Coleman12●John Nemunaitis7 Received: 14 October 2020 / Revised: 4 February 2021 / Accepted: 17 February 2021 © The Author(s), under exclusive licence to Springer Nature America, Inc. 2021 Abstract Vigil ®is a personalized vaccine that enhances tumor neoantigen expression. We investigated for the ﬁrst time safety and efﬁcacy of Vigil in combination with atezolizumab in relapsed ovarian cancer (OC) patients. This is a randomized, Phase 1 study of Vigil, an autologous tumor tissue transfected vaccine encoding for GMCSF and bi-shRNA-furin thereby creating enhanced immune activation and TGF βexpression control. Part 1 is a safety assessment of Vigil (1 × 10e7 cells/mL/21 days) plus atezolizumab (1200 mg/21 days). Part 2 is a randomized study of Vigil ﬁrst (Vigil-1st) or atezolizumab ﬁrst (Atezo-1st) for two cycles followed by the combination of both agents. The primary endpoint of the study was the determination of safety. Twenty-four patients were enrolled in the study; three patients to Part 1 and 21 to Part 2. Patients in Part 1 completed combination therapy without dose-limiting toxicity justifying expansion to Part 2. Twenty-one patients were randomized(1:1) to Part 2 to Vigil-1st ( n=11) or Atezo-1st ( n=10). Grade 3/4 treatment-related adverse events of Atezo-1st vs. Vigil- 1st were 17.2% vs. 5.1%. Median overall survival (OS) was not reached (NR) (Vigil-1st) vs. 10.8 months (Atezo-1st) (hazard ratio [HR] 0.33). The exploratory subset analysis of BRCA wtsuggested improved OS bene ﬁt [NR in Vigil-1st vs. 5.2 months in Atezo-1st, HR 0.16, p 0.027]. The Vigil-1st combination therapy with atezolizumab was safe and results in support continued investigation in BRCAwtpatients. Introduction Despite impressive evidence of immune-modulatory advantage to checkpoint inhibitor (CI) therapy in many cancer types, little advantage has been shown in OC [ 1]. Theories point to this inef ﬁciency being likely due to lack of speci ﬁcity of CI combined with signi ﬁcant hetero- genicity and genetic instab ility in OC. Recent reports detail how OC cells can acquire potential escapemechanisms to evade host immunity via several immu- nosuppressive factors, including a loss of MHC expres- sion and the upregulation of immunosuppressive factorsincluding TGF-beta (TGF β), indoleamine 2,3-dioxygen- ase (IDO), and cyclooxygenase (COX-1 and COX-2) [2,3]. Preclinical evidence supports vaccination of DNA neoantigen products prior to checkpoint inhibitor therapy [4] .U s i n ga nM C 3 8m u r i n em o d e l ,D N An e o a n t i g e n vaccination demonstrated an increase in CD4 +and CD8+ T cell generated antitumor response. Vaccination*John Nemunaitis jnemunaitis@gradalisinc.com 1University of South Alabama - Mitchell Cancer Institute,Mobile, AL, USA 2Billings Clinic, Billings, MT, USA 3Augusta University, Augusta, GA, USA 4Greenville Health System, Greenville, SC, USA 5Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA 6Josephine Ford Cancer Institute, Detroit, MI, USA 7Gradalis, Inc., Dallas, TX, USA 8ProMedica, Toledo, OH, USA 9Mayo Clinic, Rochester, MN, USA 10University of Cincinnati Cancer Center, Cincinnati, OH, USA 11Arizona Oncology, Phoenix, AZ, USA 12MD Anderson Cancer Center, Houston, TX, USA 1234567890();,: 1234567890();,: combined with anti-PD-1 therapy resulted in delayed tumor growth and a survival rate of 25%. When CD8 T cells were depleted after vaccination this effect was abolished, supporting th e role of neoantigen-speci ﬁc T cells in decreasing tumor growth. Additionally, in a preclinical prostate cancer murine model, anti-CTLA-4administered post-GVAX vaccination resulted in increased CD8 +and CD4 T cells compared to treatment with anti-CTLA-4 ab ﬁrst [5]. They hypothesize that treatment with anti-CTLA-4 antibody prior to vaccination was not effective. Verma et al. also found that the sequence of tumor vaccine and PD-1 blockade wasimportant in PD-1 resistant murine models of melanoma and lung cancer. He compared combination therapy with a variance of administration of anti-PD-1 ﬁrst and last to the vaccine and concluded that vaccination before or con- comitantly with PD-1 blockade is critical for optimal effect, and that PD-1 blockade before neoantigen educa-t i o nm a ye v e nr e s u l ti nr e s i s t a n c e[ 6]. Vigil is an autologous tumor cell vaccine in which tumor cells are harvested from patients at the time ofdebulking surgery and transfected via electroporation extracorporeally with a plasmid encoding for the GMCSF gene, an immune-stimulatory cytokine, and a bifunctional,short hairpin RNA (bi-shRNA) which speci ﬁcally knocks down the expression of furin, the critical convertase responsible for activation of two TGF βisoforms TGF β-1 and TGF β-2 (cancer immune effector suppressors). By downregulating TGF βexpression, cancer cells have a reduced ability to evade host immune responses. Vigil is apersonalized “neoantigen educating ”immunotherapy that has been administered safely at doses up to 2.5 × 10e7 cells/injection and has shown evidence of bene ﬁti nc l i n - ical testing of a variety of cancers including OC [ 7–14]. Most recently, a randomized Phase IIb trial of Vigil against placebo use at maintenance in frontline ovariancancer demonstrated statistically signi ﬁcant improvement in relapse-free survival and overall survival advantage in BRCA wtmolecular pro ﬁle patients using the 1 × 107doses per injection. No Grade 3/4 product-related toxic effect was observed [ 15]. Improved expression of clonal tumor neoantigen and reduced tumor suppressor effect of TGF β is hypothesized to synergistically enhance the activity of checkpoint inhibitor treatment. Moreover, preclinical evi- dence supports that prior adm inistration of neoantigen educating therapy prior to cycle 1 (C1) will enhance the immunotherapeutic anticancer activity of CI. We thus explored the ﬁrst clinical combination of Vigil and ate- zolizumab together and in sequence using single-agent prior clinically validated safe dose levels [ 7,9,16–18], comparing Vigil ﬁrst (Vigil-1st) vs. atezolizumab ﬁrst (Atezo-1st) in order to assess the safety and preliminary evidence of bene ﬁt.Methods Trial design and treatments This Phase 1, 2-part, open-label trial was conducted at 6 centers across the United States (NCT03073525). Part 1explored the safety of the combination of Vigil and atezoli- zumab. Part 2 was a randomized study of either Vigil-1st vs. Atezo-1st, followed by the combination of Vigil plus atezo-lizumab. Patients entering a Vigil vaccine construction pro- tocol involving ovarian cancer were eligible for this trial. Tissue for vaccine construction was collected during frontlinedebulking surgery. Tissue and peripheral blood mononuclear cell samples were collected and analyzed for BRCA1/2 molecular pro ﬁling using a call quality of 40 and a minimum allele depth of 5 (Ocean Ridge Biosciences, Deer ﬁeld Beach, FL). Patients received Vigil (1 × 10e6 –10e7 cells/dose intradermally) and atezolizumab (1200 mg/dose intravenousinfusion) once every 3 weeks for up to 12 doses. Informed consent was obtained prior to procurement and prior to main study registration/randomization. Written documentation offull IRB approval of the protocol and consent documents were required before a patient could be registered at any site. Patients Women who had relapsed OC, stable medical conditions, and failed at least one prior line of systemic therapy in the recurrent setting or platinum-resistant disease were eligible for the trial. Subjects were required to have at least 4 vials ofVigil manufactured, ECOG-performance status (PS) ≤1, and normal organ and marrow function were required and de ﬁned per-protocol (Absolute neutrophil count ≥1500/mm 3; Platelets ≥100,000/mm3; Hemoglobin ≥5.59 mmol/L; serum bilirubin ≤1.5× institutional upper limit of normal; AST/ALT ≤2.5× institutional upper limit of normal; creatinine >50 mL/min;TSH within institutional limits). Patients were excluded for prior immunotherapy and active autoimmune disease. Tumor procurement and manufacturing Gradalis, Inc. (Carrollton, TX) manufactured Vigil from the harvested tumor tissue. Manufacturing was a 2-day process. The equivalent of a “golf ball size ”mass (10 –30 g tissue, cumulative) was required for vaccine manufacturing (3 cmby radiological scan). Lesions extending into the bowel lumen were excluded due to the risk of bacterial contamination. Investigational product and placebo manufacturing Surgically excised tumor tissue was procured during the debulking procedure and cut into ¼ - ½ inch sections beforeR. P.