GEN-1 in Combination with Neoadjuvant Chemotherapy for Patients with Advanced Epithelial Ovarian Cancer: A Phase I Dose-Escalation Study

Abstract

Purpose:

GEN-1 (phIL-12-005/PPC), an interleukin-12 (IL-12) plasmid formulated with polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, has preclinical activity when combined with platinum-taxane intravenous (IV) chemotherapy and administered intraperitoneally (IP) in epithelial ovarian cancer (EOC) models. OVATION I was a multi-center, non-randomized, open-label phase IB trial to evaluate the safety, preliminary anti-tumor activity, and immunological response to GEN-1 in combination with neoadjuvant chemotherapy (NAC) carboplatin-paclitaxel in patients with advanced EOC.

Patients and Methods:

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 (DLT). GEN-1 was administered in eight weekly IP infusions starting at cycle 1 week 2 in combination with three 21-day cycles of NAC 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 NAC. No DLTs occurred. 14 patients were evaluable for response and 12 (85.7%) had radiological response (2CR + 10PR) prior to debulking; 9 were R0 at debulking and one patient had complete pathological response. IL-12 and its downstream cytokine, interferon-γ (IFN-γ), increased in peritoneal washings but not 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 (TTF) of 18.4 months (95% CI, 9.2, 24.5) was observed in the ITT population.

Conclusions:

Adding GEN-1 to standard NAC is safe, appears active and impacts the tumor microenvironment.

Introduction

Epithelial ovarian cancer (EOC) is the 5^th deadliest malignancy among women in the United States. [1] There are approximately 22,000 new cases of ovarian cancer every year and the majority, approximately 70% of cases, are diagnosed in advanced stages III and IV. EOC is characterized by dissemination of tumor in the peritoneal cavity with a high risk of recurrence (75%, stage III and IV) after seemingly successful surgery and chemotherapy. [2] Maintenance therapy following a complete or partial remission for patients with a germline or somatic BRCA 1/2 mutation utilizing poly ADP -ribose polymerase inhibitors (PARPi), olaparib or niraparib prolongs time to recurrence. These maintenance therapies have not yet demonstrated improved overall survival. [3][4] Similarly, the angiogenesis inhibitor bevacizumab improves progression free survival as a primary treatment in EOC without improving overall survival. [5] Since the 5-year survival rates of patients with stages III and IV disease at diagnosis are poor, at 41% and 20% respectively, there remains a need for a therapy that not only reduces the recurrence rate but also meaningfully improves overall survival. [1][6]

Immunotherapy interventions are considered promising candidates for the treatment of ovarian cancer considering the immunogenic nature of the malignancy. [7] The evidence of immune activation in ovarian cancer has been demonstrated in the production of antibodies or anti-tumor T-cell lymphocytes in primary tumor, ascites, and blood. [8] [9] [10] Presence of tumor-infiltrating cytotoxic T-cell lymphocytes has been linked to better prognosis while presence of immunosuppressive regulatory T cells (Tregs) has been associated with poor prognosis survival in ovarian cancer. [11] [12] [13] The peritoneal cavity of advanced ovarian cancer patients contains the primary tumor environment and is an attractive target for a regional approach to immune modulation.

Interleukin-12 (IL-12) is a pluripotent cytokine associated with stimulation of innate and adaptive immune response against cancer. Clinical responses to recombinant IL-12 have been observed in multiple cancers. [14] [15] [16] For optimal effect, cytokines must be present over an extended period of time, which is not achievable with recombinant IL-12 due to its short half-life when a single dose is administered in humans. [16] [17] Toxicity is a serious dose limiting concern with systemic exposure, leading researchers to explore alternative means of IL-12 delivery. [18]

GEN-1 is a gene therapy that produces safe and durable local levels IL-12 to stimulate innate and adaptive components of the immune system. The GEN-1 nanoparticle comprises a DNA plasmid encoding IL-12 gene and a synthetic polymer facilitating plasmid delivery. [19] Intraperitoneal administration of GEN-1 in recurrent ovarian cancer patients and in preclinical models of the disease has been shown to elevate IL-12 and its downstream cytokines IFN-γ and TNF-α levels locally at tumor site for several days after a single injection and have resulted in encouraging efficacy results. [20] [21] [22] [23] In these studies GEN-1 was well-tolerated, and unlike previous studies of IL-12 no dose-limiting toxicities (DLT) were identified up to the highest evaluated dose of 36 mg/m².

Recent preclinical and clinical studies evaluating the timing of immunotherapy suggest that the greatest opportunity for effectiveness is in the neoadjuvant setting. [24] In mouse models neoadjuvant immunotherapy generated persistent levels of tumor-specific CD8+ T cells in the blood even after the mice were tumor free and throughout life. [25] Furthermore, clinically, neoadjuvant chemotherapy (NAC) has been shown to be associated with increased concentrations of CD3+ and CD8+ T cells, CD8+ TIA-1+ T cells, and CD20+ B cells. Meanwhile, the immunosuppressive cells FoxP3+, PD-1+, IDO-1+ cells and CD68+ PD-L1 macrophages remain unchanged. [26]

The OVATION I study evaluated escalating doses of GEN-1 (36, 47, 61, and 79 mg/m²) in combination with a standard carboplatin/paclitaxel neoadjuvant regimen. This setting ensured the patient’s immune system had not been weakened by prior therapies and maximized the potential for complementary or synergistic effects of the immune stimulator and chemotherapy. Moreover, the neoadjuvant setting allowed for collection of treatment naïve and post-treatment primary tumor tissue at interval debulking for translational studies that was not possible in previous GEN-1 studies conducted in recurrent disease.

Patients & Methods

Study design and patient eligibility

The OVATION I Study was a multi-center open-label phase IB trial that enrolled newly diagnosed patients with advanced EOC who were candidates for NAC. The study used a standard 3+3 dose-escalation design to determine the safety, biological activity, and preliminary activity of GEN-1 in combination with a standard neoadjuvant dose dense paclitaxel with tri-weekly carboplatin regimen. A recommended Phase II dose would be based on a MTD or a maximum biological activity dose.

A histologic diagnosis of epithelial ovarian, fallopian tube, or primary peritoneal carcinoma with an epithelial cell type of either: high grade serous adenocarcinoma, endometrioid adenocarcinoma, undifferentiated carcinoma, clear cell adenocarcinoma, mixed epithelial carcinoma, or adenocarcinoma not otherwise specified (See Table 1) was required prior to enrollment. Patients were at least 18 years old, with adequate bone marrow, renal, hepatic, and neurologic functions and had to be free of active infection requiring parenteral antibiotics or a serious uncontrolled medical illness/disorder within four weeks of study entry. Patients were also required to have an ECOG performance score of 0, 1, or 2 and had to be free of any condition/anomaly that would interfere with the appropriate placement of the intraperitoneal (IP) catheter for GEN-1 administration.

Table 1.

Patient Characteristics (N=18)

Characteristics
Age median (range)	63.3 (48 – 79)
Race/ethnicity
White, non-Hispanic	15 (83%)
Black	3 (17%)
FIGO
IIIC	12 (67%)
IV	6 (33%)
Staging laparoscopy Findings
Omental disease	14 (78%)
Carcinomatosis	14 (78%)
Diaphragmatic carcinomatosis	10 (56%)
Mesenteric retraction	8 (44%)
Bowel infiltration	7 (39%)
Stomach infiltration	1 (6%)
Liver infiltration	4 (22%)
Histology
High grade, serous adenocarcinoma	17 (95%)
Adenocarcinoma NOS	1 (6%)
Homologous-recombination deficiency (HRD)	5 (28%)
BRCA 1	1 (6%)
BRCA 2	1 (6%)
HRD Unknown	1 (6%)

Patients who were treated previously with GEN-1 or with chemotherapy or radiotherapy for any tumor of the abdominal cavity or pelvis were excluded, as were patients who had received oral or parenteral corticosteroids within 2 weeks of study entry or required ongoing systemic immunosuppressive therapy. Additional exclusion criteria included active autoimmune disease requiring treatment, active hepatitis, other invasive malignancies (other than non-melanoma skin cancer), a history or evidence of CNS disease, and a concurrent severe medical problem unrelated to the malignancy that would expose the patient to extreme risk or decreased life expectancy.

Four centers in the United States participated in OVATION I. This study was sponsored by Celsion Corporation and registered with ClinicalTrials.gov (NCT02480374). The protocol was approved by the institutional review board (IRB) or central IRB and biological safety committees of each institution, and all patients provided written informed consent before enrollment and performance of any study-related procedures. The study was conducted in compliance with the protocol, International Conference on Harmonization Good Clinical Practice Guidelines E6 (ICH-GCP), National Institute of Health (NIH) Guidelines for Research Involving Recombinant DNA Molecules (April 2002) and with the Declaration of Helsinki and its amendments. The study was monitored for safety and dose escalation decisions by an independent Data Safety Monitoring Board (DSMB).

Treatment

This study evaluated four dose levels of GEN-1 [36, 47, 61, and 79 mg/m² q week intraperitoneal (IP) for up to 8 weeks] in combination with a standard NAC regimen, carboplatin [AUC of 6 mg/mL-min intravenously (IV) q 3 weeks for 3 cycles] and paclitaxel [80 mg/m² q week IV for 9 weeks]. The starting dose of 36 mg/m² was chosen since it was the highest dose examined in a previous study combining GEN-1 with pegylated liposomal doxorubicin without evidence of DLT in patients with platinum-resistant ovarian cancer. [22] Eighteen patients were enrolled, and an IP catheter with subcutaneous reservoir was implanted. GEN-1 dosing commenced on cycle one day eight in order to skip administration during dexamethasone pretreatment given on cycle one day one. Steroids may blunt the effects of immunotherapies such as GEN-1; therefore, concurrent administration was not permitted. [27] The DSMB met to review safety data for subjects in each cohort before the next highest dosage strength was assessed. Patients had to have at least four doses of GEN-1 in order to be evaluated for safety at each dose level. Treated patients were planned to undergo interval debulking surgery after three cycles of NAC with weekly GEN-1 followed by three additional cycles of adjuvant chemotherapy. The dose escalation was stopped at 79 mg/m² due to limitations in the manufacturing of additional doses. These limitations were overcome for a subsequent trial of GEN-1 currently ongoing where the starting dose has been determined to be 100 mg/m². [28]

Assessments

Toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 and dose limiting toxicities were defined in the Data Safety Monitoring Board charter. [29] Clinical endpoints included objective response rate (RECIST version 1.1), R0 resection rate, pathological response, chemotherapy response score (CRS) and progression free survival (PFS) per physician assessment. [30] [31] [32] All enrolled patients were assessed for toxicity however patients had to complete four administrations of GEN-1 to be considered evaluable for a dose level.

Tumor, ascites, and blood samples were collected at time of initial staging and at interval debulking surgery. Venous blood and peritoneal fluid samples (via intraperitoneal port) prior to and 24 hours after each of the first four GEN-1 treatments. Cytokine ELISAs, flow cytometry, and immunohistochemical analyses were conducted at Roswell Park Cancer Institute (Buffalo, NY).

Immunohistochemistry (IHC)

IHC assays and automated digital pathology analysis were performed on FFPE tissue blocks. Tissue sections (4–5 μm) were prepared on slides loaded on a DAKO autostainer (Dako, Glostrup, Denmark) and after serum free protein block the respective primary antibodies for CD3, CD4, CD8, FoxP3, IDO-1, PD-1 and PD-L1 were applied separately. The EnVision+ horseradish peroxidase system (Dako) and DAB chromogen were used for visualization. Slides were digitally scanned using Aperio Scanscope (Aperio Technologies, Inc., Vista, CA) with 20× bright-field microscopy. These images were then accessible using Spectrum (Aperio Technologies, Inc., Vista, CA), a web-based digital pathology information management system. Slide images are automatically associated to a digital slide created in the Digital Slide table in Aperio eSlide Manager. Once slides are scanned, Aperio ImageScope version 11.2.0.780 (Aperio Technologies, Inc., Vista, CA) was used to view images for image analysis. An outline of the tumor and the size of the analysis area were defined, and the lymphocytes were counted using an optimized algorithm for each stain and results were normalized to number of lymphocytes per square millimeter.

ELISA

Cytokine levels in plasma and peritoneal fluid were measured using ELISA kits according to the manufacturer’s protocol. IL-12 ELISA kit was purchased from R&D Systems (Minneapolis, MN) and IFN-γ ELISA kit was purchased from Thermo Fisher Scientific (Waltham, MA). For peritoneal fluid or wash samples, the cytokine data was normalized with total protein concentration which was quantified with a Pierce BCA protein assay kit from Thermo Fisher (Waltham, MA). Optical Density of triplicate wells was read by BioTek Synergy HT microplate reader (Winooski, VT) and concentration was determined from standard curve. The cytokine values generated from each treatment were used to generate the overall mean for each cohort.

Flow Cytometry

Peripheral blood mononuclear cells (PBMC) and cells from peritoneal fluid were isolated by density gradient centrifugation and cryopreserved in liquid nitrogen freezer until analysis. The cells were stained with fixable viability stain 700 (BD Horizon) and incubated with FcR block (Myltenyi Biotech) followed by staining with various antibodies for T-cell phenotype (APC/H7-CD45, PerCP/Cy5.5-CD3, BUV395-CD4, BV650-CD45RA and BV421-CCR7) and myeloid dendritic cells (APC/H7-CD45, PE-CD11c and BV650-CD123). The stained cells were acquired by BD Fortessa flow cytometer and analyzed using FlowJo software.

Statistical design

This study utilized a standard 3 + 3 design to identify the MTD of GEN-1 with NAC in patients with newly diagnosed EOC. Dose escalation would be considered by the DSMB after a cohort of at least three patients who received at least 4 doses of GEN-1 were evaluated. If no patients out of three in a specific dose level demonstrated a DLT, then the study can proceed to the next higher dose level. If one patient out of three demonstrated a DLT, an additional three patients were to be enrolled at that dose level. If two or more patients experience a DLT then the GEN-1 dose must be dropped to the previous level. Less than 2 patients of six can experience a DLT before declaring a maximum tolerated dose. PFS is defined as the duration of time from start of treatment (cycle 1 day 1) to time of progression or death, whichever occurs first. The data was expressed as the median value for the intent to treat or per protocol population. Resection scores were expressed as a percentage of total patients for a defined group. Translational data was tabulated as mean ± standard deviation except for instances where average values were plotted for trend analysis and where comparisons are expressed as a percentage of baseline. Data were analyzed using Student’s t-test. For comparison of pre-treatment and post-treatment paired samples, Wilcoxon matched pairs signed rank test was used, after checking that for most differences in cell density, the normality assumption was violated.

Results

Four centers enrolled 18 patients between September 2015 and May 2017 (ITT). Six patients did not receive the full regimen of GEN-1 treatments due to: port related infection (0 GEN-1 doses), bowel perforation (1 dose of GEN-1), bowel obstruction (1 dose of GEN-1), dosing delays of >21 days due to myelotoxicity (6 doses of GEN-1), sepsis and congestive heart failure (5 doses of GEN-1) and altered taste (5 doses of GEN-1). One patient voluntarily withdrew from GEN-1 treatment due to altered taste attributed to GEN-1. In addition, one patient died within 40 days of her first dose of chemotherapy due to complications of the ovarian cancer. As a result, 15 patients were evaluable for safety of dose (received 4 administrations of GEN-1) and 14 underwent interval debulking thus were evaluable for RECIST, for resection status, and pathological response (per protocol population). Biological samples (i.e., blood, ascites/peritoneal washes, and tumor tissue) were collected from 12 patients who received the full complement of 8 doses of GEN-1 for translational data.

Safety

The DSMB reviewed the safety data at the completion of every dosing cohort which would comprise of at least three patients who completed at least 4 doses of GEN-1. Table 2 summarizes the adverse events that were at least possibly related to GEN-1 by frequency and severity for those 15 patients treated with at least 4 doses of GEN-1. In general, all dose levels were well tolerated. Most AEs were grade 1 or 2 in nature. The most commonly reported adverse events at least possibly attributed to GEN-1 in descending order include nausea (67%), fatigue (53%), abdominal pain/cramping (40%), anorexia (40%), diarrhea (40%), and vomiting (40%). Of the grade 3 and 4 reported AEs, the following were at least possibly attributed to GEN-1: nausea (n =2), fatigue (n=2), abdominal pain/cramping (n=1), anorexia (n=1), diarrhea (n=2), dehydration (n=2), vomiting (n=1), hypokalemia (n=1), sepsis (n=1), and vasovagal reaction (n=1). Hematological toxicities were also reported as possibly being attributed to GEN-1 which were also associated with chemotherapy. There were no dose limiting toxicities detected and a MTD was not reached at the doses evaluated in this study. Limiting dexamethasone premedication to cycle one day 1 did not result in any clinically significant hypersensitivity reactions at subsequent cycles of chemotherapy administration. A supplementary table is provided which presents all adverse events for all patients (N=18) regardless of attribution.

Table 2.

Frequency and Severity^* of Adverse Events At Least Possibly Related to GEN-1 (N=15)

Term	Frequency	Percentage	Grade 1 – 2	Grade 3	Grade 4	Grade 5
Nausea	10	67%	8	2	0	0
Fatigue	8	53%	6	2	0	0
Neutropenia**	8	53%	2	1	5	0
Abdominal Pain / Cramping	6	40%	5	1	0	0
Leukopenia**	6	40%	3	2	1	0
Anorexia	6	40%	5	1	0	0
Diarrhea	6	40%	4	2	0	0
Vomiting	6	40%	5	1	0	0
Anemia**	6	40%	2	4	0	0
Thrombocytopenia**	5	33%	2	1	2	0
Weakness	5	33%	5	0	0	0
Chills	4	27%	4	0	0	0
Fever	4	27%	4	0	0	0
Dehydration	3	20%	1	2	0	0
Hypomagnesemia	3	20%	3	0	0	0
Constipation	2	13%	2	0	0	0
Dysguesia	2	13%	2	0	0	0
Hypokalemia	2	13%	1	1	0	0
Alopecia	1	7%	1	0	0	0
Creatinine Increased	1	7%	1	0	0	0
Cytokine Release Syndrome	1	7%	1	0	0	0
Dizziness	1	7%	1	0	0	0
Dyspnea	1	7%	1	0	0	0
Elevated Alkaline Phosphatase	1	7%	1	0	0	0
Elevated ALT	1	7%	1	0	0	0
Elevated AST	1	7%	1	0	0	0
Erythema (port site)	1	7%	1	0	0	0
Hot Flash	1	7%	1	0	0	0
Hyperglycemia	1	7%	1	0	0	0
Hypocalcemia	1	7%	1	0	0	0
Nasal Congestion	1	7%	1	0	0	0
Neuropathy	1	7%	1	0	0	0
Pain	1	7%	1	0	0	0
Port Infection	1	7%	1	0	0	0
Sepsis	1	7%	0	0	1	0
Sinus Disorder	1	7%	1	0	0	0
Tremor	1	7%	1	0	0	0
Vasovagal Reaction	1	7%	0	1	0	0

^*Common Terminology Criteria for Adverse Events (CTCAE) version 4.0

^**Hematological toxicities were also reported as possibly being attributed to GEN-1 which were also associated with chemotherapy

Clinical Response

All patients were evaluated for efficacy (ITT) and a per protocol assessment was conducted for patients who underwent interval debulking. Time to treatment failure (TTF) of 18.4 months with 95% CI (9.2, 24.5) (range: 0.1–48.4 months) was observed in the ITT population (N=18) while a TTF of 21 months with 95% CI (11.5, 33.8) (range: 9.3–48.4 months) in the per protocol population (n=14).

Table 3 presents radiographic tumor response, surgical outcome, pathological response, and chemotherapy response score by dose. Objective response rates (ORR) to NACT/GEN-1 appeared to favor higher doses of GEN-1 between the two high and two low dose cohorts as calculated for RECIST-evaluable patients, with 100% of the high-dose cohorts having a complete or partial response (1 CR and 7 PR) and 67% of the low-dose patients (1 CR and 3 PR). Patients in the high-dose cohort achieved 88% R0 resection, versus 33% in the low-dose cohorts. There was a single case of a complete pathological response at 36 mg/m². Similarly, pathological response favored the higher dose cohort with 50% of subjects achieving the optimal chemotherapy response score (CRS) of 3 while only 17% of patients did so at the lower doses. In addition, one patient remains progression free at 4 years of follow up.

Table 3.

Tumor Response, Surgical Outcome, Pathological Response and Chemotherapy Response Score with NAC/GEN-1 escalating doses.

Radiographic Response		Total (n)	Cohort 1 36 mg/m2	Cohort 2 47 mg/m2	Cohort 3 61 mg/m2	Cohort 4 79 mg/m2
Tumor Response	CR	2	1	0	0	1
	PR	10	0	3	3	4
	SD	2	2	0	0	0
Objective Response Rate			67%		100%
Surgical Outcome	R0	9	2	0	2	5
	R1	3	1	2	0	0
	R2	2	0	1	1	0
R0 Resection Rate			33%		88%
Pathological Response	cPR	1	1	0	0	0
	Micro	8	1	2	1	4
	Macro	5	1	1	2	1
cPR/Micro Rate			60%		63%
Chemotherapy Response Score	CRS 3	5	1	0	2	2
	CRS 2	5	2	1	0	2
	CRS 1	4	0	2	1	1
CRS 3 Rate			17%		50%

Translational Responses

GEN-1 intraperitoneal treatment increased IL-12 and IFN-γ levels in ascites in a dose-response manner, as shown in Figure 1. IL-12 levels in ascites increased 3.2.- and 23-fold at the lowest and highest GEN-1 doses (36 and 79 mg/m²) respectively, while IFN-γ levels rose 3.1- and 67-fold. The differences in IFN-γ increase between the various dose levels were statistically significant. The increase in IL-12 levels followed a similar dose pattern, but the differences between various dose levels were statistically not as significant as seen with IFN-γ. As expected, GEN-1 had a much smaller effect on IL-12 and IFN-γ expression in the blood, where both cytokines’ levels increased between 1.2- and 3.1-fold (Supplementary Figure 1). (The graphs present data from patients in RECIST-evaluable cohorts of Table 2 with one exception – a subject in the 79 mg/m² cohort could not be assayed.)

Figure 1.

Changes in IL-12 and IFN-γ levels in ascites/peritoneal wash before and 24 hours after IP administration of GEN-1 and expressed relative to total protein in the sample (pg/mg protein). Fold changes over pre-treatment values for individual samples are represented by circles and the mean for each group is represented by the solid lines. The differences between the dose groups were determined by the Student t test.

The effects of GEN-1/NAC included reductions in four immunosuppressive markers in the tumor microenvironment: forkhead box p3 (Foxp3), indoleamine 2,3-dioxygenase-1 (IDO1), programmed cell death protein-1 (PD-1), and programmed death-ligand1 (PD-L1). The pre- and post-treatment density of cells with immunosuppressive markers on an individual patient basis are shown in Fig. 2A–D, and % changes from pre-treatment values for each patient are provided in Fig. 2E. The combination regimen reduced the cell density of all four indicators of immunosuppression between the initial biopsy sample and tumor resection in majority of patients. Reduction in FoxP3, IDO1, PD-1, and PD-L1 was observed in 67%, 67%, 83%, and 67% of patients, respectively. The range of inhibition in these patients varied from 8–95% (Foxp3), 33–94% (IDO1), 20–92% (PD-1) and 37–98% (PD-L1). Although from a small sample size, these trends are instructive since the Foxp3, IDO1, and PD-1 changes occurred generally in a GEN-1 dose-dependent manner (Fig. 2F) and are consistent with the changes in IL-12 and IFN-γ levels in ascites. The ratio of CD8+ cells to each of the four immunosuppressive markers also increased in majority of patients (Fig. 3 A–D). The increase in CD8+/Foxp3, CD8+/IDO1, CD8+/PD-1 and CD8+/PD-L1 ratio was observed in 91%, 75%, 67%, and 75% of patients, respectively. The increase in CD8+/Foxp3 and CD8+/PD-1 ratio was statistically significant with p-values of 0.016 and 0.03, respectively. The percent change in the ratio of CD8+ cells to immunosuppressive markers is plotted in Fig 3E, demonstrating a positive shift in CD8+ ratios in majority of patients.

Figure 2.

Changes in immunosuppressive markers in the tumor microenvironment following GEN-1 and NAC treatment. Changes in Foxp3 (A), PD-1 (B), IDO1 (C), and PD-L1 (D) immune cell markers before treatment (pre) and after treatment and at debulking surgery (post) for individual patients. E. Percent changes in each of the cell types. F. Percent changes between low dose group (36 mg/m², 47 mg/m²) (n=4) and high dose group (61 mg/m², 79 mg/m²) (n=8). The paired comparison of pre- and post-treatment values was done by using Wilcoxon matched pairs signed rank test.

Figure 3.

Changes in the ratio of CD8⁺ cells to immunosuppressive markers following GEN-1 and chemotherapy treatment. A-D. Changes in the ratio of CD8⁺ T cells to FoxP3, PD-1, IDO1, and PD-L1 markers in tumors of individual patients before treatment (pre) and after treatment and at debulking surgery (post). E. Percent change in CD8+/Foxp3, CD8⁺/PD-1, CD8⁺/IDO1, CD8⁺/PD-L1 ratio before (pre) and after treatment (post) for individual patients. The paired comparison of pre- and post-treatment values was done by using Wilcoxon matched pairs signed rank test.

GEN-1/NAC therapy also altered the densities of CD4+ and CD8+ T cells in tumor specimens collected at enrollment and during debulking surgery. The pre- and post-treatment density of CD4+ and CD8+ cells and the CD8+/CD4+ cell ratio on an individual patient basis and the mean values (n=11 for CD4⁺ and n=12 for CD8⁺) are shown in Fig. 4. The CD8+ cell density increased in 67% of patients, CD4+ cell density decreased in 72% of patients, and the CD8+/CD4+ ratio increased in 82% of patients (Fig. 4A–C). These changes noted on an individual patient basis are consistent with the mean values where the CD8⁺ T cell density increased 53% between pre- and post-treatment versus a decline of 73% in CD4⁺ T cells (Fig. 4D). Thus, the ratio of CD8⁺/CD4⁺ T cells increased 483% from 0.63 prior to GEN-1/NACT to 3.54 after. Again, these changes, although from a small sample size, are consistent with reports of an increased CD8⁺ T cell density after NAC. [26]

Figure 4.

Changes in CD4⁺ and CD8⁺ cell density following GEN-1 and chemotherapy treatment. A-B. Changes in CD4⁺ and CD8⁺ cell density in individual patients before treatment (pre) and after treatment and at debulking surgery (post). C. CD8⁺/CD4⁺ cell ratio at pre- and post-treatment. D. The paired comparison of pre- and post-treatment values was done by using Wilcoxon matched pairs signed rank test.

An assessment of the effects of GEN-1/NAC on immune cells in ascites found favorable trends in myeloid dendritic cell (mDC) and effector memory T cell (T_EM) populations. The pre- and post-treatment density of mDC and TEM cells (mDC: CD45⁺dump (CD3/CD56/CD19/CD14) ⁻CD123⁻ CD11c⁺ and T_EM: CD45⁺CD3⁺CD4⁻CD8⁺CD45RA⁻ CCR7⁻) on an individual patient basis are shown in Fig. 5 A–B. For both mDC and TEM the cell density increased in 4 out of 5 patients, and the percentage of total cells (mean ± SE, n=5) increased approximately 3-fold in response to therapy (Fig. 5C).

Figure 5.

The effect of GEN-1 on myeloid dendritic cells and CD8⁺ TEM cells in peritoneal fluid. A-B. Changes in myeloid dendritic cells and CD8⁺ TEM cell density in individual patients (n=5) before treatment (pre) and after treatment at debulking surgery (post). C. The average pre- and post-treatment data. The paired comparison of pre- and post-treatment values was done by using Wilcoxon matched pairs signed rank test.

Discussion

Escalating GEN-1 to doses up to 79 mg/m² IP administered weekly for up to 8 dosages was safe and reasonably tolerated when administered in combination with neoadjuvant chemotherapy in newly diagnosed epithelial ovarian cancer patients. There were no dose-limiting toxicities detected and an MTD was not reached. The majority of adverse events attributed to GEN-1 were low grade, and manageable. These safety findings are consistent with previous studies of GEN-1 in patients with ovarian cancer. [20] [21] [22] Moreover, the OVATION I study data supports that GEN-1 may be safely administered with standard neoadjuvant chemotherapy.

The safety profile of GEN-1 is consistent with its local administration and appears to have a distinct advantage over recombinant IL-12 therapy which is associated with systemic dose limiting toxicity. The activity of intraperitoneally administered GEN-1 appears to be localized to the peritoneal cavity and draining lymph nodes and potentially in the resident B cells, macrophages and dendritic cells. [33] The increases detected in IL-12 and IFN-γ levels were primarily peritoneal with relatively little increases in systemic circulation resulting in this favorable safety profile.

There appears to be a suggestion of clinical activity with the addition of GEN-1 to NAC with the apparent high degree of R0 resection rates and the median times of progression free survival seen in the study subjects. Our population included five subjects that tested homologous-recombination deficient (Table 1) with three receiving niraparib during maintenance and a fourth was randomized to either niraparib or placebo in a subsequent clinical study. Historically, large, randomized studies of patients with EOC treated similarly with NAC have reported an R0 rate of about 50% and a median time to progression of 12 months. [34] [35]

The results from our translational studies show activation of a multitude of immune responses following GEN-1 + NAC treatment. First, there was a dose-dependent increase in powerful immunostimulatory cytokines IL-12 and its downstream cytokine IFN-γ in ascitic fluid. The anti-cancer effects of these cytokines have been widely recognized in human malignancies. [36] The dose dependence of the cytokine response to GEN-1 at a fixed NAC dose suggests that it is GEN-1 related. Additionally, given the accumulation of the IL-12 plasmid in mesenteric lymph nodes in an animal model, it is likely that GEN-1 had a similar effect on the stimulatory cytokines in those key secondary lymphoid organs where ovarian metastases commonly occur). [33] Second, the proportion of myeloid dendritic cells in the peritoneal fluid trended higher, by 3.1-fold, accompanied by a similar 3.0-fold rise in CD8⁺ T_EM cells. Such concomitant changes in these cell types is noteworthy, given the important role of antigen-presenting cells in stimulating a cytotoxic T cell response and fostering immunological memory. Third, GEN-1 appeared to reduce four immunosuppressive signals (Foxp3, IDO1, PD-1 and PD-L1) within the tumor microenvironment, a trend not seen with NAC therapy. [9] [10] [11] [12] [13]

Finally, the GEN-1 gene therapy was associated with an apparent increase in the cytotoxic state of T cells within the tumor microenvironment as indicated by the increases in the ratios of CD8⁺/CD4⁺ and CD8⁺/Treg cells. Indeed, higher CD8⁺/CD4⁺ T cell and CD8+/Treg ratios have been considered prognostic for prolonged survival. [12] [13] [37] The OVATION I study results are consistent with the known activities of IL-12 and its related downstream cytokines IFN-γ including a reduction in the production of IL-2 which is required for proliferation of immunosuppressive Treg cells. As such, these cytokines render ovarian cancer cells more sensitive to platinum chemotherapy by inhibiting cancer-associated fibroblasts’ production of glutathione and cysteine, and conversion of tumor-associated macrophages from the immunosuppressive M2 to the anti-tumor M1 phenotype. [38] [39] [40].

The immune changes resulting from GEN-1 + NAC treatment are distinct from those reported with NAC therapy in epithelial ovarian cancer. First, the intraepithelial CD4+ and CD8+ T cell densities following neoadjuvant chemotherapy increased or remained unchanged, and the CD8+/CD4+ ratio remain unchanged. [13] [26] [41] [42] In comparison, our study shows an increase in CD8+ cell density, decrease in CD4+ cell density and increase in CD8+/CD4+ ratio in majority of patients, although the magnitude of the increase highly varied from patient to patient. These data suggest that the addition of GEN-1 to NAC produces a different pattern of immune response that is not typically associated with the neoadjuvant chemotherapy alone. Second, the neoadjuvant chemotherapy increased or produced no change in immunosuppressive markers. [13] [26] [41] [42] In one of the studies, NAC produced a doubling of CD8+ cell density but failed to relieve the immunosuppression markers including FoxP3+, IDO1, PD-1, PD-L1 in tumor tissue. [26] In another study, NAC-treated tumors had higher PD-L1 expression on tumor infiltrating immune cells and persistent high levels of PD-1- and CTLA-4- expressing cells. [41] In a separate study, NAC therapy increased tumor infiltrating lymphocytes but did not affect FoxP3 cells. [13] In another study, NAC increased PD-L1 positive cells from 30% to 53%. [42] These studies consistently demonstrate an increase or no change in the immunosuppressive signals following neoadjuvant chemotherapy in advanced epithelial ovarian cancer. In comparison, the present study demonstrates a reduction in FoxP3, IDO1, PD-1, and PD-L1 signals and an increase in the ratio of CD8+ cells to FoxP3, IDO1, PD-1 and PD-L1 cells in majority of patients suggesting GEN-1 may have a role in the observed immune changes in NAC combination setting. A decrease in immunosuppressive markers in this study is consistent with the inhibitory action of IL-12 on PD-1 expression in malignant melanoma and peripheral lymphocytes and on FoxP3 and other regulatory T cells in lymphocyte cultures. [43] [44] [45]

A parallel increase in IFN-γ and decrease in PD-1/PDL-1 after GEN-1/NAC therapy is interesting since an increase in IFN-γ has been associated with upregulation of PD-1/PD-L1 expression in the action of some immunotherapy agents. [46] The mechanism of a parallel increase in IFN-γ and inhibition of immunosuppressive markers following GEN-1 treatment in the present study warrants further investigation. However, there is some evidence in the literature to suggest that these parallel effects could be explained by dual action of IL-12 on immune cells. IL-12 increases IL-2 receptor expression on CD4+ and CD8+ cells to produce IFN-γ but diminishes its expression on immunosuppressive T cells resulting in the starvation of immunosuppressive T cells thereby favoring the outgrowth of non-Treg cells. [45] In another study, IL-12-stimulated IFN-γ production from CD8+ cells and countered IFN-γ-mediated PD-L1 expression by downregulating IFN-γ receptors. [47] The stimulation of IFN-γ and inhibition of immunosuppressive markers in our study may be explained by dual actions of IL-12 involving IFN-γ-independent mechanisms. [48]

The multifactorial nature of GEN-1 immune response built on a durable local production of IL-12 may be activating the innate and adaptive immune system creating a unique tumor microenvironment potentially favorable to anti-tumor responses and also conducive to other therapeutic drugs that may be suboptimal as single agents due to highly immunosuppressive tumor microenvironment in ovarian cancer. For example, checkpoint inhibitors despite having demonstrated activity in some cancer types are only of limited to modestly active in ovarian cancer. [49] [50] Combination with GEN-1 could potentiate CD8+ T cell infiltration and reduce immunosuppressive tumor microenvironment to improve the efficacy of checkpoint inhibitors and produce an overall better quality of clinical response against cancer. Similarly, the efficacy of adaptive T-cell therapies may also be improved by remodeling the peritoneal cavity with GEN-1 pre-treatment reducing the tumor immunosuppressive environment and improving the T-cell survival and clinical efficacy. Similarly, other novel combinations with GEN-1 may also be investigated to improve clinical outcome in ovarian cancer.

This study was limited by its small sample size and termination of dose escalation at 79 mg/m² even though a MTD was not achieved. A maximum biological dose was not established as well. Another limitation is that a control arm was not employed to evaluate the full impact of GEN-1 on the immune response as well as therapeutic response when GEN-1 is combined with chemotherapy. Future studies will evaluate GEN-1 at higher doses in this patient population with a control group to address these limitations.

In conclusion, weekly intraperitoneal GEN-1 treatment in conjunction with standard NAC in advanced epithelial ovarian cancer patients is safe, well-tolerated and appears to be active. Repeated durable increases in lL-12 and IFN-γ levels at tumor site for an eight-week treatment period provides for an unprecedented pharmacology remodeling of the tumor microenvironment as evidenced by reduction in immunosuppressive signals Foxp3, IDO1, PD-1 and PD-L1 and potentiation of immunostimulatory signals including the increases in the ratios of CD8⁺/CD4⁺ and CD8⁺/Treg cells and increases in myeloid dendritic cells and CD8⁺ T_EM cells. These immunomodulatory effects of GEN-1 may result in an increased sensitivity of tumor microenvironment to other anti-cancer agents including cytotoxic drugs and immunotherapies such as checkpoint inhibitors and adaptive T cell therapies. OVATION II (NCT03393884) is a phase I/II study of concurrent GEN-1 at a dose of 100 mg/m² weekly for up to 17 doses administered during chemotherapy and is currently actively accruing.