1988; Tsunawaki, Sporn et al. 1988; Hirte and Cla rk 1991; Ruffini, Rivoltini et al. 1993; Naganuma, Sasaki et al. 1996). The immune suppressor functions of TGF β are likely to play a major role in modulating the effectiveness of cancer cell vaccines. TGF β inhibits GMCSF Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 16 induced maturation of bone marrow derived dendritic cells (DCs) (Yamaguchi, Tsumura et al. 1997) as well as expression of MHC Class II and co-stimulatory molecules (Geissmann, Revy et al. 1999). It has been shown that antigen presentation by immature DCs result in T cell unresponsiveness (Steinman, Hawiger et al. 2003). TGFβ also inhibits activated macrophages (Ashcroft 1999) including their antigen presenting functi on (Du and Sriram 1998; Takeuchi, Alard et al. 1998). Therefore, both the ubiquitous expression of the TGF β isoforms as well as the inhibitory effects of th ese isoforms on GMCSF immune modulatory function (see below), support a broad based tumor target range for the application of a TGFβ suppressed / GMCSF ex pressing immune enhancing therapeutic. Background on GMCSF 1.5 One factor that may limit the immunogenici ty of tumor cells is the influence of cytokines in the tumor microenvironment (Mach and Dranoff 2000). There is evidence that cytokines have a role in tumor formation and it has been demonstrated that manipulation of the cytokine balance can be exploited for cancer therapy. Forni et al. (Forni, Gi ovarelli et al. 1985) showed that modifying intracellular cytokine levels could al ter the outcome of the host response. Protective immunity against later tumor challenge was also reported in some instances (Forni, Giovarelli et al. 1985). More specifically, tumor cells genetically modified to secrete GMCSF have consistently demonstrated the most pot ent induction of anti-tumor immunity compared to other cytokines (Dranoff, Ja ffee et al. 1993). Results suggest that treatment with GMCSF protein may translate into clinical advantage, perhaps through immune stimulation (Anderson, Markovic et al. 1999; Spitler, Grossbard et al. 2000; Rini, Weinberg et al. 2003) . Interestingly, systemic cytokine administration has not induced direct anticancer response in randomized controlled trials, possibly due to a failure of the approach to recreate accurately the paracrine function of cytokines in tissue microenvironments (Dranoff 2004). Increasing evidence suggests that GMCS F is involved in the augmentation of tumor antigen presentation (Dranoff, Jaff ee et al. 1993; Huang, Golumbek et al. 1994). DCs mediate a crucial role in pr iming antigen-specific immune responses (Zeng, Wang et al. 2001). DCs express diverse receptors that allow for recognition and capture of antigens in per ipheral tissues, process this material efficiently albeit by different routes into the MHC Class I and II presentation pathways, upregulate costim ulatory molecules upon maturation, and migrate to secondary lymphoid tissues (Banchereau, Briere et al. 2000). It was observed following injection of GVAX ® in patients that an intense local reaction consisting of dendritic cells, macrophages, and granulo cytes occurs (Dranoff, Jaffee et al. 1993; Mach and Dranoff 2000). Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 17 Dendritic cells have proven to be incredibly versatile with regards to their response to inflammatory conditions and pathogens, which has made it difficult for researchers to determine the appropriate functional characteristics DCs must attain for cancer vaccination (Banchereau, Briere et al. 2000). In one study B16 melanoma cells engineered to secrete either GMCSF or Flt-3-Ligand (FL) and their immunologic effect s were reported (Mach and Dranoff 2000). Although both cytokines provoke a marked expansion of DCs locally and systemically, GMCSF stimulated greater levels of protective immunity. Three profound differences have been described, which may account for the disparity in response. First, GMCSF induces a subset of DCs that are superior for the phagocytosis of apoptotic tumor cells (Young and Inaba 1996; Pulendran, Lingappa et al. 1997; Shen, Reznikoff et al. 1997). Second, compared to FL, GMCSF evoked higher levels of costimulatory molecules, which is characteristic of greater functional maturation. This enhanced activity results in more efficient T cell stimulation, thereby broadening the arsenal of induced lymphocyte effector mechanisms (Murtaza, Kuchroo et al. 1999) . Third, GMCSF promoted uniformly high levels of CD1d on DCs, in contrast to FL, which triggered a more heterogeneous expression (Kawano, Cui et al. 1997). CD1d is a nonclassical MHC Class I molecule that presents lipid antigens (Yamaguchi, Furukawa et al. 1990). The CD1d lipid complex activates NKT cells, a population of lymphocytes that display a restricted Class I MHC-like recept or (Bendelac, Rivera et al. 1997). Importantly, natural killer T cells (NKT ) cells may play pivotal roles in both endogenous and therapeutic responses to tu mors (Smyth, Crowe et al. 2002). Background on Gene Vaccines in Cancer 1.6 Gene therapy has received considerable attention in recent years. Vaccination with tumor cells designed to augment tumor antigen presentation and induce specific anti-tumor immunity has yielded promising but limited results (Holladay, Heitz et al. 1992). Advances in our understanding of cancer biology and developments in vector technologies ar e advancing the therapeutic potential of tumor vaccine approaches. It is now possible to genetically modify tumor cells for vaccination to express specific tu mor suppressor genes, immune modulators, drug sensitive genes and antisense gene fragment s (Huber, Richards et al. 1991; Culver, Ram et al. 1992; Tr ojan, Blossey et al. 1992; Dranoff, Jaffee et al. 1993; Ram, Culver et al. 1993; Trojan, Johnson et al. 1993; Fakhrai, Dorigo et al. 1996; Swisher, Roth et al. 1999). Extensive experiences by others, as well, demonstrate safety with the use of GMCSF gene based vaccines as a potential vaccine therapeutic in cancer patients (Table 1). Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 18 Table 1: Summary of clinical trials te sting GMCSF gene-transduced vaccines Trial Cancer (stage) Vaccine Vector n Response Dose (number of irradiated tumor cells) GM-CSF production (ng/106 cells every 24 h) Concurrent treatment Simons (Simons, Jaffee et al. 1997) Renal cell (IV) Autologous Retrovirus 16 1 PR (7 mo) 4 x 106 – 4 x 108 17-149 None Chang (Chang, Li et al. 2000) Melanoma (IV) Autologous Retrovirus 5 1 CR (>36 mo) 1 x 10756-100 RhIL-2 3.6 x 10 /kg x 15 doses* Soiffer (Soiffer, Lynch et al. 1998) Melanoma Autologous Retrovirus 29 1 PR, 3 MR, 1 mixR, 3 pts DFS s/p surg/XRT, >36, >20 mo ND 84-965 None Jaffee (Jaffee, Hruban et al. 2001) Pancreatic cancer (I, II, III) Allogeneic Plasmid 14 3 pts DFS 4-5 years 1 x 10 7 – 5 x 108 ND Surgery to disease-free adjuvant XRT/chemo Simons (Simons, Mikhak et al. 1999) Prostate Autologous Adenovirus 8 No responses immune activation observed 1 x 10 7 or 5 x 107 143-1403 None Salgia (Salgia, Lynch et al. 2003) NSCL (IV) Autologous Adenovirus 35 2 pts DFS >3 years s/p surgery 1 x 10 6 – 1 x 107 Mean 233 Surgery Kusumoto (Kusumoto, Umeda et al. 2001) Melanoma (IV) Autologous Adenovirus 9 No clinical response 2 x 10 6 – 1 x 107 80-424 None Mastrangelo (Mastrangelo, Maguire et al. 1999) Melanoma (IV) Intratumoral injection Vaccinia virus 7 1 PR, 1 CR (injected lesion) NA NA None Hu (Hu, McNeish et al. 2003) Solid tumor Intratumoral injection Herpes simplex type 1 15 1 PR (injected lesion) NA NA None Soiffer (Soiffer, Hodi et al. 2003) Melanoma (IV) Autologous Adenovirus 35 1 CR, 1 PR, 3-year follow-up 10 patients alive, 4 DFS 1 x 10 6 – 1 x 107 745 None Simons (Simons, Nelson et al. 2002) Prostate Allogeneic Adenovirus 34 Survival 31 mo with 3 x 10 8 cells vs. 22 mo with 1 x 108 cells 1 x 108 – 3 x 108 None Simons (Simons, Higaro et al. 2003) Prostate Allogeneic Adenovirus 65 33 evaluable (1 PSA PR, 2 PSA MR) 1 x 10 8 – 3 x 108 None Nemunaitis (Nemunaitis, Sterman et al. 2004) NSCLC (IIIB/IV) Autologous Adenovirus 63 3/33 CR Dose-related survival advantage (p <0.05) 5-100 x 10 644-236 None Nemunaitis (Nemunaitis, Sterman et al. 2004) NSCLC (IB/II) Autologous Adenovirus 20 7/10 DFS at 16 mo 5-100 x 10 6 None Tani (Tani, Azuma et al. 2004) Renal (IV) Autologous Retrovirus 4 2 patients alive >40 and >58 mo 1.4-3.7 x 10 8 None Simons (Simons, Carducci et al. 2006) Prostate (hormone naive) Allogeneic Adenovirus 21 76% decrease PSA velocity 1.2x10 8 ND None Small (Small, Sacks et al. 2007) Prostate (hormone refractory) Allogeneic Adenovirus 55 Median survival metastatic group n=(34) Low dose-24 m High dose-34.9 m Prime: 5x10 8 Boost: 1x108 or 3x108 ND None Urba (Urba, Nemunaitis Prostate (hormone Allogeneic Adenovirus 19 PSA doubling ↑ 29 → 57 wks 1-5 x 108 ND None Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 19 Trial Cancer (stage) Vaccine Vector n Response Dose (number of irradiated tumor cells) GM-CSF production (ng/106 cells every 24 h) Concurrent treatment et al. 2008) naïve) Nemunaitis (Nemunaitis, Jahan et al. 2006) NSCL IIIB/IV Autologous and transfected K562 Adenovirus 49 None 5 x 10 6 auto 5 x 108 K562 2624 None Laheru (Laheru, Lutz et al. 2008) Pancreatic Allogeneic Adenovirus 50 Survival 4.3 mo vs. 2.3 mo when combined with cytoxan 5 x 10 8 ND Cohort 13 and cyclophosphomide Senzer (Senzer 2009) Melanoma IIIC/IV Intratumoral injection Herpes simplex 1 virus 50 61% 1-year survival 1 x 10 61 x 108 pfu/ml ND None Hofbauer (Hofbauer, Baur et al. 2008) Melanoma, leiomyosarcom a Intratumoral injection Canarypox (ALVAC) __ SD __ ND None Cornelio (G. H. Cornelio 2008) Miscellaneous Reximmune- C Retrovirus 7 Median OS 5 mo 2.5x109 or 5.0x1010 pfu ND None Higano (Higano, Corman et al. 2008) Prostate (hormone refractory) Allogeneic Adenovirus 80 Median survival Hi 35 mo. Int 20 mo. Lo 23.1 mo. Hi 100x10 6 or 200x106 (q28dx6) Int 200x106 (q14dx12) Lo 500x106 x1 300x106 (q14dx11) Serum levels only None Ho (Ho, Vanneman et al. 2009) AML Autologous 10 9/10 durable CR Allogeneic, nonmyeloablative HSCT Emens (Emens, Asquith et al. 2009) Breast Allogeneic Plasmid 28 Not reported 5.0x107 or 5.0x108 cells 16.829.4 pg/mL (5x107) 153.636.3 pg/mL (5x10*) Cyclophosphamide, doxorubicin Additional studies involving the use of adenoviral vectors to transf er the GMCSF® gene have been approved by the National Insti tutes of Health in patient with prostate, lung, breast, colon and head and nec k cancer, as well as in melanoma patients. *Following vaccine injection, four patients received ex vivo expanded lymphocytes (CD3 negative) from harvested vaccine-primed lymph nodes. chemo: Chemotherapy; CR: Complete response; DFS: Disease-free survival; GMCSF®: Granulocyte-macrophage colony-stimulating fa ctor; NA: Not available; mixR: Mixed response; mo: Months; MR: Minor res ponse; ND: Not described; NSCLC: Non-small cell lung cancer; PR: Partial response; PSA: Prostate-specific antigen; pts: Pa tients; RhIL: Recombinant human interleukin; s/ p: Status post; surg: Surgery; XRT: Rad iation therapy. Blockade of multiple TFG β isoforms is needed to reverse 1.7 immunosuppression Overexpression of two or more of the TGF β isoforms has been demonstrated in melanoma, gliomas, prostate, gastric, colorectal, ovarian and gastric cancers (Tsamandas, Kardamakis et al. 2004; Dallas, Zhao et al. 2005; Polak, Borthwick et al. 2007; Vagenas, Spyropoulos et al. 200 7). It was previously noted that expression of all three immunosuppressive TGF  isoforms is increased in ovarian tumor as compared with normal ovarian tissue (Henriksen et al, 1995; Bristow et al, 1999) with significant increases in TGF 1 in both primary (2.9 fold; p ≤ 0.002) and recurrent (4.4 fold; p ≤ 0.002) ovarian cancer (Bristow, Baldwin et al. 1999). TGF β1 and -β2 both bind to TGF β receptor 1 and suppress DC and helper T cell function through Smad and M AP kinases (Larmonier, Marron et al. 2007; Park, Letterio et al. 2007). The rec ent correlative study by Polak showed that tumor infiltrating tolerogenic DCs and suppressor T cell lymphocytes in malignant melanoma can be correlat ed with immunosuppressive TGF- β1, -β2 and IL-10 expression (Polak, Borthwick et al. 2007). This mechanism of tumor- Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 20 associated immunosuppression is likely to contribute to tumor escape to immune response. The genetic modification with a TGF β2 antisense-encoding plasmid represents one of many currently examined approaches to inhibit local TGF β activity. Others include the use of neutralizing antib odies, soluble receptors, receptor kinase antagonist drugs, ant isense reagents, and a number of less specific drugs such as angiotensin II antagonist and trani last (Prud'homme 2007). Systemic TGFβ blockade may result in severe infla mmatory disease, although this has not been observed, presumably because the neutra lization is only partial. To attain localized TGF β blockade as is applicable to achieve optimal immunostimulation with a GM-CSF-modified cancer cell vaccine, the desired outcome may be accomplished expediently thr ough the incorporation of a de novo gene modifying moiety into the vaccinating cancer cells ex vivo , thereby bypassing pharmacokinetic and toxicity concerns from systemic intr oduction of anti-TGF β neutralizing antibodies, soluble receptors, or receptor kinase antagonists. In view of the overlapping immunosuppressi ve activities of the various TGF β isoforms, it would be particular advantageous if such a gene modifying moiety can globally attenuate the activity of TGF β1, -β2, and -β 3. Background on TAG (TGF 2 antisense and GMCSF vaccine) 1.8 Based on the hypothesis that t he combination of GMCSF and TGF β2 antisense genes will enhance potency of immune stim ulation against cancer specific antigens, a combined plasmid, TAG was constructed (Gradalis, Inc. Carrollton, TX) (Kumar 2009). Components of this plasmid are the clinically utilized pUMVC3 vector backbone (University of Michigan), a bacterial origin of replication, a kanamycin resistance gene, a CMV IE promoter / enhancer and intron A driving the hGMCSF cDNA followed by a 2A linker sequence and a 930 base pair fragment of the hTGF β2 cDNA molecule in antisense orientation followed by a rabbit hemoglobin poly-A tail (Kumar 2009; Maples PB 2009). The TAG expression vector was elec troporated into the autologous cells ex vivo . Therefore, only the cells pr esent at the time of elec troporation would incorporate the transfected DNA. The vector utilized is expected to remain extra-chromosomal. Amplification of the insert by PCR suggested that the vector is non-rearranged in the cells. Our previous human and animal vaccination studies with TGF β2 antisense vaccine and GVAX® vaccine (GMCSF) had not demonstr ated deleterious effects due to unwanted gene expression. It is theoretically possible that the structure or expression of a gene near an integration site (if the vector was incorporated into a chromosome) may be affected by insertion of the added DNA. However, all Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 21 cells used for vaccinations were irradiat ed with 10,000 cGy to block their growth potential. GMCSF expression in the TAG product despi te being significant is lower with the electroporation processing (1ng per 10 6 tumor cells per 24 hours). Thus, although survival in one GVAX® study (Nemunaitis, Sterman et al. 2004) correlated with GMCSF expression ≥40ng/106 cells/24 hours, in a second study there was no correlation (see below). Further, in addition to documented variability in level of GMCSF expressi on between manufacturing processes, the levels of expression achieved with the TAG vaccine are deemed clinically relevant as 1) use of a plasmid rather than a viral vector obv iates the obfuscating effects of elicited anti-viral neutralizing ant ibodies, 2) use of a plasmid likewise prevents the development of elicited antibodies interfering with long-term gene expression, and 3) concurrent suppression of TGF β2 would minimize tumor associated inhibition of GMCSF induced den dritic cell maturation (Yamaguchi, Tsumura et al. 1997), which we hypothesiz e would improve product activity and theoretically lower minimal toxic effe ct related to GMCSF and or adenoviral presence. In addition, our experience with the bystander GVAX® vaccine with a 25-fold higher GMCSF secretion than the autologous vaccine in which no objective tumor responses were seen s uggests that levels of GMCSF protein expression from the manufac tured vaccine is not an exclusive or possibly even necessary factor in vaccine activity. Thirty-eight patients (BB- IND 13650) underwent harvest for TAG vaccine. Thirty- two were successfully manufactured under GMP conditions (Maples PB 2009). GMCSF expression and TGF β2 knockdown met product release criteria. Three (all gastrointestinal tumors with luminal access) had bacterial contaminants and could not be released. Three had insufficient cells. Twenty-two advanced refractory cancer patients have been treat ed (Maples, Kumar et al. 2009; Maples PB 2009; Nemunaitis 2009). No significant toxic effect was observed. No Grade 3/4 toxicity was demonstrated to TAG (T ables 2 and 3). Sign ificant increase in GMCSF expression and knockdown of TGF β2 but not TGF- β1 were demonstrated as components of product releas e criteria. In pr eliminary results we have observed 17 of 22 (77%) eval uable patients with stable disease or better of at least 3 months. Two withdrew early for personal reasons with stable disease after 1 cycle; two had progressive disease at the 2 month assessment; and one withdrew consent at month 3 with progressive disease (Tables 4 and 5). One patient achieved CR, confirmed by imaging studies (Figure 1) (melanoma). Three patients remain aliv e under observation following successful administration of all manufactured vaccines (008, 013, and 023). Two additional patients are continuing on trial (037 and 041). Eight of 18 evaluable patients survived > 1 year following initiation of treatment. Thus the TA G vaccine appears to be safe and has evidence of clinical efficacy (Olivares J 2011). Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 22 Table 2: Frequency of Adverse Events Possibly, Probably, or Definitely Related to the TAG vaccine Preferred Term Grade Relation to TAG Number of Events Number of Subjects Edema 1 Definitely 1 1 Fatigue 1 Possibly 1 1 Fatigue 2 Possibly 1 1 General Pain (NOS) 1 Definitely 4 1 Injection Site Reaction 1 Definitely 3 3 Injection Site Reaction 1 Probably 2 2 Injection Site Reaction 2 Probably 1 1 Rash 1 Possibly 1 1 Rash 2 Possibly 1 1 Fatigue 2 Possibly 1 1 Left Arm Soreness 1 Probably 1 1 Left Arm Weakness 1 Probably 1 1 Table 3: Serious Adverse Experiences reported on TAG vaccine ID# Reported Term Grade Drug Related Assessment Unexpected 007 Small Bowel Obstruction 3 Not Related Yes 007 Dehydration 3 Not Related No 010 Seizure 3 Not Related Yes 010 GI Bleed 3 Not Related Yes 010 Brain Metastasis 3 Not Related Yes 017 Pneumonia (resulting in death) 5 Not Related Yes 031 Pericardial effusion 3 Not Related Yes 032 Weight Loss 2 Not Related No 032 Vomiting 3 Not Related No 032 Aspiration Pneumonia 3 Not Related No Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 23 Table 4: Demographics of Subjects on TAG Age (years) 16-64 24 ≥65 14 Gender Male 17 Female 21 Cancer Adrenocortical 2 Bladder Cancer 2 Breast Cancer 4 Colon Cancer 4 Colorectal 1 Duodenal Cancer 1 Gastric 1 Leiomyosarcoma 1 Melanoma 5 Neuroendocrine 2 NSCLC 7 Ovarian 1 Rectal Cancer 1 Renal Cancer 1 Urachal Adenocarcinoma 1 Prostate 1 Hemangiopericytoma 1 Esophageal 1 Cervical Cancer 1 Table 5: Response status of subjects on TAG 2.5 x 107 cells / injection 1.0 x 107 cells / injection Stable disease ≥ 3 months after vaccine received 10 6 Unevaluable 2 0 Progressive disease 3 0 Complete response 0 1 Active on treatment < 3 months 4 0 Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 24 Figure 1: Patient 013 had diffusely metastatic melanoma and had previously failed multiple standard therapies. PET CT 11 cycles after TAG demonstrated significant response on 7/7/09 in compar ison to baseline on 7/7/08. New 2,3 FDG uptake however in upper paratracheal nodes in association with a viral syndrome confused demonstration of CR. Re-scan 3 months later with viral syndrome resolved revealed CR. Residual uptake at L 2 was followed up with a MRI scan and biopsy which revealed no malignancy. Harvested malignant tissue was processed to a single cell suspension, and cells were transfected with the TAG expressi on vector via electroporation. All processed TAG vaccines demonstra ted GMCSF secretion and low to undetectable levels of TGF β2, as shown in Figures 2A and 2B. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 25 Figure 2A: GM-CSF expression in TAG vaccines. GMCSF Expression in Pre and Post TAG Plasmid Transfected Autologous Tumor Cells 110100100010000 Pre Postpg/mlLung Lung Lung Lung Lung Lung Lung Lung B reast B reast B reast B reast B reast Colon Colon Colon Colon Re ctal Gastric Duodenal Melanoma Melanoma Melanoma Melanoma Adrenocortical Adrenocortical Liver Ovarian Neuroendocrine Re nal Leiomyosarcoma Bladde r Bladde r Prostate Hemagiopericytoma E sophageal Cervical Ur achal Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 26 Figure 2B: TGF β-2 expression in TAG vaccines. Background on Furin 1.9 Furin, an upstream regulator of TGF β activity, is a member of the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. This encoded protein is a calcium-dependent serine endoprotease that can efficiently cleave precursor protei ns at their paired basic amino acid processing sites by the consensus sequenc e –Arg-X-K/Arg-Arg (RXK/RR), with - RXXR- constituting the minimal cleav age site (Thomas 2002). Furin is synthesized as an inactive zymogen and translocated into the endoplasmic reticulum (ER), where the propeptide is cleaved autocatalytically. Furin is activated when it exits the ER and trans its to the mildly acidic trans Golgi apparatus, where it is further cleaved by the resident, activated furin or by an TGF2 Expression in Pre & Post TAG Plasmid Transfected Autologous Tumor Cells n=38 110100100010000100000 Pre Postpg/mlLung 1 Lung 2 Lung 3 Lung 4 Lung 5 Lung 6 Lung 7 Lung 8 Breast 1 Breast 2 Breast 3 Breast 4 Breast 5 Colon 1 Colon 2 Colon 3 Colon 4 Rectal 5 Gastric 6 Duodenal 7 Adre nocortica l 1 Adre nocortica l 2 Adre nocortica l 3 Melanoma 1 Melanoma 2 Melanoma 3 Melanoma 4 Bladder 1 Bladder 2 Liver (H) Ovarian (O) Neuroendocrine (N) Renal ® Leiomyosarcoma (S) Prostate (P) Hemagiopericytoma (H) Esophageal (E) Cervical © Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 27 associated molecule in trans. Its dissoci ation from the catalytic domain leads to its activation. Following release and intrac ellular trafficking, furin can be tethered at the cell membrane by the cytoskeletal pr otein filamin. Proteolytic cleavage by furin is required for TGF β convertase activation (i.e. pro-TGF β TGFβ). All three of the TGF β isoforms contain the RXXR motif at their cleavage site albeit with different amino acid sequences (Kusakabe, Cheong et al. 2008). Data suggest that the unique structure of the TGF β2 latency associated peptide (LAP) region might attenuate furin mediated cleav age but do not exclude a role for furin in its processing. Furin is the primary proconvertase in TGF β processing events. It is worth noting that there appears to be a disparity between the complete cleavage of TGF β in vivo versus the partial cleavage in vitro (Dubois, Laprise et al. 1995). TGF β in turn appears to amplify furin gene transcr iption through an amplification loop (Blanchette, Day et al. 1997; McMahon, Laprise et al. 2003). High levels of furin mRNA and furin protein are widely expressed in human tumors and, specifically, in ovarian cancer (Schalken, Roebroek et al. 1987; Cheng, Watson et al. 1997; Mbikay, Sirois et al. 1997; Bassi, Mahloogi et al. 2000; Bassi, Mahloogi et al. 2001; Bassi, Fu et al. 2005). There is evidence that furin may be an important target in the treatment of prostate cancer (Uchida, Chaudhary et al. 2003), colorectal c ancer (Scamuffa, Basak et al. 2008; Scamuffa, Siegfried et al. 2008), and breast cancer (Lapierre, Siegfried et al. 2007). In addition, furin plays an important role in immune regulation (Pesu, Muul et al. 2006; Pesu, Watford et al. 2008) . In APCs, cytotoxic T lymphocyte- sensitive epitopes in the trans-Golgi compartment were processed by furin and the less frequented TAP inde pendent pathway (Lu, Wetts tein et al. 2001). The conditional deletion of endoge nous-expressing furin in T lymphocytes allowed for normal T-cell development, but impaired the function of regulatory and effector T cells, which produced less TGF β1. Furin-deficient Tregs were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector ce lls were inherently over-active and were resistant to suppressive activity of wild -type Treg cells. Thus, furin expression by T cells appears to be indispensable in main taining peripheral to lerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF β1 production (Pesu, Watford et al. 2008). We and others have found that up to a 10-fold higher level of TGF β1 may be produced by human colorectal, lung cancer , melanoma, and ovarian cancer cells, and likely impact the immune toler ance state by a higher magnitude (Bommireddy and Doetschman 2007; Fogel-Petrovic, Long et al. 2007; Polak, Borthwick et al. 2007). The presence of furin in tumor cells likely contributes significantly to the maint enance of tumor directed, TGF β1 peripheral immune tolerance (Pesu, Watford et al. 2008). Hence, furin knockdown represents a novel and attractive approach fo r optimizing immunosensitization. Initial studies Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 28 to validate the effect of furin blockade on TGF β expression was carried out with the furin inhibitor decanoyl-Arg-Val-L ys-Arg-CMK (Dec-RVKR-CMK), a peptidyl chloromethylketone that binds irreversible to the catalytic site of furin and blocks its activity ((Henrich, Cameron et al . 2003)). Treatment with Dec-RVKR-CMK either completely or partially reduces the activity of furin substrates BASE ( β-site APP-cleaving enzyme), MT5-MMP, and Bo c-RVRR-AMC (Pearton, Nirunsuksiri et al. 2001). TGFβ1 and TGFβ2 specific immunoassays (R&D Systems Quantikine ELISA) were used to quantify the effect of furi n blockade on secreted cytokine levels of the human colorectal line CCL-247. Dec-RVKR-CMK (30 nM) reduced TGF 1 level from 250 pg/ml to below det ectable levels, and inhibited TGF β2 expression by approximately 50% (from 35 to 18 pg/ ml). Dec-RVKR-CMK was similarly effective in reducing TGF β1 and TGFβ2 production in the human melanoma line CRL-1585 (reductions of 100% and 82%, respectively) and the Human NSCLC line H460 (reductions of 100% and 93%, respec tively). These findings indicate that TGF β isoform expression can be effectivel y reduced through furin blockade. To consider the applicability of furin knockdown for inhibiting TGF β isoform expression, similar assessments were per formed following transfection with furin- specific siRNAs. Prospective siRNA ta rgeting sites in the furin mRNA were determined by the published recommendations of Tuschl and colleagues and the additional selection par ameters that integrates BLAST searches of the human and mouse genome databases (http:jura.wi.mit.edu/bioc/siRNAext). siRNAs targeting eligible translated and 3’UTRs were purchased from Ambion (A-1, -2,-3) and Integrated DNA Design Technologies/IDT (I-1,-2,-3; Figure 3). Proprietary siRNAs from Ambion were des igned with an additional algorithm that utilized a proprietary classification approach support vector machine (SVM) approach for enhanced specificity and acti vity. siRNAs from IDT utilizes a proprietary rational design al gorithm that integrates bot h traditional 21-mer siRNA design rules as well as new 27-mer Dicer -substrate specific design criteria. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 29 Figure 3: siRNA targeted regi ons of furin mRNA. Prospective siRNA targeting regions in 3’-UTR and encoding regions of furin mRNA and the targeted sequence by each siRNA construct is shown. The transfection of CCL-247 cells was carried out with Lipofectamine 2000 with 10nM -60nM siRNA and 1x105 cells/ml. Treatment with each of the 6 siRNA constructs resulted in marked reduced levels of TGF β1 and TGFβ2 in the culture supernatants. Depletion ranged from 60-72% for TGF 1 and comparable levels (57-77%) for TGF β2 (Figure 4). By comparison, the inhibitory effects of scrambled siRNA treatment were less profound (<20%). Further, siRNAs that targeted the encoding (A-1, A-2, I-1, I-2) or the 3’-UTR (A-3, I-3) of furin miRNA appeared to be equally effective for TGF β1 and β2 depletion. Similar levels of cytokine reduction were observed over the dose range tested (10-60 nM) for each siRNA. The viability of untreated CCL-247 cells was 84% at 24 hours post-culture, based on trypan blue dye exclusion viability analysis . By comparison, 70% of cells were viable after treatment with the fuin inhibitor Dec-RVKR-CMK. siRNA furin treated cultures displayed a viability of > 70% fo r all siRNA tested at the same time point, as compared to 80% following contro l siRNA treatment. There was no remarkable survival advantage following treatment with any of the 6 siRNA tested. These findings indi cate that the observed TGF β reductions cannot be explained by the marginal cytotox icity of Dec-RVKR-CMK or siRNAfurin transfection. The relatively robust viabilit y of transfected cultures implies that immunosensitization through transgene expression is likely to remain active over an extended time peri od, (i.e. > 48 hrs). siRNAfurin treatment similarly reduced TGF levels the human melanoma line CRL- 1585 (37-59% reduction of TGF- β1, 25-51% reduction of TGF- β2) and the NSCLC line H460 (78-94% reduction of TGF- β1, 72-79% reduction of TGF- β2) Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 30 (Figure 4). Our findings indicate that siRNA-mediated furin knockdown is effective for the depletion of TGF- β isoforms. a B Figure 4: TGF- β 1 (Figure 4a) and TGF-β2 (Figure 4b) expression in human cancer lines following siRNAfurin knockdown. Currently, it is unclear why TGF β reduction in CRL-1585 was comparatively modest. All three lines displayed simila r transfection efficiencies (75-85%) by assessment with a GFP expression plasmi d. Followup studies are underway to quantify furin reduction at the mRNA and pr otein levels, in order to correlate knockdown outcome with TGF  reduction outcomes. Gradalis, Inc., is at the forefront in the design of “bifunctional” vectors that embed both siRNA and miRNA functional component s in a miR 30 scaffold for optimized gene knockdown (Rao 2009; Rao D 2010). The siRNA component is encoded as a hairpin composed of complete matching sequences of the passenger and guide strands. Following cleavage of the passenger strand by Ago 2, an endonuclease with RNase H like activity, the guide strand is incorporated into the RNA-induced silencing complex (RIS C), which binds to and cleaves complementary target mRNA (cleavage dep endent process). In distinction, the miRNA component of the “bifunctional” vect or incorporates mismatches between the passenger and guide strands within the encoding shRNA hairpin in order to achieve lower thermodynamic stability. This configuration allows the passenger strand to dissociate from RISC without cleavage (cleavage i ndependent process) (Matranga, Tomari et al. 2005; Leuschner, Ameres et al. 2006), and the RISC incorporated guide strand acts bot h through translational repression and sequestration of the target mRNA in the cytoplasmic processing bodies (P-body). We have previously demonstrated t he markedly enhanced effectiveness of bi- functional shRNASTMN1 in knockdown of stathmin ( oncoprotein 18) which encodes a protein that regulates microtubule rem odeling of the cytoskeleton and shown to be upregulated in a high proportion of patient s with solid cancers (Rana, Maples Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 31 et al. 2008). The bifunctional shRNA c onstruct achieved effective knockdown against stathmin 1, with a 5 log dose enhance ment in potency of tumor cell killing compared with the identically targeted siRNA (Rao 2009; Rao D 2010). A similarly designed bifunctional shRNA was designed to effect furin knockdown. The bi-shRNA furin consists of two stem-loop st ructures with m iR-30a backbone; the first stem-loop structure has comp lete complementary guiding strand and passenger strand, while the second stem-l oop structure has two bp mismatches at positions 11 and 12 of the passenger str and. Our strategy is to use a single targeted site for both cleavage and sequestration. By the use of a proprietary algorithm, the encoding shRNAs are pr oposed to accommodate mature shRNA to be loaded onto more than one types of RISC (Azuma-Mukai, Oguri et al. 2008). Our reason for focusing on a single si te is that multi-site targeting may double the chance for “seed sequence” indu ced off-target effect (Jackson, Koduvayur et al. 2006). Figure 5: Plasmid construct of FANG™. Construction of the FANG™ expression vector is based on the TAG plasmid (Kumar 2009) with the only difference being the replacement of the TGF β2 antisense DNA sequence with the bi-shRNA furin DNA sequence (Figure 5). The two stem-loop double stranded DNA sequenc e was assembled with 10 pieces of synthetic complementing and interconnecting oligonucleotides through DNA Ori ceCMVPromoter HI BamHI Bam 2000 3000 5000 I 2A polyAsh-Furin 5139 bp Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 32 ligation. The completed 241 base pairs DN A with Bam HI sites at both ends was inserted into the Bam HI site of the TA G expression vector in place of the TGF β2 antisense sequence. Otherwise the two ex pression vectors (TAG and FANG™) are identical (confirmed by sequencing). Orientation of the inserted DNA was screened by PCR primer pairs designed to screen for the shRNA insert and orientation. The FANG™ construct has a single mammalian promoter (CMV) that drives the ent ire cassette, with an intervening 2A ribosomal skip peptide between the GMCSF and the furin bifuncti onal shRNA transcript, followed by a rabbit polyA tail. There is a stop codon at the end of the GMCSF transcript. Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the j unction of the 2A and downstream sequences, leading to the production of tw o proteins from a single open reading frame (Funston, Kallioinen et al. 2008). We have found that the 2A linker to be effective for generating approximately equal levels of GMCSF and anti-TGF β transcripts with the TAG va ccine, and elected to use th e same design for Vigil™. Electroporation of GMP FANG™ plasmid into patient tumor cells (the cGMP vaccine manufacturing process) demons trated GMCSF prot ein production and concomitantly TGF β1 and – β2 knockdown as predicted. Figure 6 depicts a FANG™-transfected NSCLC tumor’s expr ession profile over 14 days (FANG- 004). A summary of the 14 day expressi on (after manufacturing) profiles of the 23 tumors processed to date is depicted in Figure 7. Of not e is the comparison of the TAG-038 and FANG-004 vaccines pr oduced from the same patient’s NSCLC tumor. The TAG vaccine has similar GMCSF expression and TGF- β2 knockdown but TAG did not effect the TGF- β1 expression (which is about 10X that of pre-transfection TGF-β2) (Figure 8). Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 33 Figure 6: Summary of TGF β1, -β2, and GMCSF expression in FANG-004 tumor cells pre and post FANG™ cGMP plasmid tr ansfection. Cells were incubated for 14 days and media was periodically sampled for cytokine analysis. TGF beta1 Expression by FANG-004 (NSCLC) pre and post transfection/post irradiated cells 05001000150020002500300035004000 Day0 Day1 Day2 Day3 Day4 Day7 Day10 Day14 Days in culturepg/ml Pre Post TGFb2 Expression by FANG-004 (NSCLC) pre and post transfection/post irradiated cells 02004006008001000 Day0 Day1 Day2 Day3 Day4 Day7 Day10 Day14 Days in culturepg/ml Pre Post GMCSF Expression by FANG-004 (NSCLC) pre and post transfected/post irradiated cells 0.02.04.06.08.010.012.014.016.018.0 Day0 Day1 Day2 Day3 Day4 Day7 Day10 Day14ng/ml Pre Post Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 34 Figure 7: Summary of TGF β1, -β2, and GMCSF protein pr oduction pre and post FANG™ plasmid transfection. ELISA values from Day 4 of the 14-day determinations of cytokine production in manufactured autologous cancer cells. TGF1 Expression (Day 7) Pre and Post FANG Transfected Autologous Tumor Cells 110100100010000 Pre Postpg/ml TGF2 Expression (Day 7) Pre and Post FANG Transfected Autologous Tumor Cells 110100100010000 Pre Postpg/ml GMCSF Expression (Day 7) Pre and Post FANG Transfected Autologous Tumor Cells 110100100010000 Pre Postpg/mlFANG-001 M elanom a FANG-002 M elanom a FANG-005 M elanom a FANG-006 M elanom a FANG-007 Colon FANG-008 Colorectal FANG-003 Gallbladder FANG-004 NSCLC FANG-009 Breast Can FANG-01 0 Colorectal FANG-01 1 SCLC FANG-01 2 M elanom a FANG-013 Breast Can FANG-01 4 M elanom a FANG-01 5 NSCLC FANG-01 6 Colon FANG-01 7 NSCLC FANG-01 8 Colon FANG-01 9 M elanom a FANG-020 M elanom a FANG-021 Colorectal FANG-022 M elanom a FANG-023 Pituitary Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 35 Data represents autologous vaccines in dependently generated from 23 patients who underwent Vigil™ processing (FANG 001 – 023). Figure 8: Manufactured consecutively from the same patient tumor. Values represent ELISA determinati ons of cytokine production at Day 7 post transfection in harvested autologous cancer cells transfe cted with either Vigil™ (blue) or TAG plasmid (red). The TGF β2 post transfection values are the same for TAG (ACTV-038) and Vigil™ (FANG-004), hence the black line. Phase I Results 1.10 The Vigil™ vaccine is comprised of autologous tumor cells as a source of TAA’s (tumor associated antigens) and two genetic modifications in order to optimize a “triad” functionality--pati ent tumor-specific antigen presentation, dendritic cell activation (GMCSF), and tolerance escape (blocking TGF 1, 2 activation) (Maples PB 2009; Nemunaitis 2011). To construct Vigil™, autologous cancer cells are transfected with a multiple component expression vector encoding GMCSF for recruitment and differentiation of antigen presenting dendritic cells (DCs) and a downstream bi-functional small hairpin RNA for specific knockdown of furin, a proprotein convertase critical for maturational proteolytic processing of immune relevant TGF  isoforms. We recently completed a Phase I trial with Vigil™ vaccine in which 56 cancer patients underwent successful vaccine cons truction and 33 patients (1 of them being a pediatric patient 15 years of age) received ≥1 vaccination. No significant toxic effect was observed. A marked el evation of interferon gamma producing T cells was observed in a subset of patients which correlated with survival. The capacity of Vigil™ to deplete TGF 1 and TGF 2 isoforms is hypothesized to 1) effect silencing of a well doc umented, primarily endogenous family of immunosuppressive cytokines and 2) specifically inhibit TGF -suppression of GMCSF induced maturation of DCs, ex pression of MHC Class II and co- stimulatory molecules. TGF1 Expression 05001000150020002500 Pre Post Day 7pg/mlTGF2 Expression 0100200300400500 Pre Post Day 7pg/mlGMCSF Expression 0500100015002000 Pre PostDay 7pg/ml Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 36 Product transgene expression of GMCSF a nd downregulation of expression of TGF1, -2, and Furin are shown in Figure 9. Mean post-transfection GMCSF expression value was 1135 pg/106 cells/ml (Day 4 of QC assay), the GMCSF release specification of ≥30pg GMCSF/106 cells/ml was met for each vaccine manufactured). Mean post-transfection TGF 1 and  2 knockdown were 93% and 95%, respectively (Day 4 of QC assay). Furin knockdown was 78%. There was no difference in the rate of adverse events across the 2 dose cohorts. Two possibly related Grade 3 treatm ent-related events were observed: abdominal pain and neutropenia. There were no treatment-related serious adverse events. The most common Gr ade 1, 2 adverse event occurring at ≥5% frequency related to study medication wa s injection site erythema. No relationship between dose and adverse event frequency or severity was observed. Of the 33 patients who have received ≥ 1 vaccine dose, 31 were evaluable for tumor response (1 adult and 1 pediatric patient progressed following first injection). Mean and median survival of all Vigil™ treated adult patients (n=32) from time of procurement was 584 days and 562 days, respectively. Their mean and median survival from time of treatment was 477 days and 490 days, respectively. Factors such as age, sex, dose level, pre-treatment expression levels of TGF 1 and  2 and Furin and vaccine transgene expression or knockdown did not correlate with surviv al. However, breakdown of survival comparing Vigil™ treated adult patients receiving ≥ 4 vaccines (n=24) vs. <4 vaccines (n=8) was significantly different (p<0.001) as shown in Figure 10. Although not randomized, comparison to patients fulfilling the same inclusion criteria who did not receive Vigil™ fo rmerly known as FANG™ from time of procurement (no FANG™) is shown in Figure 11. Difference in survival (median 122 days for no FANG™, n=29 and 562 da ys for FANG™, n=32) achieved statistical significance (p<0.00001). Conservative assessment of only patients who survived 4 or more months since proc urement revealed a median survival of 251 days for the no FANG™ patients (n=11) and 572 days for FANG™ patients (n=30) (p=0.005). Interestingly, all four patients with advanced metastatic melanoma achieved ≥1 year of survival follo wing treatment with Vigil™ (specifically, 967, 835, 560, and 490 days each). Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 37 a b Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 38 c d Figure 9. Protein data results for GMCSF gene expression (a), TGFβ 1 knockdown (b), TGF β2 knockdown (c), and Furin knockdown (d) from 56 successful vaccines and 4 vaccines not meeting the minimum dose requirement but for which protein data were collected. Failed vaccines (n=5) did not have protein data collected. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 39 Figure 10. Survival comparison of treat ed adult patients who received <4 FANG™ vaccinations (red) vs. ≥4 vaccinations (blue), n=32, p<0.001. (Data as of 01/08/13) Figure 11. Survival comparison of adult pat ients receiving FANG™ (blue) to those not receiving FANG™ (red), n= 61, p<0.00001. (Data as of 01/08/13) Sequential ELISPOT analysis at baseline and prior to t he 4th Vigil™ vaccine was performed in 24 patients. Results are shown in Figure 12. Twenty-two had no Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 40 detectable autologous tumor specific cytotoxic CD8+ T-cell activity at baseline. Two patients showed ELISPOT reaction at baseline (>10 spots), but while one showed reactivity >2 times that of basel ine at the Month 3 assessment, the other did not have a >2 times increase in ELI SPOT spots, and thus was considered a negative responder. Twelve patients had an increase in immune response from a baseline mean of 6 IFN producing T-cell spots to an end of Cycle 3 mean of 106 IFN producing T-cell spots (p = 0.011) . The other 12 patient showed neither reactivity nor enhancement of immune response from a baseline mean of 3 spots through Cycle 3. These 2 populatio ns were statistically different from each other at the end of Cycle 3 in EL ISPOT response (106 spots vs. 3 spot, p=0.007). Comparison of survival between the 12 patients demonstrating positive ELISPOT respons e and those 12 not demons trating response at completion of Cycle 3 demonstrated a statistically significant increase in survival from time of procurement (p=0.040) and ti me of treatment star t (p=0.023, Figure 13a, b) in the former group. A dditionally, patients who received ≥3 doses of Vigil™ and had a positive ELISPOT had bette r survival than patients who did not receive Vigil™ (p<0.001). Figure 12. IFN- (ELISPOT) in FANG™ vacci ne treated patients peripheral blood mononuclear cells in response to non-transfected autologous tumor cells. Blue lines indicate patients achieving ≥ 10 IFN- producing lymphocytes (positive response) after 3 cycles of FANG™ vacci ne. Red lines indicate patients not achieving positive ELISPOT response after 3 cycles of the FANG™ vaccine. (Data as of 01/08/13) Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 41 All patients demonstrating positive respons e after 3 complete cycles of Vigil™ vaccine who had long term assessment continued to show positive ELISPOT response during vaccine treatment and afte r discontinuation of vaccine therapy (Figure 12). Two patients who were in the non-responsive group at the end of Cycle 3 demonstrated a late positive res ponse after 5 cycles of Vigil™ vaccine. One of these patients had adv anced metastatic melanoma with survival of 490 days since treatment start, and the other had hepatocellular cancer with a survival of 738 days since treatment start. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 42 a b Figure 13: Survival comparison of pati ents achieving positive ELISPOT response prior to the 4th injection (blue) vs. those not achieving positive ELISPOT response prior to the 4th injection (red) from time of procurement, p=0.40 (a) and time of first vaccinatio n, p=0.023 (b) (n=24). This Phase I study was designed to assess the safety of a RNAi mediated, GMCSF expressing autologous tumor cell vaccine, Vigil™, and to evaluate the p = 0.040 p = 0.023 Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 43 triad immunotherapeutic concept (Nemunai tis 2011) of concu rrent autologous tumor cell antigen provision, immunostimulation and inhibition of autologous whole cell component endogenous immunosuppression. Enhanced effectiveness of the bi-shRNA vis-à-vis downregulation of expression as compared with siRNA and shRNA has been previously demonstrated (Liu, Rao et al. ; Rao, Maples et al. 2010). In the cont ext of comparative Vigil™ plasmid functionality with the prior generation TAG vaccine, the median GMCSF expression was similar but TGF 2 knockdown was more effe ctive, i.e., 92.5% as compared to slightly more than 54% k nockdown with TAG. However, most significantly, TGF 1 knockdown, which is not affected in TAG, was 93% with Vigil™. These results validate the ra tionale and confirm the effectiveness of inhibition of expression of immunosuppressive TGF  isoforms via a bi-shRNA mediated knockdown of the proprotein conver tase Furin as well as the feasibility of an integrated GMCSF + RNAi moiety. The high degree of TGF 1 and  2 knockdown herein achieved combined with the clinical and immunological data presented justify advancement of the current manufacturing process. A 3+ year follow up of 74 Phase I patients with successful vaccine manufacture, of whom 35 received Vigil™, confirmed tolerability and safety with no evidence of long- term toxicity. In addi tion, survival continues to co rrelate with ELISPOT response (median 836 days vs. 440 days with posit ive and negative ELISPOT respectively) (Senzer N 2013) Results support the hypothesis that the Vi gil™ vaccine is safe and, in addition, provide a Phase I database justifying cont inued clinical ev aluation and expansion of immune assessment assays. STUDY RATIONALE 2.0 This study adopts an alternative approac h to immune therapy assessment by eschewing the treatment of late st age disease and evaluating adjuvant-based disease prevention in patients with minimal disease but at high risk for recurrence. According to the serial immune function analyses by Coleman (Coleman, Clayton et al. 2005), five of nine patients with Stages III-IV ovarian adenocarcinoma who achieved post-c hemotherapy remission retained the capacity of CD8 + T-cell responses to a panel of 11 viral peptides restricted by at least six common HLA class I allele s. Chemotherapy produced no apparent effect on naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+), or effector memory (CD45-CCR7-) T-cells. These findings a ffirm the strategy of vaccine deployment following chemotherapy, and justif y selection of Stage III/IV epithelial ovarian cancer patients who have achiev ed complete clinical or pathological remission following cytoreductive surgery and front-line chemotherapy for this Vigil™ vaccine study. Exploratory anal ysis of two Phase III studies (D9901 and D9902) with the recently FDA approved Provenge® has i dentified prolonged Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 44 survival benefits in prostate cancer patients initially tr eated with Provenge® and subsequently received docetaxel (median su rvival of 34.5 months, compared to 25.4 months in patients treated with placebo followed by docetaxel) (Petrylak 2006). Thus the potential for vacci ne mediated immune modulation both posterior and anterior to chemotherapy is a c linically attractive strategy worthy of exploration. With regard to the appropriateness of a placebo arm in this group of patients, to date, in conjunction wit h platinum/taxol base regimens, maintenance therapies have shown neither a DFS endpoint advantage (Sabbatini, Harter et al. 2013) nor an OS (overall survival) endpoint advantage low dose taxol (Markman, Liu et al. 2009), Abagovomab (Sabbatini, Harter et al. 2013), and bevacizumab (Burger, Brady et al. 2011; Perren, Swart et al. 2011) while giving rise to a spectrum of extended treatment related side effe cts and questions of results-related economic justification (Lesnock, Farris et al. 2011). In the two recent randomized Phase III studies of bevacizumab, GOG-0218 (15 mg/kg x 16 cycles) and ICON7 (7.5 mg/kg x 12 cycles), in which t here was a RFS endpoint advantage, the bevacizumab and control curves converged after discontinuation of bevacizumab prior to progression at 24 months and ~22 months, respectively, implying that continuation of bevacizumab until progression be considered, consistent with the results of the OCEANS trial in women wit h platinum-sensitive recurrent disease (for a significant RFS endpoint albeit ther e was still no significant difference in OS at first interim analysi s) (Aghajanian, Blank et al . 2012). This must be weighed against the added exposure to bevacizumab associated adverse effects. As subsequently pointed out, the 3.8 month gain in PFS came at the patient cost of “23% risk for devel oping Grade 2 hypertension, 10% risk for Grades 3 to 4 hypertension, and 2.3% ri sk for Grade 3 or worse GI perforation, hemorrhage, or fistula formation”(Hensley 2011). In addition, based on Quality Adjusted Life Year (QALY), the cost of chemotherapy plus just 16 cycles of bevacizumab is $326,530/QALY vers us $13,402/QALY for the same chemotherapy and maintenance low-dose paclitaxel (Lesnock, Farris et al. 2011). Likewise in a cost-effectiveness analysis of GOG-0218, the cost effectiveness ratio (i.e. the cost per y ear of PFS) was $2 47,616, $1,800,000 and $5,500,000 for the paclitaxel (P) and carbopl atin (C); P+C and bevacizumab (B); and P+C+B and maintenance B, respectively (Cohn, Kim et al. 2011). In the absence of a confirmed OS advantage, at l east for front-line therapy for ovarian cancer, the additional cost is hard to just ify (Hensley 2011; To mao, Tomao et al. 2013); particularly so without FDA approval. Similarly Grade ≥2 toxic effects including peripheral neuropathy (15.5), infection/fever (19.9% ), and dermatologic events (70.8%), are observed with maint enance therapy involving taxol (Mannel, Brady et al.). Despite a recurrence- free-interval delay in the SWOGS9761/GOG 178 study of 12 vs. 3 cycles of maintenanc e Taxol (22 vs. 14 months), long-term follow up revealed no survival advantage ( 53 vs. 48 months; p=0.34) (Markman, Liu et al. 2009). In addition, a Phase III study of maintenance Taxol x 6 cycles vs. observation revealed neither a progression-free survival nor overall survival advantage (Conte 2007). The at tendant significant morbidity and lack of survival Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 45 benefit qualified the National Comprehensive Cancer Network (NCCN) commentary on use of Taxol maintenance as 2B with the further proviso recommending use in a clinical trial. OBJECTIVES 3.0 Primary objective(s) 3.1  To assess time to recurrence (TTR) following the administration of bi- shRNAfurin and GMCSF autologous tumor cell (Vigil™) vaccine. Secondary objective(s) 3.2  To identify and determine the effect of Vigil™ autologous tumor cell vaccine on immune surrogate markers in this group of patients.  To assess the predictive potential of initial tumor infiltrating lymphocyte (TIL) and tumor associated macrophage (TAM) phenotypes.  To enlarge the safety database of Vigil™ autologous tumor cell vaccine in patients with miminal disease. STUDY DESIGN 4.0 Design 4.1 This is a Phase II open-label trial of ma intenance Vigil™ autologous tumor cell vaccine. Tumor will be harvested at the time of surgical debulking (standard of medical care). Subsequently, patients ac hieving clinical CR following primary surgical debulking and doublet chemotherapy will be stratified for i) surgical stage (Stage IV or suboptimal debulking (>1 cm residual) Stage III disease versus Stage III patients with optimal debul king (<1 cm residual)) and ii) post-op chemotherapy, pre-vaccine CA-125 >10 ≤ 20 U/mL versus 0 ≤10 U/ml, then randomized 2:1 (Note: patients with Stage IIIc ovarian cancer will be additionally evaluated as a subset using descriptive st atistics only). Patients will receive 1.0 x 10 7 cells / intradermal injection of gene transfected autologous tumor cells, Vigil™, once a month for up to 12 doses as long as sufficient material is available. Enough harvested tissue to provide a minimum of 4 monthly injections will be required for entry into the st udy. These patients will be managed in an outpatient setting. Hematologic func tion, liver enzymes, renal function and electrolytes will be monitored monthly. Immune function analysis including Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 46 ELISPOT analysis of cytotoxic T cell func tion to autologous tumor antigens will be monitored at ( ≤24 hours before) tissue harvest, ≤24 hours before the first cycle of chemotherapy (post debulking), ≤24 hours before the third cycle of chemotherapy (post debulking), baseline (screening), prior to Vigil™ injection Months 2, 4, 6 and at EOT. The dates of the last dose of chemotherapy and the administration of Vigil™ vaccine #1 will be recorded. Treatment will be continued until disease recurrence or exhaustion of the patient’s vaccine supply. If ≥ Grade 2 toxicity by NCI Common Toxicity Criteria (excluding Grade 2 fever ≤ 24 hours and Grade 2 and 3 injection site reactions) develops related to study treatment the vaccine dose will be reduced by 50% and continued on a monthly basis. If a single patient develops ≥ Grade 3 toxicity (other than injection site reaction) rela ted to study treatment the trial will be placed on hold for reevaluation of design in discussion with FDA. During this hold, no new subjects will initiate dosi ng, but subjects already being dosed may continue dosing as scheduled if deemed clinically appropriate by the PI. STUDY POPULATION 5.0 Sample Size 5.1 Approximately fifty (50) treatment naï ve patients with high risk Stage III/IV ovarian cancer who achieve clinical CR following surgical debulking and chemotherapy will be randomized in this trial. Inclusion Criteria 5.2 Patients will be eligible for tissue procurement for the Vigil™ vaccine manufacturing process if they meet all of the following critieria: 1. Presumptive Stage III/IV papillary serous or endometrioid ovarian cancer. 2. Per Amendment #8, treat ment naïve, high risk ovarian cancer will no longer be stratified, but the follow ing information will be collected: a. Stage IV or suboptimal (>1 cm residual) Stage III disease versus Stage III patients with optimal ( ≤1 cm residual) disease, b. CA-125 ≤10 U/ml versus CA-125 greater than 10 but less than or equal to 20 U/ml c. IP chemotherapy versus IV chemotherapy 3. Availability of “golf-ball” size 10-30 grams tissue at time of primary surgical debulking. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 47 4. ECOG performance status ( PS) 0-2 prior to tumor debulking laparotomy. 5. Ability to understand and the willi ngness to sign a written informed consent document for tissue harvest. Patients will be registered for inclusion in this study if they meet all of the following criteria: 1. Histologically confirmed Stage III /IV papillary serous or endometrioid ovarian cancer. 2. Clinically defined CR (no cancer related symptoms, normal physical examination and CT scan abdomen/ pelvis and CXR, and CA-125 ≤20 U/ml) following completion of prim ary surgical debulking. Patients enrolled must complete at least 5 but no more than 6 cycles platinum/taxane adjuvant or inte rval debulking + chemotherapy (or chemotherapy as per recommendations of NCCN guidelines, category 1 (IP chemotherapy included)). (Patients who complete surgery/chemotherapy with a CA-125 >20 U/mL pre-registration have the option of being followed up to 2 months if serial CA-125 values continue to decrease at a ra te of CA-125 decrease of 50% per month.) 3. Successful manufacturing of 4 vials of Vigil™ vaccine. 4. Recovered from all clinically relevant toxicities related to prior protocol specific therapies (including neuropathy to ≤Grade 2). 5. ECOG performance status (PS) 0-1. 6. Normal organ and marrow function as defined below: Absolute granulocyte count ≥ 1,500/mm 3 Absolute lymphocyte count ≥ 200/mm3 Platelets ≥ 75,000/mm3 Total bilirubin ≤ 2 mg/dL AST(SGOT)/ALT(SGPT) ≤ 2x institutional upper limit of normal Creatinine < 1.5 mg/dL 7. Patients must be off all “statin” drugs for ≥ 2 weeks prior to initiation of therapy. 8. Ability to understand and the willi ngness to sign a written informed protocol specific consent document. Exclusion Criteria 5.3 Patients will be excluded from this study if they meet an y of the following criteria: Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 48 1. Surgery involving general anesthes ia, radiotherapy, or immunotherapy within 4 weeks prior to randomizati on. Chemotherapy within 3 weeks prior to Vigil™ vaccine administrati on. Steroid therapy within 1 week prior to vaccine administration. 2. Patients must not have received any other investigational agents within 4 weeks prior to Vigil™ vaccine administration. 3. Patients with history of brain metastases. 4. Patients with compromised pulmonary disease. 5. Short term (<30 days) c oncurrent systemic steroids ≤ 0.25 mg/kg prednisone per day (maximum 7. 5 mg/day) and bronchodilators (inhaled steroids) are permitted; patients requiring other steroid regimens and/or immunosuppressives at randomization are excluded. 6. Prior splenectomy. 7. Prior malignancy (excluding nonmelanoma carcinomas of the skin and carcinoma in situ cervix) unless in remission for ≥ 2 years. 8. Kaposi’s Sarcoma. 9. Uncontrolled intercurrent illness including, but not limited to ongoing or active infection, symptomatic conges tive heart failur e, unstable angina pectoris, cardiac arrhythmia, or psychia tric illness/social situations that would limit compliance wi th study requirements. 10. Patients with known HIV. 11. Patients with chronic Hepatitis B and C infection. 12. Patients with uncontrolled autoimmune diseases. Withdrawal 5.4 Off Study Treatment Patients will be taken off study treatment if any of the following occur: 1. The patient experiences unacceptable ( ≥Grade 3) toxicity felt to be related to treatment with the tumor cell vaccination that persists for >1 week. 2. Persisting Grade 3 or 4 toxicity unrelated to treatment, defined as failing to normalize within 4 weeks. 3. ≥ Grade 2 allergic reactions related to the study agent. 4. Grade 2 autoimmune reactions unless there is evidence of clinical benefit. 5. ≥ Grade 3 autoimmune reactions. 6. An intercurrent illness, which would in the judgment of the investigator, affects assessments of clinical st atus to a significant degree or requires discontinuation of study treatment. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 49 7. Non-protocol therapy (including, but not limited to, chemotherapy not listed as Level 1 Category of Evidence per NCCN Guidelines Version 3.2012, radiotherapy, hormonal therapy , immunotherapy, or surgery) is administered during study treatment. 8. Non-compliant with protocol or treatment. 9. Patient refuses to continue treatment. The date of and reason for discontinuati on must be noted in the Case Report Form (CRF). Every effort should be made to complete the appropriate assessments. Stopping Rules Patients with disease recurrence will not receive further vaccine treatment. Withdrawal of patients from the study treatment will be in the event of unexpected and unacceptable vaccine-related toxicity, ≥ Grade 2 allergic reactions, Grade 2 autoimmune reactions un less there is evidence of clinical benefit, ≥ Grade 3 autoimmune reactions related to the study agent, or clinically significant disease progression. INVESTIGATIONAL PLAN 6.0 Patient Registration and Enrollment 6.1 Written documentation of fu ll, non-contingent IRB approval of the protocol and consent document must be on file before a patient can be registered. Patients will be assigned a tissue procurem ent number upon harvest and a vaccine number / patient number will be assigne d upon completion of the manufacturing process. Once eligibility has been confirmed, t he site Research Nurse or Clinical Research Coordinator will email or fa x the Registration Form and applicable de- identified records to MCCRO@marycrowley.org or 214-658-1990. If sent by fax, and email should be sent to the above em ail address to aler t the CRO of the incoming fax. The received documents will be reviewed for completeness and sent to the Medical Monitor for review and randomization (if applicable ). If records are not complete or if question arise, the site Research Nurse or Clinical Research Coordinator will be contact ed for more information. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 50 Please allow 48 hours for patient registrati on / randomization. Once confirmed, the completed Registration Form will be returned to the site indicating the assigned patient cohort. Tumor Procurement 6.2 The investigative sites will ensure pati ents are scheduled for surgery for tumor procurement. Gradalis, Inc. will manufac ture vaccine from the procured tissue, and thus will need to be notified at least 24 hours before the scheduled procurement in order to prepare for vaccine manufacturing (when possible). Additionally, tumors are to be procured on Mondays, Tuesdays, Wednesdays and Thursdays only due to the manufacturing being a 2-day process. Please contact the Sponsor if scheduling adjus tments need to be made. Refer to the Tissue Procurement Study Refer ence Manual for instructions. The equivalent of a “golf ball size” mass (10-30 gm tissue) and/or ascites fluid (preferably first paracentesis) is necessary for vaccine manufacturing. Radiological scans should confirm the pr esence of a lesion >3cm. If surgeons have the option of collecting more tissue, more doses of vaccine may be prepared (up to 12 doses). Vaccine manufac turing is rarely successful with small tumor masses. Lesions extending into bowel lumen cannot be processed. Once the procured tissue is received at Gr adalis, Inc. samples will be processed for autologous vaccine manufacture. No tissue or vaccine will be given to the participant or site apart from t he outlined clinical protocol. If available without compromising vaccine manufacture or pathology requirements, an aliquot of tumor will be collected for TIL and TAM immunophenotyping. Any excess tu mor tissue, not used for vaccine manufacture will be used towards Vigil™ vaccine research and process development assays. Vaccine Manufacturing 6.3 Gradalis, Inc. will manufacture vacci ne from the procured patient tumors. Gradalis, Inc. will release vaccine once all release criteria have been met and eligibility has been confirmed. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 51 Study Treatment Administration 6.4 Treatment will be admin istered on an outpatient basis. The site must contact the Mary Crowley CRO to obtain parti cipant randomization assignment. Recommended Chemotherapy Patients will receive 6 cycles of chemot herapy as either adjuvant or interval debulking + chemotherapyschedule. Recommended regimens are those classified as Level 1 Category of Evi dence per NCCN Guidelines Version 3.2012. Regimens other than these should be discussed with the sponsor. Patients will not receive maintenance therapy other than that specified by protocol. Vaccination Schedule and Dose Patients will be vaccinated according to the schedule outlined in Appendix B. Patients will receive Vigil™ vaccine at 1 x 107 cells via intradermal injection for a minimum of 4 doses and a ma ximum of 12 doses starting ≥3 weeks following completion of chemotherapy (no longer than 2.5 months post chemotherapy). Patients with viable cells in sufficient num bers (i.e. at least 4 doses of 1.0 x 107 cells / injection) will receive monthly intradermal injections of the tumor cell vaccine for up to 12 months as long as suffici ent material is available. Patients whose vaccine manufacturing fails to achieve a minimum of 4 doses will not undergo treatment. Administration of Vigil™ vaccine The sites of injection will be rotated bet ween the right and left upper arms. If the ipsilateral axillary lymph nodes were radiated or surgically removed during prior therapy, alternative sites (e.g., anterior th igh) will be used. The patient will be observed for at least 30 minutes (wit h a 10% window) following vaccination. During this observation period, vital signs will be taken every 10 minutes (with a 10% window). If clinically stable, vacci ne administration may continue for up to 12 doses given on a monthly basis as long as sufficient material is available. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 52 Vigil™ Vaccine Transfer All manufactured vaccine wi ll be stored in the vapor phase of liquid nitrogen until ready for use. The site will contact Gradalis, Inc. when the study agent is needed for patient vaccine administration. Gradalis will complete a Drug Transfer and Administration Form to release the patient vaccine. The clinic will si gn off on the form upon receipt of the vaccination. Please reference the Pharmacy Reference Manual for vaccine preparation and handling information. Treatment Delay 1. Treatment may be delayed no more than 4 weeks to allow recovery from toxicity. 2. Patients who delay treatment for mo re than 4 weeks due to toxicities will be considered off study treatment (see Section 4.4 Off Study Treatment). 3. Treatment delay not related to to xic events (including subjects unable to adhere to monthly injection) will not be delayed for more than three days. Unless a delay due to symptoms related to disease or infection in which case up to a 2 week delay is allowed. 4. If ≥ one 2 week delay due to disease or infection occurs, patient status must be reviewed by sponsor. 5. If patients miss doses due to toxici ties, the doses will be made up the following week and continue on a revi sed monthly schedule thereafter. Dose Modification for Toxicity If ≥ Grade 2 toxicity by NCI Common Toxici ty Criteria (excluding Grades 2 and 3 injection site reactions) develops related to study treatment the vaccine dose will be reduced by 50% and continued on a m onthly basis. If a single patient develops ≥ Grade 3 toxicity (other than site r eaction) related to study treatment the trial will be placed on hold for re-evaluation of design in discussion with FDA. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 53 Toxicity 6.5 Toxicities will be graded and reported a ccording to the NCI Common Toxicity Criteria for Adverse Events (CTCAE) Vers ion 3.0 as linked in Appendix C. This document can also be downloaded from the Cancer Therapy Evaluation Program (CTEP) home page < http://ctep.info.nih.gov >. Should a report of an infection (any grade) arise, please identify the bacteria or fungus related and report the event via Serious Adverse Event form. In particular, if any organisms are identified as anaerobic, infectious disease should be consulted for management using standard of care relevant to anaerobic bacterium. Schedule of Assessments 6.6 The schedule of assessments fo r the trial is shown in Appendix B. If a required observation or procedure is missed, docum entation is required on the Protocol Deviation Form, to explain the re ason for this protocol deviation. Prestudy Assessments The following evaluations will be perfo rmed within 4 weeks of the tumor debulking laparotomy (unless otherwise specified): 1. A signed Patient Informed Consent Form for tissue harvest must be obtained. 2. It has been confirmed that the pat ient meets all ti ssue procurement inclusion criteria and none of the exclusion criteria. 3. A complete medical hi story must be obtained. 4. A physical examinatio n (including vital signs, height, and body weight) must be obtained. 5. Assessment of PS on the ECOG scale (see Appendix A) must be obtained. 6. Radiological assessment of tumors (i.e., chest X-ray, (chest CT or MRI only if indicated), pelvic/abdominal CT or MRI) used to stage the extent of disease must be performed within 6 weeks. The methods used for prestudy assessments (e.g., CT or MRI) should be used throughout the study. If possible, the same equipment should be used each time. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 54 7. A complete blood count (CBC) with differential and platelet count must be performed. (HIV testing is not required if the subject has no medical history of HIV). 8. CA-125 tumor assessment (at init ial diagnosis per medical history). 9. Routine pre-operative serum chemis tries (including but not limited to creatinine, total bilirubin, alkaline phosphatase, and aspartate transaminase (AST) and/or alanine transaminase (ALT). 10. Immune function analysis consisting of ELISPOT analysis of cytotoxic T cell reaction to autologous tumor antigens will be obtained at tissue harvest. (within 24 hour s before tissue procurem ent or on the day of harvest) Following tissue procurement, patients will be contacted every 6 weeks  2 weeks to assess interest / eligibility of th e main randomized portion of the trial and survival status. Blood for immune function analysis will be collected prior to the first and third cycles of ( ≤24 hours before) chemotherapy post debulking. Medical records should be provided to support continued eligibility. The following evaluations will be perform ed on all subjects within 2 weeks of registration / randomization ( unless otherwise specified): 1. A signed protocol specific Patient Informed Consent Form must be obtained. 2. It has been confirmed that the patient meets all inclusion criteria and none of the exclusion criteria. 3. An interval medical history must be obtained within 4 weeks. 4. A physical examination (including vital signs, ox ygen saturation, height, temperature and body weight) must be obtained. 5. Assessment of concomitant medications 6. Assessment of PS on the ECOG scale (see Appendix A) must be obtained. 7. A tumor clinical assessment of th e patient’s disease (i.e., by physical examination) must be per formed within 4 weeks. 8. Radiological assessment of disease status (i.e., chest X-ray, (chest CT or MRI only if indicated), pelvic/abdom inal CT or MRI) used to stage the extent of disease must be perf ormed within 4 weeks. The methods used for prestudy assessments (e.g., CT or MRI) should be used throughout the study. If possible, the same equipment should be used each time. 9. A complete blood count (CBC) with differential and platelet count must be performed. (HIV testing is not required if the subject has no medical history of HIV). 10. CA-125 tumor assessment withi n 4 weeks following completion of surgery chemotherapy. If the CA- 125 is >20 U/mL, the patient can continue with serial CA-125 evaluations up to 2 months if the levels Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 55 continue to decrease and there is no objective evidence of disease recurrence. 11. Serum chemistries (creatinine, glucose, total protein, blood urea nitrogen (BUN), total carbon dioxide (CO 2), albumin, total bilirubin, alkaline phosphatase, and aspartate transaminase (AST) and/or alanine transaminase (ALT)) and electrol ytes (total calcium, chloride, potassium, sodium) must be performed. 12. Immune function analysis consisting of ELISPOT analysis of cytotoxic T cell reaction to autologous tumor antigens. 13. Urinalysis must be performed. 14. EKG must be performed. 15. FACT-O, Version 4 assessment Assessments During Treatment The following evaluations will be perform ed monthly (every 28 days ±3 days) during the therapy (unle ss otherwise specified): 1. A physical examinat ion, including vital signs and body weight. 2. A toxicity assessm ent (adverse events). 3. Assessment of concomitant medications taken. 4. Tumor response by clinical assess ment of the patient’s disease (i.e., by physical examination). 5. Radiological assessment of disease status (i.e., chest X-ray, (chest CT or MRI only if indicated), pelvi c/abdominal CT or MRI) must be performed ≤ 1 week prior to Cycle 4) and then at standard of care intervals. The methods used for pr estudy assessments (e.g., CT or MRI) should be used throughout the study. If possible, the same equipment should be used each time. 6. A CBC with differential and platelet count. 7. CA-125 tumor assessment until recurr ence. (monthly for the first year, then every 3 months ± 2 weeks for the second and third year.) 8. Serum chemistry and electrolytes. 9. Immune function analysis consisting of effector T cell response (ELISPOT assay will be monitored - M onths 2, 4, 6 (prior to Vigil™ administration). 10. Assessment of PS on the EC OG scale (see Appendix A). 11. Vaccine administration. The patient will be observed for at least 30 minutes (with a 10% window) follo wing vaccination. During this observation period, vital signs will be taken every 10 minutes (with a 10% window). 12. Day 2 assessment of injection site. 13. FACT-O, Version 4 assessment at baseline, Month 3, Month 6, Month 12 and Month 18 (or until recurrence). Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 56 Radiological assessment of disease status (i.e., chest X-ray, (chest CT or MRI only if indicated), pelvic/abdominal CT or MRI) must be collected if CA-125 >35 U/mL after two consecutive meas urements taken one month apart. Off Treatment Assessments The following evaluations will be perform ed within 30 days following the last Vigil™ administration and within 30 days of disease recurrence (unless otherwise specified): 1. A physical examinat ion, including vital signs and body weight. 2. Toxicity assessment (adverse ev ents) (Vigil™ administered patients only). 3. Assessment of concomitant medications taken. 4. Assessment of PS on the EC OG scale (see Appendix A). 5. A tumor clinical assessment of th e patient’s disease (i.e., by physical examination). 6. Radiological assessment of disease status (i.e., chest X-ray, (chest CT or MRI only if indicated), pelvic/abdom inal CT or MRI) used to stage the extent of disease. (within 45 da ys of the last injection or disease recurrence) 7. A CBC with differential and platelet count. 8. CA-125 tumor assessment. 9. Serum chemistry and electrolytes. 10. Serum for immune function analysis consisting of ELISPOT analysis of cytotoxic T cell function to autologous tumor antigens. Follow Up Assessments If the Vigil™ vaccine is discontinued (for reasons such as completion of all available doses of vaccine, intolerable to xicity, treatment interruption of more than 4 weeks, intercurrent illness, protocol deviation at PI’s discretion), the patient will be contacted by phone every 3 months after the end of study visit for:  disease status  request of medical records, when applic able (laboratory findings, radiological scans, and progress report)  documentation of 2 nd line therapy  recording of response and duratio n of response to that therapy  survival  assessment of any additi onal cancer treatments) Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 57 Note: Patients who die or withdraw consent are considered off study and no further information except survival data will be collected. Based on findings during the study or duri ng the follow up portion of the trial, Gradalis may request for additional bloo d and / or tissue samples from the research participant. Collection of whole blood (40ml) and / or tissue samples (via biopsy or clinically indica ted surgical removal) will be optional and used to study the effects of the st udy agent (included, but not limited to testing of biomarkers, predictors or biological re sponses, toxicity, relationship between genotype and study agent responses). Should Gradalis request for addi tional blood or tissue, the c linical site will present the option of the procur ement to the participant and obtain written informed consent. CONDUCT OF THE STUDY 7.0 Ethics and Regulatory Considerations 7.1 This study must have the approval of a pr operly constituted Institutional Review Board (IRB) or Independent Ethics Commi ttee (IEC), and the Institutional Biosafety Committee (IBC). Before the investigational drug is shipped to the investigator, the investigator will provide Gradalis, Inc. with a copy of the IRB or IEC and IBC approval letter stating that the study protocol and informed consent form have been reviewed and approved. IRB and IBC 7.2 This trial can be undertaken only after revi ew and full approval of the protocol and a Patient Informed Consent Form has been obtained from a properly constituted IRB. This written approval must be dated and it must clearly identify the protocol, any amendments, the Pa tient Informed Consent Form, and any applicable recruiting materials and subject compensation programs approved. Since this trial will also be reviewed by a properly constituted IBC, this trial will not be undertaken until the protocol has been reviewed and received full approval from a properly constituted IBC. This written approval must be dated and it must clearly identify t he protocol and any amendments. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 58 The decision concerning the conduct of the study will be made in writing to the sponsor. Copies of this decision and of all IRB and IBC correspondence will be kept on file at the study site; copies will be provided to the Mary Crowley Research Office. During the trial, the PI is required to send various documents to the IRB and IBC for review: 1. All protocol amendments and Pa tient Informed Consent Form revisions. 2. Reports of all Serious Adverse Events. The PI provides Gradalis, Inc. with the necessary assurance that an IRB and IBC is responsible for the initial and cont inuing review and approval of the proposed clinical study in accordance with 21 CFR 312.60. At least once a year, the IRB and IBC will be asked to review and re- approve the clinical trial protocol; the request must be documented in writing. At the end of the trial, the PI will notify the IRB and IBC that the trial has been completed. Written Informed Consent 7.3 The informed consent document should meet the requirements of the latest version of the Declaration of Hels inki and any applicable regulations and guidelines. It must be approved by an IRB or IEC. Prior to entry into the trial and befor e any protocol-required procedures are performed, the Investigator must explai n the nature of the trial, its intended purpose, and the implications of participati on to potential patients or to their legal representatives. They will be told about the possible risks and benefits, and the possible adverse experiences. They will be informed that patients’ participation is voluntary, and that they may withdraw consent to participate at any time. They will also be informed that if patients choose not to participate in the trial alternative treatment s are available; such refusa l will not prejudice further treatment of their disease. Potential pat ients or their legal representatives must be given the opportunity to ask questions about the trial protocol and the procedures involved. Finally, each patient will be told that his or her records may be accessed by authorized personnel of Mary Crowley, Gradalis, Inc. and other authorized individuals without violating the patient’s confidentiality, to the extent permitted by the applicable laws and/or regulations. By signing the written Patient Informed Consent Form, the patient or his or her legal represent ative is authorizing such access. Following this explanation and prior to entry into the trial, the written, Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 59 dated, and signed Patient Informed Consent Form must be obtained from each patient or his or her legal representati ve; a copy will be given to the person signing the form. Confidentiality of Records 7.4 The Investigator is required to reta in, in a confidential manner, sufficient information on each patient (i.e., full nam e, current address, and social security number) so that the patient may be contac ted by the FDA, Gradalis, Inc., or by Mary Crowley should the need arise. Modification of Protocol 7.5 Any changes to this protocol that affe ct study objectives, study design, study procedures, patient population, or signifi cant administrative procedures will require a formal amendment to the protocol. Any proposed protocol amendments must be sent in writing to the applicable IRB. Prior to implementation, an amendm ent must be approved by the Gradalis, Inc., and approved by the applicable IRB or IEC and IBC. General administrative changes to the protocol are minor corrections and/or clarifications that do not affect the manner in which the study is to be conducted. Such administrative changes will be agr eed upon by the Gradalis, Inc., and will be documented in a memorandum. The applicable IRB or IEC and IBC will be notified of administrative changes according to applicable IRB guidelines. Protocol Questions and Deviations 7.6 When evaluating a potential patient or while a patient is on study, protocol questions can be sent to MCCRO@marycrowley.org. Mary Crowley CRO will work with Gradalis in obtaining an answer / clarification. Please allow 24 hours for a response. For urgent questions affe cting patient safety, please contact the Gradalis personnel by the phone provided in the Gradalis Contact Information section of the Study Reference Manual. For Planned Protocol Deviation requests, th e site will complete the Eligibility Inquiry/Planned Protocol Deviation Form and email or fax it to MCCRO@marycrowley.org or 214-658-1990. If sent by fax, an email should be sent to the above email address to alert Ma ry Crowley of the fax. Mary Crowley CRO will work with Gradalis and t he Medical Monitor to review the Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 60 request. Please allow 48 – 72 hours for review . Once complete, the form will be returned to the site. EVALUATION OF TUMORS 8.0 Disease Evaluation 8.1 Disease recurrence will be evaluated in this study using the Response Evaluation Criteria in Solid Tumors Version 1.1 (R ECIST) (unidimensi onal measurement) of the tumor lesions are used in the RECIST criteria (Eisenhauer, Therasse et al. 2009). Disease recurrence is defined as the appearance of any measurable or evaluable lesion or as asymptomatic CA-1 25 levels greater than 35 U/mL at two consecutive measurements, at least one m onth apart. The time to recurrence is measured from date of first treatment unt il the first date that recurrence is objectively documented whether local, regional, or distant. DRUG INFORMATION 9.0 Investigational Product 9.1 The Vigil™ vaccine is made up of irradi ated autologous tumor cells which have been electroporated ex vivo with the Vigil™ plasmid designed to suppress expression of both the TGF β1 and TGF β2 proteins while simultaneously expressing rhGMCSF protein. Vigil™ Vaccine Production The Vigil™ Phase II ovarian vaccine cGMP manufacturing process is identical to prior Vigil™ vaccine cGMP manufact uring (BB-IND 14205) (Maples 2010). Surgically excised tumor is collected in the surgical field and placed in sterile saline and packaged for transport to the m anufacturing facility. The tumor is mechanically and enzymatically dissociated into a single cell suspension. The cells are counted and then transfected with the FANG™ plasmid. The cells are incubated overnight to allow transcription of the FANG™ plasmid. The following morning the cells are harvested, washed, and then irradiated at 10,000cGy in a standard Blood Bank irradiator. The i rradiated cell suspension is then enumerated, aliquoted and frozen (1 x 107 cells). The freeze media consists of Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 61 10% DMSO (dimethyl sulfoxide; Cryo serv USP; Bionichepharma US), 1% Human Serum Albumin (ABO Pharmaceuticals) in Plasma-Lyte A at pH 7.4 (Baxter). After freezing the cells are stored in the vapor phase of liquid nitrogen until all release testing is complet ed, all necessary approvals are obtained and the patient is ready for treatment. Safety Analysis 9.2 The FANG™ plasmid employed in the gener ation of this autologous cell vaccine has been tested for identity, ster ility, purity and strength. Irradiated Gene Modified Tumor Cells To ensure safety, all gene-modified tumor ce lls to be used in patient vaccinations must be irradiated 10,000 cGy prior to freez ing. This is the same irradiation process as for the TAG vaccine, BB-IND 13650 and prior vaccines (Belagenpumatucel-L and GVAX® publishe d trial results and BB-IND 13401 and BB-IND 12118) (Kumar 2009; Maples PB 2 009; Maples PB 2009). The selection of this radiation dose is based on the desire to utilize the lowest possible radiation dose for the transfected cell s to optimize the level and duration of bifunctional shRNAfurin transcription and GMCSF protein production and maximize the safety of vaccine cell inje ctions at the same time. In addition, investigators have demonstrated that irradi ating cultured tumor cells of different histologic origins at 10,000 cGy complete ly arrests tumor colony formation. Preparation Reference the Study Reference Manual for vaccine prepar ation and handling information. Drug concentrate: 1.0 x 10 7cells per injection in a volume of 1mL. Route of administration: Intradermal injection Storage Frozen, unopened vials are stored in the vapor phase of Liquid Nitrogen. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 62 Administration of vaccine Patients with viable cells in suffi cient numbers for at least 1.0 x 107 cells / injection for 4 doses will receive monthly intradermal injections of the tumor cell vaccine (as assigned cohort and available cell yield following tumor harvest and processing) as long as sufficient material is available. Patients who fail to achieve successful manufacturing of a minimum of 4 doses will not undergo treatment. The sites of in jection will be rotated bet ween the right and left upper arms. If the ipsilateral axillary lymph nodes were radiated or surgically resected during prior therapy, alternative sites (e.g., anterior thigh) will be used. The patient will be observed for at least 30 minutes (with a 10% window) following vaccination. During this observation peri od, vital signs will be taken every 10 minutes (with a 10% window) . If clinically stable vaccine administration may continue for up to 12 doses given on a monthly basis as long as sufficient material is available. Concomitant Therapy 9.3 All concomitant treatments, including blood and blood products, must be reported on the source documentation. The follow ing concomitant medications must be documented on the Con Meds page of the CRF:  Prophylactic antibiotics  Antifungals  Premedications  Antinauseants, antipyretics Short term (<30 days) conc urrent systemic steroids ≤ 0.25 mg/kg prednisone per day (maximum 7.5 mg/day) and bronchodila tors (inhaled steroids) are permitted; other steroid regimens and/or immunosuppressives are excluded. OCCUPATIONAL SAFETY 10.0 Study medications are not expected to pos e significant occupational safety risks to investigational staff under normal c onditions of use and administration. However, precautions should be taken to avoid direct contact with study medication. Biosafety Level 2 practi ces shall be employed with this study medication. Reference the Study Reference Manual. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 63 ADVERSE EVENTS 11.0 Adverse Event and Serious Adverse Event Definitions 11.1 Adverse Event An AE is any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and that does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally asso ciated with the use of a medicinal (investigational) product, whether or not re lated to the medicinal (investigational) product. Serious Adverse Event An AE (experience) or reaction occurring at any dose should be classified as a serious adverse event (SAE) if any of the following occur:  Initial or prolong ed hospitalization  A life-threatening condition (i.e. an event in which the patient was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it was more severe)  Significant disability/incapacity (i.e. t he AE resulted in a substantial disruption of the subject’s ability to carry out normal life functions)  Congenital anomaly/birth defect  It does not meet any of the above seri ous criteria, but may jeopardize the subject or may require surgical or m edical intervention to prevent one of the outcomes listed above.  Death Unexpected Adverse Event An unexpected event is any AE that is not identified in nature, severity or frequency in the Clinical Investigator’s brochure or the drug package insert. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 64 Grading Adverse Events Adverse events (AEs) will be recorded th roughout the trial. Toxicities and AEs will be graded and reported using the Co mmon Toxicity Criteria for Adverse Events (CTCAE) Version 3.0 as linked in Appendix C. All AEs, regardless of severity, will be followed by the Treating Physician until resolution is satisfactory. Attribution of Causality 11.2 The relationship of each event to treat ment will be assessed by the Treating Physician and recorded on the CRF. Expected Side Effects 11.3 Tumor cell vaccines have been previously ad ministered to patients with cancer. Side effects were minimal, the most frequent of which included local reactions at the site of injection. Potential adverse events are listed below. Local skin reactions at th e site of injection: Erythema, tenderness, induration, urticaria/rash, pruritus. Other expected adverse events: Fever, myalgias/arthralagias, chil ls/rigors, nausea, fatigue, headache, thrombocytopenia and other cytopenias, hyperglycemia, vomiting, hypotension, infection at the immunization site. In addition there may also be a ri sk of autoimmune disease development, although to date no evidence of this has been seen in any vaccination study. There may also be worsening of tumor related symptoms secondary to immune mediated attack on patient’s tumor. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 65 Recording of an Adverse Event 11.4 Adverse events will be recorded for the duration of a patient’s study treatment, and for up to 30 days following the last st udy treatment. All AEs, regardless of causal relationship are to be recorded in the CRF and source documentation. Additional information about each event, such as treatment required, eventual outcome, and whether or not therapy had to be interrupted or dosages reduced, will also be recorded on the CRF. Pre-existing conditions will be recorded at baseline on the Medical History Form. If a pre-existing condition does not change, it does not have to be reported as an AE on subsequent cycles. Serious Adverse Event Reporting 11.5 All SAEs will be reported by email or fa csimile upon becoming aware of the event within 24 hours to Gradalis, Inc. This includes any death from any cause while a patient is receiving treatment (Vigil™) on this protocol, or  30 days following the last dose of protocol treatment (Vigil™). The site will supply as much information as is available at the time of the initial facsimile (study number, patient initia ls, patient study number, onset date, relationship, patient demographics, event, dosing regimen of study agent) to: Gradalis, Inc. 2545 Golden Bear Drive, Suite 110 Carrollton, TX 75006 Vigil@gradalisinc.com Direct: (214) 442-8124 Fax: (214) 442-8101 Gradalis, Inc. will report adverse events to the FDA in compliance with 21 CFR 312.32. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 66 PATIENT COMPLETION AND WITHDRAWAL 12.0 Indication for Taking Patients Off Study 12.1 The Investigator must notify the sponsor at any time following discontinuation of a patient on study for the occurrence of a serious or unexpected AE associated with the use of the study medication. DATA EVALUATION 13.0 Statistics and Estimated Sample Size 13.1 The primary objective of this study is to investigate whether maintenance Vigil™ vaccine after a clinically defined complete response to induction platinum/paclitaxel chemotherapy results in prolonge d time to recurrence (TTR) compared with standard of care observa tion. TTR was used for the purpose of sample-size estimation. Based on prio r published experience (Markman, Liu et al. 2003), the median RFS after a clinical complete response to induction therapy for the control arm was estimated to be approximately 16 months for those with stage IV or suboptimal (> 1 cm residua l) stage III disease and 24 months for stage III patients with optimal (< 1 cm residual) disease. Given an average RFS of 24 and 16 months, respectively, a 10% increase in RFS (i.e. TTR) would provide adequate statistical power to s upport expanding the study (Table 6). This analysis was performed using a statistical power calculator (http://www.dssresearch.com/KnowledgeCenter /toolkitcalculators/statisticalpower calculators.aspx ) to conduct a 1-tail test of a sample average in comparison to the historical value, a standard deviation of 1.0, and an alpha error level of 5%. In order to expand the protocol treatment base and increase the rate of accrual while maintaining the objectives of the protocol, the study, which was initially limited to patients with stage IIIc ovarian cancer, is being expanded to patients with Stage III/IV disease. In the SEER database: 1988-2001(Kosary 1988-2001), women with Stage IIIc diseas e comprised 19.4% of the ovarian cancer population; those with Stage III inclusive, 35.3%; and those with Stage IV, 31.7%. Thus, the eligibility expansion increases the base catchment population from 19.4% of women with ovarian cancer to 67% of which only a subset will meet protocol criteria. In the completed Phase I study of Vigil™ vaccine in patients with solid tumors experiencing progressive disease followi ng prior therapy, 50% of patients with serial assessments (n=18) demonstrated conversion from γIFN ELISPOT Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 67 negative to positive by Month 4 (Senzer N 2012). The percent γIFN ELISPOT conversions and the timing thereof will be recorded in the current patient population of patients with minimal or no residual disease using descriptive statistics to assess the hypothesis that there is an inverse relationship between tumor volume and immune responsiveness based in part on the quantitative relationship between tumor volume and t he production of immunosuppressive factors. Although all patients will be evaluatat ed for RFS, subgroup analyses will be separately perfomed on patients with negative ELISPOT at baseline and those with positive ELISPOT at baseline. Table 6: Statistical Power of 2:1 Random ization Trial per Surgical Stage Stratification Group (1 tailed t test) Surgical Stage Stratification Vigil™ Vaccine (TTR in months)Observation Control (TTR in months) Power n Stage III patients with optimal (< 1 cm residual) 26.4 24 1.0 30 Stage IV or suboptimal (> 1 cm residual) stage III disease 17.6 16 1.0 30 Exploratory analysis of two Phase III studies (D9901 and D9902) with the recently FDA approved Provenge documentated prolonged survival benefits in Provenge-treated prostate cancer patient s (25.9 months vs. 21.4 months and 25.8 vs. 21.7 months, respectively). S upporting our clinical objective, patients who subsequently received docetaxel experienced a median survival of 34.5 months compared to 25.4 m onths in patients treated with placebo followed by docetaxel. Descriptive statistics will be employed to format the response rates and response durations to 2 nd line therapy in patient s that relapse in both FANG™ and observational groups. Definition of Evaluable Patients 13.2 All measurements should be taken and recor ded in metric notation using a ruler or calipers. All baseline ev aluations will be performed as closely as possible to the beginning of treatment and never more than 2 weeks before the beginning of the treatment. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 68 The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination when both methods have been used to assess the antitumor effect of treatment. Clinical Lesions: Clinical lesions will only be considered measurable when they are superficial (e.g., skin nodules, pal pable lymph nodes). In the case of skin lesions, documentation by color photograph y including a ruler to estimate the size of the lesion is recommended. Chest X-rays: Lesions on chest X-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable. Conventional CT and MRI: These techniques should be performed with cuts of 10mm or less in slice thickness contiguously. Spiral CT: Spiral CT should be performed using a 5mm contiguous reconstruction algorithm. Ultrasound: When the primary endpoint of the study is objective response evaluation, ultrasound should not be used to measure tumor lesions. It is, however, a possible alternative to clinical measurements of superficial palpable lymph nodes, subcutaneous lesions, and thyroid nodules. Ultrasound may also be useful to confirm the complete disappearance of superficial lesions usually assessed by clinical examination. Time to Recurrence of Disease (TTR) 13.3 Time to recurrence will be measured from the treatment start date (date of first dose) to either the date the patient is first recorded as having disease recurrence (even if the patient went off treatment because of toxicity ), or the date of death if the patient dies due to any causes before recurrence. Time to Treatment Failure 13.4 Time to treatment failure will be meas ured as the date from when the patient started treatment (fir st dose) to the date the patient is withdrawn due to: AE(s), progressive disease/insufficient therapeutic response, death, failure to return, refused treatment/did not cooperate/withdrew consent, or started a new Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 69 treatment. Patients who do not fail treatment will be cens ored at the date of last follow-up. Survival 13.5 Survival will be measured, as the time from start of treatment to the date of death or the last date the patient was known to be alive. Long-term survival informat ion will be collected. STUDY RECORDS 14.0 Documentation 14.1 A log of all patients evaluated for this protocol must be maintained at each site. Patients excluded from admission will be pr ovided with a clear explanation of the specific reasons why they have been excl uded from the study. Patients who are included will be assigned a pat ient identification number. For each patient treated with the study drug(s), the Research Coordinator is required to prepare and maintain case hi stories that include all observations and other data pertinent to the in vestigation. This will include all source documents needed to verify the accuracy of all obser vations and other data contained in the CRFs on each study patient. The Investigator or his/her designee is r equired to retain the records related to the trial for a period of 2 years follow ing the date a marketing application is approved for the indication being investigated. If no application is to be filed or if the application is not approved for such indication, the records must be retained until 2 years after the investigation is discontinued and the regulatory agencies are notified. The Investigator shall retain study drug disposition records and source documents for the maximum period require d by the country and institution in which the study will be conducted, or fo r the period specified by Gradalis, which ever is longer. The Investigator must contact Gradalis, Inc. prior to destroying any records associated with the study. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 70 If the Investigator withdraws from the st udy (e.g., relocation, retirement), the records shall be transferred to a mut ually agreed upon designee (e.g., another investigator, IRB). Notice of such transfe r will be given in writing to Gradalis, Inc. Case Report Form (CRF) Procedures 14.2 CRFs will be supplied by Gradalis, Inc. for recording all data from each patient. CRFs must be typewritten or printed l egibly using black ballpoint pen or completed electronically. The investigat or or his/her designee is responsible for recording all data relating to the trial on t he CRFs. The investigator must verify that all data entries on the CRFs are accurate and correct. If an item is not available or is not appl icable, it should be documented as such; no blank spaces should be left on a CRF. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 71 APPENDIX A ECOG Performance Status Scale ECOG Performance Status Scale Grade Description 0 Normal activity. Fully active, able to carry on all pre-disease performance without restriction. 1 Symptoms, but ambulatory. Restricted in physically strenuous activity, but ambulatory and able to carry out work of light or sedentary nature—(e.g., light housework or office work). 2 In bed <50% of the time. Ambulatory and capable of all self care, but unable to carry out any work activities; up and about > 50% of waking hours. 3 In bed >50% of the time. Capable only of limited self-care, confined to bed or chair >50% of waking hours. 4 100% bedridden. Completely disabled; cannot carry out any self care; totally confined to bed chair. 5 Dead Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 APPENDIX B Schedule Of Assessments Procedure Prestudy Screening Monthly (q 28±3 days) End of TX Follow-Up Informed consent X X Phone Contact X1 Medical History X Interval Medical History within 4 weeks Physical Examination X X X X Toxicity (adverse events) X X Concomitant medications X X X Performance Status X X X X Clinical Tumor Assessment X X X Radiological Tumor Assessment (abdomen/pelvis)2 within 6 weeks within 4 weeks X CBC with differential X X X X HIV testing, if applicable X CA-125 tumor assessment3 X X X X Serum Chemistry X4 X X X Immune Function Analysis At tissue procurement and prior to the 1st and 3rd cycles of chemotherapy post debulking X Months 2, 4, 6 prior to vaccine administration X Urinalysis X EKG X Vaccine administration5 X Injection Site Assessment Day 2 only FACT-O6 X X Survival Status X X X7 1 Following tissue procurement, patients will be contacted every 6 weeks 2 weeks to assess interest / elig ibility of the main randomized portion of the trial and survival status. 2 Radiographic assessments taken at baseline, ≤ 1 week prior to Cycle 4 , at SOC intervals, and when CA-125 >35U/mL. 3 CA-125 taken at baseline, monthly for the first year, ev ery 3 months ± 2 weeks for the second and third year. 4 Obtain from medical records from standard preoperative hematology and chemistry panels. 5 The patient will be observed for at least 30 minutes (with a 10% window) following vaccination. During this observation period, vital signs will be taken every 10 minutes (with a 10% window). 6 FACT-O assessment will be collected at baselin e, Month 3, Month 6, Month 12, and Month 18. 7 After EOT, subjects will be contacted by phone quarterly for di sease status, medical records (w hen applicable), documentation o f 2nd line therapy and recording of response and duration of response to that therapy, additional cancer treatments as well as survival. Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 73 APPENDIX C NCI Common Toxicity Criteria For Adverse Events (CTCAE), Version 3.0 Publish Date: MARCH 31, 2003 As of April 02, 2003 NCI has introduced versi on 3.0 of the Common To xicity Criteria fo r Adverse Events. These may be obt ained at the fo llowing web link http://ctep.cancer.gov/reporting/ctc.ht ml or on the Mary Crowley CTMS. DO NOT USE CTC VERSION 2.0 TO GRADE TOXICITIES IN THIS STUDY! Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 74 APPENDIX D FACT-O, VERSION 4 Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 75 Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 76 Protocol # CL-PTL 105 CONFIDENTIAL AM #9 09/25/15 Page 77 REFERENCES 1. Aghajanian, C., S. V. Bl ank, et al. (2012). "OCEANS: a randomized, double-blind, placebo-controlled phase III trial of chemotherapy wit h or without bevacizumab in patients with platinum-sensitiv e recurrent epithelial ovari an, primary peritoneal, or fallopian tube cancer." J Clin Oncol 30(17): 2039-45. 2.