An open label Phase-1 proof-of-concept study evaluated the safety and immunogenicity of IMNN-101, a DNA vaccine based on an expression plasmid and a synthetic delivery carrier (the PlaCCine vaccine), in healthy human adults previously vaccinated or infected with SARS-CoV-2. Study participants received a single intramuscular dose of 0.5 mg, 1.0 mg, or 2.0 mg of IMNN-101 DNA vaccine (n = 8 participants per group). Mild to moderate reactogenicity events were observed in 67% (16/24) of participants, including tenderness, hardening, pain, redness, swelling, and itching at the injection site, as well as fatigue, muscle aches, headache, and nausea/vomiting. Fourteen grade 1 or 2 treatment-emergent adverse events were reported: none of those attributed to IMNN-101. There were no reports of myocarditis or pericarditis. The immunization with IMNN-101 resulted in an increase in neutralizing antibody (NAb) titers against XBB.1.5 and other circulating variants of SARS-CoV-2, with 2-3-fold increase in NAb titers that were maintained during the 6-month follow up. This first-in-human evaluation of a PlaCCine-based DNA vaccine demonstrates a favorable safety profile and durable humoral immune response. The PlaCCine vaccine is thermostable, does not require a device or virus for delivery, and offers a viable alternative approach to immunization.

INTRODUCTION:IMNN-001 is designed for local and durable delivery of a pluripotent anti-tumor cytokine, IL-12, using an expression plasmid and a synthetic lipopolymer delivery system. IMNN-001, delivered intraperitoneally in combination with chemotherapy, is currently in a Phase 3 trial for the front-line treatment of advanced epithelial ovarian cancer. AREAS COVERED:This report details IMNN-001 preclinical and clinical development, demonstrating local and durable production of IL-12, minimal systemic exposure and manageable safety profile, as well as its antitumoral effects in a total of six completed trials in ovarian cancer. In the OVATION-2 Phase 2 randomized trial, neo- and adjuvant chemotherapy combined with IMNN-001 produced a numerical 13 month increase in overall survival, with even greater benefit in tumors that lacked DNA homologous repair activity. In translational studies, IMNN-001-induced changes in the tumor microenvironment are consistent with the observed induction of IL-12 and IFN-γ levels at the tumor site and support the hypothesis that IMNN-001 treatment alters the tumor microenvironment in favor of broad immune stimulation and inhibition of immunosuppressive mechanisms. EXPERT OPINION:IMNN-001 gene therapy could add clinically meaningful IL-12-driven immunotherapy to newly diagnosed ovarian cancer patients. IMNN-001 holds promise for synergistic combinations with immunotherapies requiring intrinsic immune activity.

Cancer vaccine approaches targeting neoantigens are gaining credence due to their specificity for tumors while sparing healthy tissues. Cancer vaccines based on DNA technology are attractive anti-cancer therapeutic tools due to their ability to elicit an effector T-cell-based immune responses. We report the development of a novel DNA-based cancer vaccine that relies on the delivery of a plasmid DNA vector encompassing immune epitopes of multiple neoantigens delivered with a novel synthetic DNA delivery system with the aim to elicit a broader immune response against cancer. The formulated vaccine was also investigated in combination with different adjuvants to maximize the anti-cancer responses. The vaccine vector encoding the immunogenic epitopes of multiple melanoma neoantigens and flanked by signal peptide at N-terminus and an MHC-targeted domain at the C-terminus end was formulated with a functionalized novel synthetic polymer with or without the adjuvants Poly I:C, diABZI, a STING agonist, or a mouse IL-12 plasmid. Mice were implanted subcutaneously with 0.5 x 106 B16F10 tumor cells on Day 0 and vaccinated intramuscularly with the DNA formulations on Day 2, 9, 16 and 23, and monitored for tumor growth and survival . For cellular responses, mice were injected intramuscularly with the DNA formulations on Day 0, 21, 42 and sacrificed 7 days later for cellular immune response by ELISPOT assay. Vaccination with formulated DNA alone yielded a significant inhibition (60%, p<0.01) of tumor growth and improved survival. Addition of Poly I:C, diABZI, or IL-12 plasmid further augmented the tumor inhibition to 90%(p<0.0001), 94%(p<0.0001), and 88% (p<0.0001), respectively, and improved the survival outcome. All animals in the adjuvant groups also lived longer. The anti-tumor efficacy results are supported by the onset of robust T-cell responses in all treatment groups supporting the involvement of immune activation in the action of this novel DNA vaccine that does not require a virus or device and can be manufactured rapidly with a simple and cost-effective manufacturing process critical for the viability of cancer vaccines. Kempaiah Rayavara, Subeena Sood, Majed Matar, Jessica Kim, John Henderson, Jeff Sparks, Meredyth Kinsella, Olivia Signer, Joseph Rogers, Chelsey Bellmon, Khursheed Anwer. A novel formulated neoantigen DNA vaccine with promising anticancer efficacy in B16F10 mouse melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5842.

Nucleic acid vaccine approaches have proven successful in the context of the SARS-CoV-2 pandemic, however challenges with delivery remain. Here we describe PlaCCine, a DNA-based vaccine platform that utilizes a device- and vector-free chemical delivery system. This system includes a DNA plasmid encoding the target antigen and generates robust immune responses, offering significant protection against live viral challenges in both non-human primates and mice. We designed spike plasmid immunogens representing early SARS-CoV-2 strains and found that parental spike PlaCCine vaccination induced SARS-CoV-2 specific cellular and humoral responses in non-human primates and supported significant viral control following challenge. To evaluate immunogenicity and protective efficacy against emerging variants, we further advanced the platform to incorporate the SARS-CoV-2 XBB1.5 variant and observed robust, dose-dependent cellular and humoral responses in mice. When mice were immunized and intranasally challenged with 1×105 TCID50 of SARS-CoV-2 XBB1.5 virus, all immunized animals survived the challenge and displayed undetectable lung viral loads. Together these data demonstrate the efficacy of the PlaCCine platform for the delivery of vaccine antigens and support the continued translation of this platform for infectious diseases.

OBJECTIVE:OVATION-2, a randomized, controlled, open label phase 1/2 study, evaluated the safety and efficacy of IMNN-001, an IL-12 immune gene therapy, with neo/adjuvant chemotherapy (N/ACT) compared to N/ACT in newly-diagnosed advanced epithelial ovarian cancer (EOC). METHODS:IMNN-001 is an immunotherapeutic nanoparticle comprising a DNA plasmid encoding the IL-12 gene encased in a lipopolymer. High-grade EOC patients were randomized 1:1 to carboplatin/paclitaxel IV every 21 days for 3 cycles, before and after interval debulking surgery (IDS) or to intraperitoneal (IP) IMNN-001, given weekly concurrently with chemotherapy for 8 weeks before and 9 weeks after IDS. RESULTS:54 and 58 patients with predominantly Stage IIIC/IV EOC were evaluated in the control and experimental arm, respectively. Primary endpoints were safety and PFS. Overall, the experimental arm was well tolerated with gastrointestinal and cytopenias as the most common TEAEs with no CRS or elevated risk of immune events. PFS was 14.9 months (mo) for the experimental arm vs 11.9 mo; HR 0.79 (95 % CI: 0.51-1.23). Secondary endpoints included OS (46.0 mo for experimental arm vs 33.0 mo; HR 0.69 (CI: 0.40-1.19)) and surgical response R0 rate (64.6 % experimental arm vs 52.1 %). For patients who received PARPi maintenance, PFS was 33.8 mo vs 22.1 mo; HR 0.80 (CI: 0.31-2.12) and OS was NE vs 37.1 mo with a HR of 0.38 (CI: 0.13-1.06) both favoring the experimental arm. CONCLUSION:The addition of IMNN-001 to N/ACT shows a promising numerical 13-mo benefit on survival with an acceptable safety profile in patients with newly-diagnosed advanced EOC.

Abstract The development of effective cancer vaccines that can trigger strong and enduring memory T cell responses against specific tumor associated antigens (TAAs) is crucial for advancing immunotherapy. DNA-based cancer vaccines have shown promise in generating precise and long-lasting immune responses. In particular DNA vaccines are capable of inducing both humoral and cellular immune responses. In this study, we aimed to investigate the effectiveness of DNA vaccines targeting the mouse Tyrosine-related protein-2 (Trp2) and human New York esophageal squamous cell carcinoma 1 (NY-ESO-1) TAAs in mouse melanoma model. These DNA vaccines were formulated with a functionalized polymer designed to protect DNA from degradation and improve its bioavailability. DNA vaccines targeting both mouse Trp2 (pVACTR) and human NYESO-1 (pVACNY) were constructed. A mouse melanoma cell line (B16F10-NY) overexpressing human NYESO-1 was also generated and demonstrated to induce tumors in C57BL/6 mice. Female C57BL/6 mice received two doses of intramuscular (i.m.) injection of formulated pVACTR or pVACNY or a combination thereof, formulated with the synthetic functionalized polymer, with a 3-week interval. The DNA vaccination induced a potent T cell response, as evidenced by ELISPOT assays showing strong IFN-γ secreting T cells against NYESO-1 and Trp2. Flow cytometry analysis of spleenocytes showed strong IFN-γ and TNF-α secreting CD4 T cells against NYESO-1. We further evaluated the prophylactic and therapeutic efficacy of DNA vaccines targeting both Trp2 and NYESO-1 using a murine syngeneic model. Animals were challenged with a lethal dose of B16F10-NY two weeks after the second immunization exhibited a significant (p<0.0001) delay in tumor progression and a substantial increase in survival compared to the mock control group. In the therapeutic testing, animals challenged with B16F10-NY and subsequently vaccinated at 4, 11, and 25 days also demonstrated delayed tumor growth and prolonged survival. These findings indicate that formulated DNA vaccine delivered via i.m. triggers robust T cell responses and exhibit both prophylactic and therapeutic efficacy, leading to significantly prolonged survival and delayed tumor growth. A DNA-based vaccine independent of viral vector or device has potential to elicit potent anti-tumor responses with better safety and compliance. Citation Format: Kempaiah Rayavara, John Henderson, Meredyth Kinsella, Jessica Kim, Majed Matar, Subeena Sood, Olivia Signer, Chelsey Bellmon, Corinne Le Goff, Khursheed Anwer, Jean Boyer. Intramuscular delivery of tumor associated antigen DNA vaccines elicits strong cellular immune response, delays tumor growth and prolongs survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6748.

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 10 5 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90% along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.

Changes in IL-12 and IFN-γ levels in blood samples

Adverse Events by Body System and Grade

Introduction: The purpose of this study was to investigate the effect of dosing frequency on the antitumor activity of intraperitoneal GEN-1, an interleukin-12 (IL-12) immune gene therapy in a mouse model of peritoneally disseminated ovarian cancer. Procedures: Three GEN-1 dosing regimens were examined for efficacy in ID-8 tumor-bearing mice: weekly, every 2 weeks and every 3 weeks. 2.5 million cancer cells were bolus injected into 4 groups (B, C, and D) of 10 mice each. A control group (A) of 6 mice had PBS injected IP without tumor and an untreated control (E) of 15 mice were injected with 2.5 million cancer cells. Groups B, C, and D were injected with GEN-1 IP weekly, every 2 weeks, and every 3 weeks respectively. Six animals from each group B, C, and D were harvested for translational research (TR) after 5 weekly, 3 every 2-week and 2 every 3-week treatments respectively. The remaining 4 animals in each group were followed for weight change (tumor burden) and survival. Additionally, TR evaluated change in ascites T-cell, B-cell and myeloid cell populations. Results: There was a gradual rise in tumor burden and mortality in all treatment groups with comparable rate between once every week and once every 2-week regimens. Once every 3-week regimen had relatively higher mortality rate and higher tumor burden. There were similar or higher increases in T-cell and B-cells with reduced treatment frequency with lesser increases in myeloid cell density with reduced treatment frequency. Conclusions: Once every 2-week dosing of GEN-1 in human studies is warranted. Future combination studies of Gen-1 with immune checkpoint inhibitors will evaluate the safety and efficacy of this regimen. Citation Format: Subeena Sood, Jean D. Boyer, Jessica Kim, Majed M. Matar, Olivia Signer, Jennifer S. Rice, Alanna M. Smith, Nicholas Borys, Khursheed Anwer. Efficacy of Gen-1, an interleukin-12 immune gene therapy, at different dose frequencies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4440.

PURPOSE:To inform continued development of the novel immune agent GEN-1, we compared ovarian cancer patients' end points from a neoadjuvant single-arm phase IB study with those of similar historic clinical trial (HCT) patients who received standard neoadjuvant chemotherapy.METHODS:Applying OVATION-1 trial (ClinicalTrials.gov identifier: NCT02480374) inclusion and exclusion criteria to Medidata HCT data, we identified historical trial patients for comparison. Integrating patient-level Medidata historic trial data (N = 41) from distinct neoadjuvant ovarian phase I-III trials with patient-level OVATION-1 data (N = 18), we selected Medidata patients with similar baseline characteristics as OVATION-1 patients using propensity score methods to create an external control arm (ECA).RESULTS:Fifteen OVATION-1 patients (15 of 18, 83%) were matched to 15 (37%, 15 of 41) Medidata historical trial control patients. Matching attenuated preexisting differences in attributes between the groups. The median progression-free survival time was not reached by the OVATION-1 group and was 15.8 months (interquartile range, 11.40 months to nonestimable) for the ECA. The hazard of progression was 0.53 (95% CI, 0.16 to 1.73), favoring GEN-1 patients. Compared with ECA patients, OVATION-1 patients had more nausea, fatigue, chills, and infusion-related reactions.CONCLUSION:Comparing results of a single-arm early-phase trial to those of a rigorously matched HCT ECA yielded insights regarding comparative efficacy prior to a randomized controlled trial. The effect size estimate itself informed both the decision to continue development and the randomized phase II trial (ClinicalTrials.gov identifier: NCT03393884) sample size. The work illustrates the potential of HCT data to inform drug development.

Background: Innovations in data science and trial design have catalyzed novel clinical research methods which may inform the future success of new experimental therapies more efficiently than previously believed. To guide development of the novel immune agent GEN-1, we compared endpoints experienced by patients from a recent neoadjuvant single-arm phase Ib study of GEN-1 plus standard chemotherapy to those of similar historical clinical trial patients in receipt of standard chemotherapy alone. Methods: To compare safety and efficacy endpoints following first-line neoadjuvant weekly GEN-1 immunotherapy and carboplatin and paclitaxel to standard first-line neoadjuvant carboplatin and paclitaxel in women with advanced ovarian cancer, we first applied key OVATION-1 trial (NCT02480374) inclusion and exclusion criteria to the Medidata Enterprise Data Store (MEDS) data to identify candidate historical clinical trial patients for comparison. We standardized and integrated patient-level MEDS data (N=41) from distinct phase I-III trials (enrollment years 2015-2016) with patient-level OVATION-1 data (N=18). Standard propensity score methods were used to identify MEDS patients who appeared similar to OVATION-1 patients to create a synthetic control arm (SCA). Results: Fifteen OVATION-1 patients (15/18, 83%) were matched to 15 (37%, 15/41) MEDS historical trial control patients. Matching attenuated pre-existing differences in attributes between the OVATION-1 and MEDS patients. The median progression-free survival time was not reached by the OVATION-1 group and was 15.8 months for the SCA. The hazard of progression for the OVATION-1 group relative to the SCA was 0.53 (95% CI 0.16, 1.73). Fourteen of 15 OVATION-1 patients (93.3%) and 15 of the SCA patients (100%, 15/15) had at least one MedDRA toxicity. Compared to SCA patients, OVATION-1 patients had a slightly higher incidence in nausea (OVATION-1 73.3%; SCA 53.3%), fatigue (OVATION-1 73.3%; SCA 33.3%), anorexia (OVATION-1 46.7%; SCA 13.3%), chills (OVATION-1 26.7%; SCA 6.7%), and infusion-related reaction (OVATION-1 26.7%; SCA 0%). Conclusions: The comparison of patient endpoints from a single-arm phase Ib trial to a historical clinical trial SCA provided informative and relatively reliable estimates of efficacy endpoints which were used to inform GEN-1’s expected effect study size in the phase II setting. This information led to a decrease in the number of planned patients for the subsequent randomized phase II trial. More broadly, this approach supports the ability of historical clinical trial patient comparisons to inform drug development via trial design, something which may further increase the scientific value of early phase trials. Citation Format: Xiang Yin, Ruthanna Davi, Elizabeth B. Lamont, Premal H. Thaker, William H. Bradley, Charles A. Leath, Kathleen M. Moore, Khursheed Anwer, Lauren Musso, Nicholas Borys. Phase Ib trial single-arm efficacy estimates via comparison to a historical clinical trial synthetic control arm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1025.

Abstract Purpose: GEN-1 (phIL-12-005/PPC), an IL12 plasmid formulated with polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, has preclinical activity when combined with platinum-taxane intravenous chemotherapy and administered intraperitoneally in epithelial ovarian cancer (EOC) models. OVATION I was a multicenter, nonrandomized, open-label phase IB trial to evaluate the safety, preliminary antitumor activity, and immunologic response to GEN-1 in combination with neoadjuvant chemotherapy (NACT) carboplatin-paclitaxel in patients with advanced EOC. Patients and Methods: A total of 18 patients with newly diagnosed stage IIIC and IV EOC were enrolled. A standard 3+3 dose-escalation design tested four GEN-1 doses (36, 47, 61, 79 mg/m2) to determine the maximum tolerated dose and dose-limiting toxicities (DLTs). GEN-1 was administered in eight weekly intraperitoneal infusions starting at cycle 1 week 2 in combination with three 21-day cycles of NACT carboplatin AUC 6 and weekly paclitaxel 80 mg/m2. Results: The most common treatment-emergent adverse events at least possibly related were nausea, fatigue, abdominal pain/cramping, anorexia, diarrhea, and vomiting. Eight patients experience grade 4 neutropenia attributed to NACT. No DLTs occurred. A total of 14 patients were evaluable for response and 12 (85.7%) had radiological response (two complete response and 10 partial response) prior to debulking; nine were R0 at debulking and one patient had complete pathologic response. IL12 and its downstream cytokine, IFNγ, increased in peritoneal washings but not as much in blood. Increased levels of myeloid dendritic cells and T-effector memory cells in peritoneal fluid, plus elevated CD8+ T cells and reduced immunosuppression within the tumor microenvironment were found. A median time to treatment failure of 18.4 months (95% confidence interval, 9.2–24.5) was observed in the intention-to-treat population. Conclusions: Adding GEN-1 to standard NACT is safe, appears active, and has an impact on the tumor microenvironment.

2 Background: This study evaluated weekly intraperitoneal (IP) GEN-1, an IL-12 plasmid formulated with polyethyleneglycol-polyethyleneimine-cholesterol lipopolymer, with intravenous (IV) weekly taxane (T) and carboplatinum (C) every 3 weeks in epithelial ovarian, fallopian tube or primary peritoneal cancer (EOC) patients undergoing neoadjuvant therapy (NAC). The primary objective was to evaluate the tolerability and safety of GEN-1 with NAC. Secondary objectives included objective clinical response and pathological response at interval debulking surgery (IDS). Methods: Newly diagnosed EOC patients with no prior therapies were eligible. The trial utilized a 3+3 design with dose escalation in ~30% increments at GEN-1 IP dose levels of 36 mg/m2, 47 mg/m2, 61 mg/m2, and 79 mg/m2 weekly for 8 treatments with concurrent IV T/C. Dose-limiting toxicity (DLT) was based on the first 4 doses of GEN-1 administered. Results: 18 patients were enrolled into the study and 12 of those patients received all 8 treatments with no DLTs. 14 patients underwent IDS. Most common related toxicities were Gr 1 nausea, abdominal pain and fatigue. One patient experienced Gr 2 fevers associated with GEN-1 but responded to acetaminophen and fluids. Conclusions: Adding GEN-1 to T/C is safe and appears to be active in EOC patients receiving NAC. Dose limiting toxicity was not reached and further dose escalation and safety and activity is being evaluated in an ongoing phase I/II study. Clinical trial information: NCT02480374. [Table: see text]

GEN-1 is a gene-based immunotherapy, comprising a human IL-12 gene expression plasmid and a synthetic plasmid delivery system, delivered intraperitoneally (ip.) to produce local and persistent levels of a pleiotropic immunocytokine, IL-12, at the tumor site in patients with advanced ovarian cancer. The goal of local and persistent IL-12 delivery is to remodel the highly immunosuppressive tumor microenvironment to favor immune stimulation while avoiding serious systemic toxicities, a major limitation of recombinant IL-12 therapy. Safe and sustained local production of IL-12 and related immunocytokines at the tumor site could produce potentially more favorable immunological changes in the tumor microenvironment and antitumor responses than a bolus systemic delivery of recombinant IL-12. Treatment safety, clinical benefits and biological activity of GEN-1 ip. in patients with ovarian cancer and in representative animal models are described.

Abstract Background: This abstract describes the translational component of a phase I study of weekly intraperitoneal (IP) GEN-1, an IL-12 plasmid formulated with polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, in combination with standard intravenous (IV) weekly taxane (T) and carboplatinum (C) every 3 weeks in epithelial ovarian, fallopian tube, or primary peritoneal cancer (EOC) patients undergoing neoadjuvant chemotherapy (NAC). Local administration of GEN-1 could provide persistent IL-12 secretion to induce type-1 immune responses in the immunosuppressive tumor microenvironment. Our hypothesis is that the GEN-1 plus NAC treatment will reprogram the tumor immune microenvironment towards a potent antitumor immune response. We have previously reported the clinical finding that GEN-1 is safe up to 79 mg/m2 IP weekly up to eight treatments. The most common related toxicities were Grade 1 nausea, vomiting, abdominal pain, and fatigue. Patients continue to be followed for progression and any drug-related safety events. Methods: Newly diagnosed advanced-stage EOC patients being treated with NAC were eligible. The trial utilized a 3+3 design with GEN-1 IP dose levels ranging from 36 mg/m2 to 79 mg/m2 weekly for 8 treatments with concurrent IV T/C. Tumor samples were collected at time of diagnostic laparoscopy and interval debulking (IDB). Blood and peritoneal ascites were collected before and after treatment. The immunologic analysis included frequency of immune cell populations and cytokine levels in tumor, blood, or peritoneal ascites. The levels of cytokines IL-12, IFN-γ, TNF-α, TGF-β, IL-10, and VEGF were quantified in blood plasma and ascites samples with commercially available cytokine-specific ELISA kits. The tumor tissue specimens were paraffin embedded and serial sections were stained with specific antibodies for various T-cell markers using immunohistochemistry. The density of various T-cells, dendritic cells, and myeloid-derived suppressor cells in blood or ascites was determined by flow cytometry. Results: The interim results obtained from Cohorts 1–4 (stage 1) show post-treatment changes in immune cell populations, including an increase in cytotoxic CD8+ T-cells and decrease in several immunosuppressive markers including FoxP3+, PD-1, PDL-1, and IDO-1. The cell ratio of CD8+ to all immunosuppressive markers appeared to be skewing beneficially towards more cytotoxic T lymphocyte and away from suppressive cells in most patients. There were no significant changes in the frequency of circulating T cells (or cytokine response). Analysis of ascites before and 24 hours after IP administration of GEN-1+NAC showed evidence of IL-12 gene transfer and activation of downstream signaling pathways, including increases in IFN-γ levels and inhibition of peritoneal VEGF levels. The cytokine response was predominantly in ascites while comparatively little changes were observed in blood plasma. Thus, the immunomodulatory and therapeutic effect of GEN-1 treatment is primarily localized within the tumor microenvironment and does not appear to have any untoward effect on systemic immune responses. The changes in cytokine levels appeared to be GEN-1 dose dependent. Additional analyses from this study are in progress. Conclusions: Adding GEN-1 to neoadjuvant T/C is safe and biologically active in EOC patients. Based on preliminary analyses of Cohorts 1–4 (stage 1), the treatment of EOC patients with GEN-1 and NAC has resulted in favorable immunologic responses. Final translational results will be presented at the meeting. Clinical trial information: NCT02480374. Citation Format: Khursheed Anwer, Junko Matsuzaki, Wiam Bshara, Amit Lugade, Angela Omilian, Premal H. Thaker, William H. Bradley, Rebecca Arend, Camille Gunderson, Jason Fewell, Nicholas Borys, Lauren Musso, Adekunle Odunsi. Immunologic changes following intraperitoneal administration of a formulated IL-12 plasmid in combination with standard neoadjuvant chemo in newly diagnosed advanced stage ovarian cancer patients. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A76.