Rocconi et al. being placed in up to four specimen containers containing sterile 0.9% sodium chloride (Baxter) supplemented with gentamicin (Fresenius Kabi) and packaged on wet ice for overnight transport to the manufacturing facility. On day 1, the transport medium and tumor specimen in each container were tested for sterility (BacT/Alert 3D Microbial Identi ﬁ- cation System, BioMerieux). The tumor tissue was trimmed to remove fat, connective/necrotic tissue, sutures/staples, and mechanically dissociated by scalpel followed byenzymatic dissociation (Type I collagenase solution) and incubated at 37 °C for up to 45 min to form a single cell suspension. The cell suspension was ﬁltered across a sterile 100μm strainer (Corning) to separate the cells from the debris, and the liberated cells were washed with PlasmaLyte (Baxter) supplemented with 1% Human Serum Albumin(Octapharma) and manually counted via hemocytometer (InCyto). Quality control (QC) samples were removed for retaining, immune monitoring, and a pre-transfection cul-ture initiated to obtain baseline cytokine levels by ELISA for GMCSF (R&D Systems) and TGF β1 (R&D Systems). The cell suspension was adjusted to a concentration of 40million cells/mL and electroporated using a Gene Pulser XL (BioRad) to insert the plasmid DNA into cells. The trans- fected cells were plated into sterile T-225cm 2ﬂasks (Corning) at 1 × 10e6 cells/mL in X-VIVO 10 media (Lonza) supplemented with gentamicin (Fresenius Kabi) and incubated overnight (14 –22 h) at 37 °C with 5% CO 2 overlay (Sanyo) to allow for incorporation of bi-shRNA furin and GMCSF mRNA into the tumor cells. On day 2, the overnight culture was harvested by scraping cells from each ﬂask, collecting media containing free-ﬂoating cells, and resuspending in fresh X-VIVO media. The cells were manually counted to determine that aminimum of 80 million cells were available for QC samples and a minimum of 4 doses before proceeding. Two 1-mL mycoplasma samples containing cells were taken and frozenat−80 °C. The cells were irradiated 4 × 25 Gy cycles using gamma-ray irradiator to arrest replication/growth. The cells were washed with PlasmaLyte (Baxter) supplemented with1% human serum albumin (Octapharma) and QC samples were removed for retaining immune monitoring, and a post- transfection culture initiated to obtain transfected cytokinelevels for GMCSF and TGF β1 by ELISA (or ELLA machine for one patient). The cells were placed into freeze media consisting of 10% DMSO (dimethyl sulfoxide;Cryoserv USP; Mylan), 1% Human Serum Albumin (Octapharma) in Plasma-Lyte A at pH 7.4 (Baxter), and aseptically aliquoted at 1 × 10e6 cells/mL or 1 × 10e7 cells/mL into sterile 2.0 mL borosilicate glass vials (Algroup Wheaton Pharmaceutical and Cosmetics Packaging); closed with a butyl rubber stopper coated with Flurotec ®barrier ﬁle (West Pharmaceutical Services) with a ﬁnalﬁll volume of 1.2 mL. The ﬁnal product vials were frozen at a controlledrate using CoolCell ®freezing containers (Biocision) placed into−80 °C freezers (Sanyo). After freezing, the cells were stored in the vapor phase of liquid nitrogen tanks pending release testing. Frozen product vials were used for sterility (USP <71>) and endotoxin testing by gel-clot (Limulus Amoebocyte Lysate, Lonza). Atezolizumab was provided by Roche/Genentech and distributed by Gradalis, Inc. Atezolizumab was formulated as 60 mg/mL in 20 mM histidine acetate, 120 mM sucrose,0.04% polysorbate 20, pH 5.8 (Phase III formulation). Atezolizumab was preservative-free and provided for single-use only. Atezolizumab in formulation F03 (1200 mgper vial) was administered in 250 mL 0.9% NaCl IV infu- sion bags and prepared and diluted under aseptic conditions. Disease evaluation Subjects remained on treatment until disease progression or death or product toxic effect. Disease progression was determined radiographically by local investigators using the Response Evaluation Criteria in Solid Tumors Version 1.1(RECIST 1.1). In Part 1, disease progression was accessed at baseline and every third cycle thereafter. In Part 2, the disease was assessed at baseline, at the end of cycle 2 ofsingle-agent therapy, and every third cycle thereafter. End points and statistical assessment The data cut-off date for the primary analysis was arbitraryat December 20, 2019. The primary endpoint was safety.Efﬁcacy assessments and endpoints included PFS and overall survival (OS) between the Vigil-1st group and the Atezo-1st group. The time to progression was calculatedfrom the date of randomization until the ﬁrst date of documented progression or death. Exploratory analysis of PFS and OS were sequentially analyzed in the BRCA wt subgroup and compared between Vigil-1st and Atezo-1st. Median follow-up time was calculated from the data cut-off date subtracted from the time of treatment start. A one-sidedp-value of 0.05 or less (log-rank) was considered to indicate statistical signi ﬁcance in analyses. The HRs of PFS and OS were estimated via a Mantel –Haenszel hazards model. The distribution of PFS and OS was estimated using the Kaplan –Meier method. Toxicity was assessed using NCI CTCAE v 4.03. Results Patients From June 2, 2017, through February 13, 2019, three patients were entered into Part 1 of the study and 21 wereProof of principle study of sequential combination atezolizumab and Vigil in relapsed ovarian cancer randomized to Part 2 of the study ( n=11 Vigil-1st; n=10 Atezo-1st) as shown in Fig. 1. All patients failed at least one prior systemic therapy (Part 1/Vigil-1st/Atezo-1st: 0/5/2 received one regimen, 1/4/3 received two regimens, 1/2/4received three regimens and 1/0/1 received four regimens). Individual (Part 2 only) and all patients summarized demographics are shown in Tables 1and2. Germline and somatic BRCA (g/sBRCA) status was determined for all 24 patients. The median follow-up time for Part 1 was 29.3 months, and for Part 2 was 21.3 months. Safety Most commonly reported (>1 event and ≥1 Grade) treatment-related AE ’s are shown in Table 3. No Grade 3 or 4 treatment-related toxicities were observed in Part 1.Therefore, accrual was opened to Part 2. There were 30 treatment-related adverse events (Table 3) in the Atezo-1st group and 83 events in the Vigil-1st group.Twenty- ﬁve of the 30 (83.3%) Atezo-1st events were Grades 1, 2 with a majority attributed to atezolizumab treatment (24/25). Eighty-one out of 83 (97.6%) events inthe Vigil-1st treatment group were Grades 1, 2 events with 61.7% (50/81) attributed to atezolizumab treatment and 38.3% (31/81) related to Vigil treatment. A comparison of all and Grades 3, 4 atezolizumab- related events between Vigil-1st and Atezo-1st groups iss h o w ni nF i g . 2. Grades 3/4 treatment-related events were higher in the Atezo-1st arm (17.2%) vs, Vigil 1st (5.1%). Grade 3 atezolizu mab-related events in the Atezo-1st group included blood and lymphatic disorders(6.9%), general disorders (3.5%), injury (3.5%), respiratory, thoracic, and mediastinal disorders (3.5%). No Vigil-related Grade 3 events were observed in theAtezo-1st group. In the Vigil-1st group, two Grade 3 events were urinary disorders (1 related to atezolizumab and 1 related to Vigil). Moreover, atezolizumab-relatedadverse events (any Grade) were higher in the Atezo-1st group (96.7%) compared to the Vigil-1st group (61.3%) (Fig. 2). Product release results Vigil vaccine from twenty subjects (83.3%) met all pro- duct release criteria. The median tumor mass harvested was 51.65 g (range 11.52 –114.23 g). Four subjects (16.7%) did not demonstrate suf ﬁcient increase in GMCSF expression but did meet TGF β1 knockdown release criteria following plasmid transfer and were pro-vided exception onto the study ( n=1P a r t1 , n=3P a r t2 ) . Key product release results are shown in Table 4.S i g - niﬁcant TGF β1 knockdown was detectable in 23/24 (95.8%) patients. Fifteen (62.5%) patients had ≥90% TGFβ1 knockdown demonstrating robust activity related Fig. 1 Consort diagram. One patient was not evaluated for BRCA status (*).R. P. Rocconi et al. Table 1 Individual patient demographics (Part 2). Subject ID Treatment arm BRCA status wt/g/g/sPD-L1 status (%)Cancer type Histology FIGO stage Lines of prior systemictherapiesCA-125 at baselineDisease status at baselinePlatinum status Notes 112_0119 aVigil-1st BRCAwt<1 Primary peritonealHGSC IVB 3 ≥35 Target & non-targetResistant 112_0007 Vigil-1st BRCAwt<1 Fallopian tube HGSC IIIC 2 <35 No disease Sensitive 1L: Carbo/taxol 2L: VITAL study (placebo, NED) PD 2 yrs later (CT: Labdominal wall, vaginal mass| tumor debulking to NED| path con ﬁrm HGSC) 206_0021 Vigil-1st sBRCA mut1 Fallopian tube HGSC IIIC 1 <35 Target & non-targetResistant 206_0034 Vigil-1st sBRCAmut2 Fallopian tube HGSC IIIC 2 ≥35 Target & non-targetSensitive 173_0044 Vigil-1st BRCAwt3 Fallopian tube HGSC IIIC 1 ≥35 No disease Sensitive 1L: Carbo/taxol Relapse after 1 year (CT:perihepatic nodule, pelvic nodule resected, CA-125 at 60 U/ml) 136_0024 Vigil-1st BRCA wt5 Fallopian tube HGSC IIIC 2 ≥35 Target & non-targetSensitive 102_0011 Vigil-1st BRCAwt10 Ovarian HGSC IIIC 1 ≥35 Target & non-targetSensitive 103_0031 Vigil-1st BRCAwt10 Ovarian HGSC IV 3 <35 Target & non-targetResistant 136_0026 Vigil-1st gBRCAmut0 Ovarian HGSC IIIC 1 ≥35 Target & non-targetSensitive 206_0011 Vigil-1st gBRCAmut0 Fallopian tube and ovaryHGSC IIIC 2 ≥35 Target only Sensitive 153_0012 Vigil-1st gBRCAmutN/A Fallopian tube HGSC IVA 1 <35 Target & non-targetResistant 112_0013 Atezo-1st BRCAwt0 Primary peritonealHGSC IIIC 4 <35 Target & non-targetSensitive 102_0008 Atezo-1st sBRCAmut0 Ovarian HGSC IIIC 1 ≥35 Non- target onlySensitive 136_0029 Atezo-1st BRCAwt<1 Primary peritonealHGSC IIIC 3 ≥35 Target & non-targetSensitive 136_0025 Atezo-1st BRCAwt<1 Ovarian HGSC IIIC 3 ≥35 Target & non-targetResistantProof of principle study of sequential combination atezolizumab and Vigil in relapsed ovarian cancer Table 1 (continued) Subject ID Treatment arm BRCA status wt/g/g/sPD-L1 status (%)Cancer type Histology FIGO stage Lines of prior systemictherapiesCA-125 at baselineDisease status at baselinePlatinum status Notes 206_0028 Atezo-1st BRCA wt5 Ovarian HGSC IIIC 1 ≥35 Target & non-targetResistant 206_0031 Atezo-1st BRCAwt10 Fallopian tube HGSC IIIC 3 ≥35 Target & non-targetSensitive 206_0016 Atezo-1st BRCAwt20 Ovarian HGSC IIIC 2 ≥35 Target & non-targetResistant 153_0006 Atezo-1st BRCAwt60 Ovarian HGSC IV 2 ≥35 No disease Resistant 1L: Carbo/Taxol 2L: Taxol/AvastinPD within 6 mo of 2L b (CA-125 at 594 U/ml) 112_0012 Atezo-1st gBRCAmut0 Primary peritonealHGSC IIIC 3 <35 Target & non-targetSensitive 173_0006 Atezo-1st BRCAmutN/A Ovarian HGSC IIIC 2 <35 No disease Sensitive 1L: Platinum/taxol 2L: VITAL study (placebo) PD 2 years later (new hepatic target lesion with surgicalresection to NED) HGSC high-grade serous carcinoma, NED no evidence of disease, PDprogressive disease. aVigil constructed from recurrent disease lesion.