bCT scan showing positive.R. P. Rocconi et al. to furin bi-shRNAi knockdown. Baseline TGF β1p r o - duction prior to plasmid trans fection of all patients with detectable levels revealed a median TGF β1 expression of 158 pg/10e6 cells (mean 206, range 93 –445). BRCAwt TGFβ1e x p r e s s i o n( n=13 Part 2) revealed a median of 139 pg/10e6 cells (mean 188, range 93 –361) for Atezo-1st and Vigil-1st groups, and BRCAmutTGFβ1 expression (n=8 Part 2) revealed a median of 223 pg/10e6 cells (mean 215, range 111 –307).Efﬁcacy The median OS in the Vigil-1st cohort was not reached and was 10.8 months in the Atezo-1st cohort (HR 0.33; 95%conﬁdence interval (CI) [0.064 –1.7]; p0.097) (Fig. 3A). The median duration of follow-up was 21.3 months for Part 2. Exploratory subset analysis by BRCA status demon-strated improvement of OS in BRCA wtpatients in Vigil-1st compared to Atezo-1st (HR 0.16, 95% CI [0.026 –1.03];Table 2 Summarized patient demographics (Part 1, Part 2). Study part Part 1 Part 2 Part 2 Characteristic ITT ITT BRCAwt Treatment arm Vigil +Atezo Atezo-1st Vigil-1st Atezo-1st Vigil-1st Patients 3 10 11 7 6 Age-years Median 61 59.5 67 63 65 Mean 65 59.7 63.9 62.3 63.5 Range 59 –75 44 –77 50 –75 50 –77 50 –75 Race—no. (%) White 3 (100) 8 (80.0) 9 (81.8) 5 (71.4) 6 (100) Asian 0 (0) 1 (10.0) 0 (0) 1 (14.3) 0 (0) Black or African American 0 (0) 1 (10.0) 2 (18.2) 1 (14.3) 0 (0) Staging —no. (%)a III 1 (33.3) 9 (90.0) 8 (72.7) 6 (85.7) 4 (66.7) IV 2 (66.7) 1 (10.0) 3 (27.3) 1 (14.3) 2 (33.3) ECOG-performance-status-score —no. (%)b 0 2 (66.7) 2 (20.0) 7 (63.6) 0 (0) 5 (83.3) 1 1 (33.3) 8 (80.0) 4 (36.4) 7 (100) 1 (16.7) No. of prior lines —no. (%) Median 3 2.5 2 3 2 Mean 3 2.4 1.73 2.6 2 Range 2 –41 –41 –31 –41 –3 Receipt of frontline chemo —no. (%) Neoadjuvant 2 (66.7) 4 (40.0) 3 (27.3) 4 (57.1) 2 (33.3) Adjuvant 1 (33.3) 6 (60.0) 8 (72.7) 3 (42.9) 4 (66.7) Platinum status —no. (%) Resistant 2 (66.7) 4 (40.0) 4 (36.4) 4 (57.1) 2 (33.3) Sensitive 1 (33.3) 6 (60.0) 7 (63.6) 3 (42.9) 4 (66.7) BRCA status —no. (%)c BRCAwt3 (100) 7 (70.0) 6 (54.5) 7 (100) 6 (100) g/sBRCAmut0 (0) 3 (30.0) 5 (45.5) 0 (0) 0 (0) Vaccine dose (cells/dose) 1 × 10e6 0 0 1 0 1 (16.7) 1 × 10e7 3 10 10 7 5 (83.3) Percentages may not total 100 because of rounding. aStaging based on the International Federation of Gynecology and Obstetrics (FIGO) guidelines. bEastern Cooperative Oncology Group (ECOG) performance-status scores are assessed on a 5-point scale, with higher scores indicating greater disability. cDeleterious BRCA mutations summarizing germline (blood) BRCA1 andBRCA2 mutations and somatic mutations.Proof of principle study of sequential combination atezolizumab and Vigil in relapsed ovarian cancer Table 3 Treatment-related adverse events. Study part Part 1 Treatment arm Vigil +atezolizumab (number of patients =3) no. of events (%by arm and relationship)a Study drug relationship Atezolizumab-related Vigil-related Event Grade 1 Grade 2 Grade 3 Grade 4 Grade 1 Grade 2 Grade 3 Grade 4 Abdominal distension 2 (22.2) 3 (33.3) 0 0 0 0 0 0 Nausea 0 1 (11.1) 0 0 0 0 0 0Fatigue 1 (11.1) 0 0 0 1 (33.3) 0 0 0 Anxiety reaction 1 (11.1) 0 0 0 1 (33.3) 0 0 0 Other b1 (11.1) 0 0 0 1 (33.3) 0 0 0 Study part Part 2 Treatment arm Atezo-1st (number of patients =10) no. of events (% by arm and relationship)aVigil-1st (number of patients =11) no. of events (%by arm and relationship)a Study drug relationship Atezolizumab-related Vigil-related Atezolizumab-related Vigil-related Event Grade 1 Grade 2 Grade 3 Grade 4 Grade 1 Grade 2 Grade 3 Grade 4 Grade 1 Grade 2 Grade 3 Grade 4 Grade 1 Grade 2 Grade 3 Grade 4 Anemia 0 1 (3.5) 0 0 00000 0 000 000 Leukopenia 0 1 (3.5) 1 (3.5) 0 00000 0 000 000 Neutropenia 0 1 (3.5) 1 (3.5) 0 00000 0 000 000 Hypothyroidism 1 (3.5) 1 (3.5) 0 0 00000 0 000 000 Abdominal pain 1 (3.5) 0 0 0 00001 (2.0) 0 0 0 0 0 0 0 Diarrhea 1 (3.5) 0 0 0 00001 (2.0) 0 0 0 1 (3.1) 0 0 0 Mucositis oral 0 0 0 0 00001 (2.0) 0 0 0 1 (2.0) 0 0 0 Nausea 0 1 (3.5) 0 0 00001 (2.0) 1 (2.0) 0 0 3 (9.4) 1 (3.1) 0 0 Axillary pain 0 0 0 0 00000 0 000 1 (3.1) 0 0 Fatigue 2 (6.9) 2 (6.9) 1 (3.5) 0 00003 (5.9) 3 (5.9) 0 0 2 (6.3) 2 (6.3) 0 0 Fever 0 0 0 0 00000 1 (2.0) 0 0 0 0 0 0 Flu-like symptoms 0 0 0 0 00002 (4.0) 2 (4.0) 0 0 0 2 (6.3) 0 0 General body pain 0 0 0 0 00000 0 000 1 (3.1) 0 0 Infusion-related reaction 0 0 1 (3.5) 0 00000 0 000 000 Injection-site tenderness 0 0 0 0 00000 0 001 (3.1) 0 0 0 AST increased 2 (6.9) 0 0 0 00000 0 000 000 Creatinine increased 0 0 0 0 00003 (5.9) 3 (5.9) 0 0 0 0 0 0 Platelet count decreased 1 (3.5) 0 0 0 00001 (2.0) 0 0 0 0 0 0 0 Weight loss 0 0 0 0 00002 (3.9) 0 0 0 2 (6.3) 0 0 0R. P. Rocconi et al. Table 3 (continued) Study part Part 2Treatment arm Atezo-1st (number of patients =10) no. of events (% by arm and relationship) aVigil-1st(number of patients =11) no. of events (%by arm and relationship) a Anorexia 0 0 0 0 00002 (4.3) 1 (4.3) 0 0 0 0 0 0 Myalgia 0 0 0 0 00001 (2.0) 0 0 0 1 (3.1) 0 0 0 Neck pain 1 (3.5) 0 0 0 00001 (2.0) 0 0 0 0 0 0 0 Dysgeusia 0 0 0 0 00002 (3.9) 0 0 0 1 (3.1) 0 0 0 Headache 1 (3.5) 0 0 0 00002 (3.9) 0 0 0 1 (3.1) 0 0 0 Acute kidney injury 0 0 0 0 00000 0 000 01 (3.1) 0 Chronic kidney disease 0 0 0 0 00000 0 1 (2.0) 0 0 0 0 0 Nephritis 0 0 0 0 00000 1 (2.0) 0 0 0 0 0 0 Pelvic pain 0 0 0 0 00001 (2.0) 1 (2.0) 0 0 1 (3.1) 1 (3.1) 0 0 Allergic rhinitis 0 0 0 0 00001 (2.0) 0 0 0 1 (3.1) 0 0 0 Cough 0 1 (3.5) 0 0 00000 0 000 000 Dyspnea 0 1 (3.5) 0 0 00001 (2.0) 0 0 0 0 0 0 0 Pneumonitis 0 0 1 (3.5) 0 00000 0 000 000 Night sweats 0 0 0 0 00001 (2.0) 0 0 0 1 (3.1) 0 0 0 Rash macular 0 0 0 0 00001 (2.0) 0 0 0 1 (3.1) 0 0 0 Urticaria 0 0 0 0 00001 (2.0) 1 (2.0) 0 0 1 (3.1) 1 (3.1) 0 0 Otherb5 (17.2) 0 0 0 1 (100) 0007 (13.7) 0 0 0 4 (12.5) 0 0 0 TOTAL 15 (51.7) 9 (31.0) 5 (17.2) 0 (0) 1 (100) 0 (0) 0 (0) 0 (0) 36 (70.6) 14 (27.5) 1 (2.0) 0 (0) 22 (28.8) 9 (28.1) 1 (3.1) 0 (0) aPercentages may not total 100 because of rounding. bThe determination of whether an adverse event was related to a trial treatment was made by the Investigator. Part 1: one Grade 1 event occurred in atezol izumab-related for hot ﬂashes and in Vigil-related for injection-site tenderness. tenderness. Part 2: one Grade 1 event occurred in Atezo-1st related to atezolizumab for ALT increase, ST segment elevation, appetite impaired, arthralgia, back pain and to Vigil for lymphocyte decrease, and in Vigil-1st related to atezolizumab for abdominal distension, constipation, tooth ache, intermittent fever, incision site pain, productive cough, wheezing and to vigil for acidemia, hyponatremia, hypophosphatemia, itching.Proof of principle study of sequential combination atezolizumab and Vigil in relapsed ovarian cancer p0.027) (Fig. 3B). There was no bene ﬁti nO Si nt h e BRCAmutcohort between Vigil-1st and Atezo-1st cohorts. No signi ﬁcant bene ﬁt was observed in PFS (Fig. 3C, D). Discussion Despite the fact that most ovarian cancer patients achieve a complete clinical response with primary surgery and com- bination chemotherapy, the overwhelming majority (75%) of these patients will, unfortunately, relapse within16–24 months and ultimately succumb to their disease [19–21]. Treatment for those patients who recur is largely based upon the recurrence-free interval. Women who recur6 months or more after completing platinum-based che- motherapy have traditionally been considered to be plati- num-sensitive, and are usually retreated with a platinumdoublet and often maintenance therapy with bevacizumab or a PARP inhibitor. Patients who relapse within 6 months of completing platinum-based therapy (platinum-resistant)are usually managed with non-platinum-based therapy with or without bevacizumab. PARP inhibitors have shown improvement in progression-free survival (PFS) as main-tenance therapy following initial chemotherapy and fol- lowing platinum-sensitive recurrent OC therapy. Bevacizumab has been found to improve PFS when com-bined with initial chemotherapy or with chemotherapy fol- lowing relapse and is commonly used in combination regimens in relapsed OC [ 22–25]. Checkpoint inhibition has shown no clinical bene ﬁti n ovarian cancer so far. In the JAVELIN Ovarian 200 study (NCT02580058), results indicate the combination of ave-lumab plus PLD achieved a better overall response (ORR, 13.3%) compared to either avelumab (3.7%) or pegylated liposomal doxorubicin (PLD) alone (4.2%). However, OSwas not signi ﬁcantly improved with avelumab alone (11.8 months) or avelumab plus PLD (15.7 months)compared to PLD alone (13.1 months). Median PFS also did not differ between avelumab plus PLD (3.7 months) andPLD alone (3.5 months) [ 26]. However, in patients with PD-L1 > 1%, both ORR and OS were improved in patients receiving avelumab and PLD. These results indicate theremay be a sensitive patient population that has not been identi ﬁed. Results demonstrated here to justify the continued combination of Vigil and atezolizumab testing in recurrent OC and suggest sequential Vigil prior to the combination therapy of Vigil/atezolizumab has a clinical advantage overAtezo-1st. The more favorable toxicity pro ﬁle with encouraging clinical bene ﬁt suggests a superior therapeutic index with Vigil-1st delivery, particularly in the BRCA-wild-type patients. These results are consistent with results recently reported from a Phase 2b trial which also demon- strated signi ﬁcantly improved bene ﬁt in RFS and OS in somatic and germ lineage BRCA-wildtype ovarian cancer patients [ 15]. This is a particularly encouraging direction as a combination of Vigil and checkpoint inhibitor therapy. Given the increased expression of TGF βin OC tissue compared to many other cancers [ 27,28] particularly involving recurrent disease [ 28] and direct activity of Vigil acting to knockdown TGF β1 and TGF β2 via furin inhibi- tion, Vigil may be directly addressing a mechanism of resistance in OC that is mediated through TGF β1 expres- sion. Results support Vigil may be a viable mechanism of improving neoantigen exposure and T cell activation thereby enabling optimal function for checkpoint inhibitor therapydespite TGF βexpression. Secreted TGF βfrom OC cells generates immunosuppressive Treg cells (CD4 +CD25+) from peripheral CD4+CD25−cells within the tumor microenvironment [ 29]. Treg tumor in ﬁltration is associated with poor outcomes in patients with high-grade serous ovarian carcinoma treated with neoadjuvant chemotherapy[30,31]. Accordingly, the CD8/Treg ratio is also a prog- nostic indicator [ 32]. Similarly, CD8 +T cell in ﬁltration into Fig. 2 Treatment-related adverse events. Comparison of all atezolizumab-related adverse events in Vigil-1st vs. Atezo-1st in study Part 2.R. P. Rocconi et al. Table 4 Summary of product release. Study arm Part 1 (N=3) Parameter TGF β1 knockdown (%) GMCSF production (pg/10e6 cells) Viability (%)Gel-clot endotoxin (vaccine < EU/mL) Counta32c33 Average 84 479 91% 0.29 StDev 11 291 1% 0.08Median 79 479 91% 0.24 Min 74 188 90% 0.24 Max 100 769 93% 0.40 Range 74 –100 188 –769 90% –93% 0.24 –0.4 Study arm Part 2 (N=21) Parameter TGF β1 knockdown (%) GMCSF production (pg/10e6 cells) Viability (%)Gel-clot endotoxin (vaccine < EU/mL) Count a20b18c21 21 Average 94 1382 86 0.34 StDev 8 1480 6 0.10 Median 100 550 87 0.40 Min 74 114 72 0.20 Max 100 4593 96 0.50Range 74 –100 114 –4593 72 –96 0.2 –0.5 Cell dose 1 × 10e6 or 1 × 10e7; USP <71> sterility negative for all patients; mycoplasma DNA negative for all patients. aCount represents no. of patients that met product release criteria speci ﬁcations. TGF β1 (% KD) Spec: ≥30%; GMCSF production (pg/10e6 cells) Spec: ≥30; Viability Spec: ≥70%. b1 patient did not meet TGF β1 release. c4 patients did not meet GMCSF release.Proof of principle study of sequential combination atezolizumab and Vigil in relapsed ovarian cancer the tumor microenvironment is a positive prognostic bio- marker in many cancer types including ovarian [ 33–36]. Previous work has also shown a Vigil-induced increase in CD3+CD8+T cell peripheral blood circulation [ 37]. It was hypothesized at the initiation of this study that Vigil-1st treatment before Vigil and atezolizumab combi- nation would focus immune effector cell visualization ofrelevant tumor neoantigen before checkpoint functional activity, thereby limiting off-target toxicity and potentially maximizing immune effector cell availability and response.These observed results support our hypothesis and work by others in preclinical models [ 38], including evidence of selective TGF β1 knockdown in overcoming resistance to checkpoint blockade [ 39]. To mount an effective antitumor immune response, T cells need to be educated to the neoantigen repertoire ofthe tumor [ 40,41]. T cells further differentiate into memory T cells, thus providing long-term immunologic memory and presumably durable disease control [ 42–44]. It has been reported that tumors with low neoantigen heterogeneity respond better to checkpoint inhibition with pembrolizumab[45]. The same report also showed that T cells were unable to recognize subclonal neoantigens, indicating that the abundance of neoantigens visible for recognition is impor- tant and may dilute the visibility of clonal neoantigens [ 45]. T cell recognition of tumor-speci ﬁc clonal neoantigens to the same patient disease is more likely when utilizing autologous tumor tissue (personalized) as opposed to allo-geneic tumor tissue. Over 1400 doses of Vigil have been administered in clinical trials. The most frequently reported adverse reac-tions attributed to Vigil engineered cell administration are injection-site reactions which are mild to moderate in intensity, consist primarily of redness and swelling at theinjection site, which is thought to be related to immune activation at the injection site. There have been no severe or life-threatening (CTC Grade 3 or 4) adverse events attrib-uted to Vigil treatment [ 7,9,16]. No new or unexpected toxic effects were observed with Vigil in combination with atezolizumab. In conclusion, further clinical exploration of the combi- nation of Vigil, a personalized autologous OC vaccine0 3 6 9 12 15 18 21 240255075100 MonthsOverall survival (%)Vigil-1stAtezo-1st No. at Risk Atezo-1st Vigil-1st10 9 6 5 4 3 11 10 9 9 9 4 2 20 3 6 9 12 15 18 21 240255075100 MonthsOverall survival (%)Vigil-1stAtezo-1st No. at Risk Atezo-1st Vigil-1st7 6 3 3 2 2 6 6 5 5 5 1 0369 1 2 1 5 1 8 2 1 2 40255075100 MonthsProgression Free Survival (%)Vigil-1stAtezo-1st No. at Risk Atezo-1st Vigil-1st7 3 6 6 1 1 1AB CD 0369 1 2 1 5 1 8 2 1 2 40255075100 MonthsProgression Free Survival (%)Vigil-1stAtezo-1st No. at Risk Atezo-1st Vigil-1st10 5 2 11 9 3 3 3 2 2 2 Fig. 3 Efﬁcacy analysis from time of study randomization. A OS of all study subjects in Vigil-1st (blue, n=11) and Atezo-1st (red, n= 10), mOS NR vs. 10.8 months, HR 0.33 (95% CI [0.064 –1.7], p0.097); BOS of BRCAwtcomparing Vigil-1st (blue, n=6) and Atezo-1st (red, n=7), mOS NR vs. 5.2 months, HR 0.16 (95% CI [0.026 –1.03], p0.027); CPFS of all study subjects in Vigil-1st (blue, n=11) and Atezo-1st (red, n=10) investigator-assessed by RECIST1.1, mPFS 3.4 months vs. 2.8 months, HR 0.76 (95% CI [0.28 –2.0],p 0.29). DPFS of BRCAwtcomparing Vigil-1st (blue, n=6) and Atezo- 1st (red, n=7), mPFS 3.5 months vs. 2.8 months, HR 0.60 (95% CI [0.16 –2.2], p0.022); OS Overall Survival, PFS Progression-free sur- vival, mOS median overall survival, mPFS median progression-freesurvival, HR hazard ratio, Vigil-1st Vigil First, Atezo-1stAtezolizumab First.R. P. Rocconi et al. followed by a checkpoint inhibitor (atezolizumab) is justiﬁed. Acknowledgements The authors would like to thank Brenda Marr for her help in manuscript preparation and submission. Author contributions All authors made signi ﬁcant contributions to the manuscript and have read and approved the ﬁnal manuscript. Compliance with ethical standards Conﬂict of interest The authors declare no competing interests. Publisher ’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional af ﬁliations. References 1.