602] maintain

[0603] All subjects will receive BEV every 21 days for up to 18 additional cycles until resistance or unacceptable toxicity occurs. Subjects who are BRCA+ / HRD (HRD status as determined by the Myriad MyChoice assay) will receive olaparib for up to 24 months until resistance or unacceptable toxicity occurs. GEN-1 will be administered every 21 days to non-BRCA+ / HRD subjects in the experimental arm with BEV every 21 days for up to 18 additional cycles until resistance occurs.

[0604] safety

[0605] The run-in phase to ensure the safety of the NACT+BEV+GEN-1 combination will evaluate at least six subjects randomized to the experimental arms 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 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 principal investigator.

[0606] Subjects will be monitored for safety (with 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 the follow-up period until they resolve to Grade 2 or less (CTCAE v5.0).

[0607] Efficacy assessment

[0608] The primary endpoint for evaluating efficacy will be the presence of histopathologically confirmed MRD in SLL. Secondary endpoints include PFS and OS.

[0609] This protocol allows for the enrollment of subjects with 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.

[0610] Second Look Laparoscopy (SLL)

[0611] SLL should be performed approximately 6–8 weeks after day 1 of the last cycle of adjuvant therapy. SLL should be performed at least 4 weeks after the last dose of bevacizumab / biosimilar.

[0612] Detection of MRD at the time of SLL will be based on surgical pathology findings from multiple biopsies and peritoneal cytology obtained during the SLL procedure (see the SLL Surgical Manual). The presence of viable tumor by histopathology in any biopsy or peritoneal cytology is considered the presence of MRD (MRD+). The absence of MRD (MRD-) is classified as achieving a complete pathological response (no evidence of disease). As an exploratory analysis, MRD+ cases are subdivided into microscopic MRD or gross (surgically evident) MRD based on whether macroscopic tumor is present at SLL.

[0613] Radiation measurement of antitumor effects

[0614] The study will assess response and progression using the revised RECIST guidelines (version 1.1) and the new international criteria proposed by immune-related response criteria, which use the change in the longest diameter (unidimensional measurement) of tumor lesions and, in the case of malignant lymph nodes, the change in the shortest diameter.

[0615] Disease parameters

[0616] Measurable disease. A measurable lesion is defined as one that can be accurately measured in at least one dimension (the longest diameter to be recorded) by chest x-ray ≥ 20 mm, by CT scan ≥ 10 mm, or by caliper by clinical examination ≥ 10 mm. All tumor measurements must be recorded in millimeters (or fractions of a centimeter).

[0617] Note: Tumor lesions located in previously irradiated areas are not considered measurable unless progression is documented or a biopsy is obtained to confirm persistence at least 90 days after completion of radiation therapy.

[0618] Malignant lymph nodes. To be considered pathologically enlarged and measurable, lymph nodes must measure ≥15 mm in the short axis when assessed by CT scan (a CT scan slice thickness of ≤5 mm is recommended). At baseline and follow-up, only the short axis is measured and tracked.

[0619] Nonmeasurable disease. Any other lesion (or site of disease), including small lesions (longest diameter <10 mm or pathologic lymph nodes with a short axis of ≥10 mm to <15 mm), is considered nonmeasurable. Leptomeningeal disease, ascites, pleural / pericardial effusion, lymphangitis cutis / pneumonia, inflammatory breast disease, and abdominal masses (identified by physical examination but not by CT or MRI) are considered nonmeasurable.

[0620] Bone Lesions: Lytic bone lesions or mixed lytic-blastic lesions with an identifiable soft tissue component that can be assessed by CT or MRI may be considered measurable lesions if the soft tissue component meets the definition of measurability above. Blastic bone lesions are not measurable.

[0621] Cystic lesions that meet the criteria for a radiographically defined simple cyst should not be considered malignant lesions (neither measurable nor nonmeasurable) because they are, by definition, simple cysts. "Cystic lesions" thought to represent cystic metastases can be considered measurable lesions if they meet the definition of measurability above. However, when noncystic lesions are present in the same patient, these are preferred for selection as target lesions.

[0622] Target Lesions. All measurable lesions, representing every involved organ, with a maximum of two lesions per organ and up to a total of five lesions, should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected based on their size (lesions with the longest diameter) and should represent every involved organ, but should also be amenable to reproducible repeated measurements. In some cases, the largest lesion may not be amenable to reproducible measurement, and the next largest lesion that can be reproducibly measured in that situation should be selected. The sum of the diameters of all target lesions (longest axis for nonnodal lesions, short axis for nodal lesions) is calculated and reported as the baseline sum diameter. If lymph nodes are included in the sum, only the short axis is added to the total. The baseline sum diameter is used as a reference to further characterize any objective tumor regression in the measurable dimensions of disease.

[0623] Non-target lesions. Any other lesions (or disease sites), including any measurable lesions beyond the five target lesions, should be identified as non-target lesions and recorded at baseline. Measurement of these lesions is not required, but their presence, absence, or, in rare cases, definite progression should be noted throughout the follow-up period.

[0624] result

[0625] Figure 8A shows an analysis of progression-free survival for all subjects using Kaplan-Meier survival plots and a Log-rank test. Figure 8B shows an analysis of progression-free survival for subjects with known BRCA status using Kaplan-Meier survival plots and a Log-rank test. Figure 8C shows an analysis of progression-free survival for BRCA-positive subjects using Kaplan-Meier survival plots and a Log-rank test. Figure 8D shows an analysis of progression-free survival for BRCA-negative subjects using Kaplan-Meier survival plots and a Log-rank test.

[0626] Figure 8E shows the hazard ratio for NACT+GEN1 treatment versus NACT treatment, without stratification, for all subjects. Figure 8F shows the hazard ratio for NACT treatment versus NACT+GEN1 treatment, without stratification, for subjects with known BRCA status. Figure 8G shows the hazard ratio for NACT treatment versus NACT+GEN1 treatment by BRCA status. Figure 8H shows the hazard ratio for NACT treatment versus NACT+GEN1 treatment stratified by BRCA status.

[0627] The results show that at the upper 95% confidence level, survival was better with NACT+GEN1 compared with NACT alone.

Claims

1. 1. A method of treating a subject suffering from cancer, comprising administering to the subject a nucleic acid vector comprising a polynucleotide encoding interleukin-12 (IL-12), the nucleic acid vector being formulated in a lipopolymer, wherein the cancer comprises a BRCA-negative genotype and the cancer is homologous recombination proficient (HRP).

2. 1. A method of treating a subject, comprising: (i) determining whether the subject is (a) positive or negative for a BRCA-negative genotype, and (b) whether the subject is homologous repair proficient (HRP) or homologous repair deficient (HRD); and (ii) if the subject is BRCA-negative and HRP (BRCA- / HRP), administering to the subject a therapy comprising a nucleic acid vector comprising a polynucleotide encoding interleukin-12 (IL-12), formulated in lipopolymer.

3. The method of claim 1 or 2, wherein the cancer and / or subject is BRCA1 negative.

4. The method of any one of claims 1 to 3, wherein the cancer and / or subject is BRCA2 negative.

5. The method of any one of claims 1 to 4, wherein the BRCA-negative genotype comprises wild-type BRCA1 and / or BRCA2 genes.

6. The method of any one of claims 1 to 5, wherein a genotyping assay is performed to determine whether the subject is BRCA positive or BRCA negative, homologous repair proficient and / or homologous repair deficient.

7. The method of any one of claims 1 to 6, further comprising administering to the subject an anticancer agent, an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody), an immune checkpoint inhibitor, or any combination thereof.

8. 8. The method of claim 7, wherein the anti-cancer agent is a chemotherapeutic agent.

9. The method of any one of claims 1 to 8, further comprising administering an anti-cancer agent.

10. 10. The method of claim 9, wherein the anti-cancer agent is a chemotherapeutic agent.

11. The method of any one of claims 1 to 10, further comprising administering an antibody or antigen-binding fragment thereof that specifically binds to vascular endothelial growth factor (VEGF) (anti-VEGF antibody).

12. The method of any one of claims 1 to 11, further comprising administering an immune checkpoint inhibitor.

13. 13. The method of any one of claims 1 to 12, further comprising administering a maintenance dose of said polynucleotide encoding IL-12 formulated in a lipopolymer.

14. The method of any one of claims 1 to 13, wherein the polynucleotide encodes human IL-12.

15. 15. The method of any one of claims 1 to 14, 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.

16. The method of any one of claims 1 to 15, wherein the nucleic acid vector comprises an intron, a 3'UTR, an antibiotic resistance gene, or any combination thereof.

17. 17. The method of claim 16, wherein the 3'UTR is an hGH 3'UTR.

18. 18. The method of any one of claims 1 to 17, wherein the lipopolymer comprises polyethyleneimine (PEI) independently covalently attached to a cholesterol group and a polyethylene glycol (PEG) group.

19. The method of any one of claims 9 to 12, wherein the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, carboplatin, docetaxel, nab-paclitaxel, olaparib, and any combination thereof.

20. The method of any one of claims 9 to 19, wherein the anti-cancer agent comprises paclitaxel.

21. The method of any one of claims 9 to 19, wherein the anticancer drug comprises carboplatin.

22. The method of any one of claims 9 to 19, wherein the anticancer agent comprises docetaxel.

23. The method of any one of claims 9 to 19, wherein the anti-cancer agent comprises nab-paclitaxel.

24. The method of any one of claims 9 to 19, wherein the anticancer agent comprises olaparib.

25. The method of any one of claims 9 to 24, wherein the anti-VEGF antibody is selected from the group consisting of bevacizumab and ranibizumab.

26. 26. The method of claim 25, wherein the anti-VEGF antibody comprises bevacizumab.

27. 27. The method of claim 26, wherein the anti-VEGF antibody comprises Avastin or a biosimilar thereof.

28. 26. The method of claim 25, wherein the anti-VEGF antibody comprises ranibizumab.

29. 29. The method of claim 28, wherein the anti-VEGF antibody comprises Lucentis or a biosimilar thereof.

30. 30. The method of any one of claims 9 to 29, 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: 85, and a variable light chain (VL) comprising an amino acid sequence having at least about 85% identity to SEQ ID NO:

86.

31. 31. The method of claim 30, 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: 85, 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:

86.

32. 32. The method of any one of claims 9 to 31, wherein the anti-VEGF antibody is administered intratumorally, intraperitoneally, intravenously, intravesically, or any combination thereof.

33. 33. The method of any one of claims 9 to 32, wherein the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-VEGF antibody.

34. 34. The method of any one of claims 9 to 33, wherein the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

35. 35. The method of any one of claims 9 to 34, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from the group consisting of CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), and / or LAG-3.

36. The method of any one of claims 9 to 35, wherein the immune checkpoint inhibitor is an antibody.

37. 37. The method of any one of claims 9 to 36, wherein the immune checkpoint inhibitor is a small molecule inhibitor.

38. 38. The method of any one of claims 9 to 37, wherein the immune checkpoint inhibitor comprises a PD-1 antagonist selected from the group consisting of nivolumab, pembrolizumab, dostallimab, and cemiplimab.

39. 39. The method of any one of claims 9 to 38, wherein the immune checkpoint inhibitor comprises a PD-L1 antagonist selected from the group consisting of atezolizumab, durvalumab, and avelumab.

40. 40. The method of any one of claims 9 to 39, wherein the immune checkpoint inhibitor comprises a CTLA-4 antagonist.

41. 41. The method of claim 40, wherein the CTLA-4 antagonist comprises ipilimumab.

42. 42. The method of any one of claims 9 to 41, wherein the immune checkpoint inhibitor comprises a LAG-3 antagonist.

43. 43. The method of claim 42, wherein the LAG-3 antagonist comprises leratolimab.

44. 44. The method of any one of claims 9 to 43, comprising administering two or more immune checkpoint inhibitors.

45. 45. The method of any one of claims 9 to 44, wherein the immune checkpoint inhibitor is administered intratumorally, intraperitoneally, intravenously, intravesically, or any combination thereof.

46. 46. ​​The method of any one of claims 9-45, wherein the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the immune checkpoint inhibitor.

47. 47. The method of any one of claims 1 to 46, wherein the lipopolymer-formulated nucleic acid vector is administered intratumorally or intraperitoneally.

48. 48. The method of any one of claims 1 to 47, wherein the lipopolymer-formulated nucleic acid vector is administered intravenously.

49. 49. The method of any one of claims 9 to 48, wherein the lipopolymer-formulated nucleic acid vector is administered before, simultaneously with, or after the anti-cancer agent.

50. The nucleic acid vector comprising a polynucleotide encoding interleukin-12 (IL-12) formulated with the lipopolymer is administered at a concentration of about 35 mg / m ２ ~about 80mg / m ２ 50. The method of any one of claims 1 to 49, wherein the dose is

51. The lipopolymer is about 60 mg / m ２ 51. The method of any one of claims 1 to 50, wherein the dose is

52. 52. The method of any one of claims 9 to 51, wherein the anti-VEGF antibody is administered at a dose of about 10 to 20 mg / kg IV.

53. The administration of the anticancer agent is about 25 to 250 mg / m ２ 53. The method of any one of claims 9 to 52, comprising administering paclitaxel at a dose of about AUC 4-6 IV, optionally followed by administering carboplatin at a dose of about AUC 4-6 IV.

54. The administration of the anticancer agent is 25 to 250 mg / m ２ 54. The method of any one of claims 9 to 53, comprising administering docetaxel at a dose of about AUC 4-6 IV, optionally followed by administering carboplatin at a dose of about AUC 4-6 IV.

55. The administration of the anticancer agent is 25 to 350 mg / m ２ 55. The method of any one of claims 9 to 54, comprising administering nab-paclitaxel at a dose of about AUC 4-6 IV, optionally followed by administration of carboplatin at a dose of about AUC 4-6 IV.

56. 56. The method of any one of claims 1 to 55, wherein the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, bladder cancer, brain cancer, lung cancer and any combination thereof, and metastasis of any of the foregoing cancers.

57. 57. The method of claim 56, wherein the brain cancer is glioblastoma.

58. 58. The method of any one of claims 1 to 57, wherein the cancer is selected from the group consisting of ovarian cancer, fallopian tube cancer, primary peritoneal cancer, and any combination thereof.

59. 59. The method of any one of claims 1 to 58, wherein the cancer is ovarian cancer.

60. 60. The method of any one of claims 1 to 59, wherein the subject is a human.

61. 61. The method of any one of claims 1 to 60, wherein the nucleic acid vector is a plasmid.

62. 62. The method of any one of claims 1 to 61, wherein the lipopolymer is a nanoparticle.

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