Work leading to the demonstration of ENTPD1/CD39 as a relevant biomarker of response of ovarian cancer patients undergoing treatment with Vigil in the VITAL study  goes back 10 years. Vigil is a novel platform technology designed to activate 3 anticancer immune mechanisms at once (immune functional enhancement, silencing of cancer suppression, clonal neoantigen exposure) . We have known for some time that GMCSF protein functionally enhanced anti-infectious and cancer (to a lesser degree) immune response, but when intracellularly expressed via plasmid transfer to autologous cancer cells, it facilitated MHC expression of neoantigens. However, clinical trials exploring GMCSF plasmid immunotherapy were limited in demonstrating clinical response. One explanation for the limited response was thought to be related to cancer produced immune suppression cytokines (i.e., TGFβ1, TGFβ2). Another independent cellular (autologous tumor) technology generating knockdown of TGFβ2 via antisense technology separately demonstrated enticing clinical response data and conducted a registration trial in non-small cell lung cancer. Unfortunately, the primary endpoint was not achieved in part possibly related to limited anticancer functional immune enhancement. Our rationale for Vigil was conceived in part from the concept that if anticancer functional enhancement with GMCSF or silencing of tumor suppressive activity with inhibition of TGFβ individually was insufficient for clinical commercial justification separately, then the combination of these immune functional activities along with provision of relevant cancer neoantigen stimulus (via irradiated autologous tumor tissue) may achieve sufficient anticancer activity. We considered that knockdown of furin, the proprotein convertase required for export/secretion of TGFβ1 and TGFβ2 may provide intracellular control of both TGFβ1 and TGFβ2 export by tumor cells . In vitro testing was done using a newly discovered proprietary bifunctional short hairpin RNA interference plasmid technology targeting the furin expression which demonstrated a robust inhibition of TGFβ1 and TGFβ2 secretion . We constructed a dual plasmid that expressed both wildtype GMCSF and bi-shRNAi-furin for engineering transfected cells to produce GMCSF and to inhibit excretion of TGFβ1 and TGFβ2, respectively. We inserted (via electroporation) this dual plasmid into each patient’s surgically harvested autologous malignant cells to preserve relevant (personal) neoantigen targets and to create a triad of anticancer immune activity. We initiated a Phase 1 trial and successfully demonstrated an optimal dose, remarkable safety and predicted immune response (ELISpot assay) in correlation with clinical benefit [overall survival (OS)] [2, 5]. We then initiated Phase 2a testing of Vigil as a maintenance treatment in patients with newly diagnosed Stage IIIb-IV resectable ovarian cancer. Relevant cytokine expression (GMCSF↑, TGFβ1↓, TGFβ2↓) and ELISpot confirmed the mechanism of action in all Vigil treated patients compared to no change in controls. Moreover, relapse free survival (RFS) was shown to be significantly better in Vigil treated patients compared to control patients not receiving Vigil [6, 7]. After discussion of these results with FDA, CBER, we moved forward with Phase 2b testing in the same ovarian cancer population as entered into the Phase 2a study. Results in the randomized placebo controlled Phase 2b VITAL study involving 91 patients (47 Vigil vs. 44 placebo) confirmed safety and suggested benefit in both OS and RFS in the BRCA-wt  and HRP molecular profile positive subpopulations [8, 9]. Moreover, the effect appeared durable with continued significant separation in Kaplan Meier curves for both RFS and OS at 3 year follow up . As part of our program, we have studied molecular biomarkers that may relate to sensitivity or resistance of Vigil. We found no relationship of Vigil response to tumor mutational burden or PD-L1 expression but with use of NanoString analysis and the NanoString PanCancer Immuno-Oncology 360™ CodeSet using the nCounter SPRINT platform (NanoString Technologies, Seattle, WA) and assistance from Dr. Rodney Rocconi at University of Alabama, Birmingham we studied correlation to RFS and OS benefit to determine an optimal biomarker of sensitivity. Genes that were significant for both RFS and OS advantage at the 1% significance level were identified. The Cox proportional hazards model with interaction term for each gene identified in the univariate Cox model was used to identify genes that were predictive of response to Vigil by analyzing data of both Vigil and placebo patients. The Cox model included the treatment groups, gene and treatment-by-gene interaction term. The model was run using the gene as a continuous variable or using binary high or low gene assignment. The median gene value for all 91 patients was calculated for each of the 750 cancer expressive pathway genes. Further model selection was also performed using a multivariate Cox model. We also used the my.stepwise.coxph function in R (open source, R Core Team), which employed both forward selection and backward elimination methodology. One gene of significance of the 750 tested that was identified using our NanoString Statistical Algorithm (NSA) was ENTPD1/CD39. ENTPD1/CD39 is a plasma membrane protein that hydrolyzes extracellular ATP and ADP to AMP and functions as a master regulator to maintain the balance between proinflammatory and immune suppressive regulatory function within the cancer microenvironment. It controls the rate limiting step in the conversion of ATP to ADP . Vigil treated patients with high (> median) ENTPD1/CD39 (Figure 1) demonstrated markedly improved RFS over high ENTPD1/CD39 patients receiving placebo regardless of HRP status (p=0.00007), OS results demonstrated similar benefit (p=0.013). Further analysis suggested continued or enhanced advantage in HRP positive patients. In light of these results, it is likely that collective use of HRP profiling, other molecular biomarkers (i.e., p53 mutation ) and ENTPD1/CD39 signaling level will provide support to assessment of Vigil response in patients with ovarian cancer.
- Rocconi, R. P. et al. Gemogenovatucel-T (Vigil) immunotherapy as maintenance in frontline stage III/IV ovarian cancer (VITAL): a randomised, double-blind, placebo-controlled, phase 2b trial. Lancet Oncol 21, 1661-1672, doi:10.1016/S1470-2045(20)30533-7 (2020).
- Senzer, N. et al. Phase I trial of "bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell" vaccine (FANG) in advanced cancer. Mol Ther 20, 679-686, doi:10.1038/mt.2011.269 (2012).
- Maples, P. et al. FANG Vaccine: Autologous Tumor Cell Vaccine Genetically Modified to Express GM-CSF and Block Production of Furin. BioProcessing Journal 8, 4-14, doi:10.12665/J84.Maples (2010).
- Rao, D. D. et al. Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference. Cancer Gene Ther 17, 780-791, doi:10.1038/cgt.2010.35 (2010).
- Senzer, N. et al. Long Term Follow Up: Phase I Trial of “bi-shRNA furin/GMCSF DNA/Autologous Tumor Cell” Immunotherapy (FANG™) in Advanced Cancer. Journal of Vaccines & Vaccination 4, 209 (2013).
- Oh, J. et al. Phase II study of Vigil(R) DNA engineered immunotherapy as maintenance in advanced stage ovarian cancer. Gynecol Oncol 143, 504-510, doi:10.1016/j.ygyno.2016.09.018 (2016).
- Oh, J. et al. Long-term follow-up of Phase 2A trial results involving advanced ovarian cancer patients treated with Vigil(R) in frontline maintenance. Gynecol Oncol Rep 34, 100648, doi:10.1016/j.gore.2020.100648 (2020).
- Rocconi, R. P. et al. Gemogenovatucel-T (Vigil) immunotherapy demonstrates clinical benefit in homologous recombination proficient (HRP) ovarian cancer. Gynecol Oncol 161, 676-680, doi:10.1016/j.ygyno.2021.03.009 (2021).
- Walter, A. et al. Gemogenovatucel-T (Vigil) maintenance immunotherapy: 3-year survival benefit in homologous recombination proficient (HRP) ovarian cancer. Gynecol Oncol 163, 459-464, doi:10.1016/j.ygyno.2021.10.004 (2021).
- Takenaka, M. C., Robson, S. & Quintana, F. J. Regulation of the T Cell Response by CD39. Trends Immunol 37, 427-439, doi:10.1016/j.it.2016.04.009 (2016).
- Sliheet, E. et al. Network based analysis identifies TP53m-BRCA1/2wt-homologous recombination proficient (HRP) population with enhanced susceptibility to Vigil immunotherapy. Cancer Gene Ther 29, 993-1000, doi:10.1038/s41417-021-00400-x (2022).