Do not make unauthorized copies. administration may remain a reasonable and acceptable alternative standard of care option (Wagner 2015) . 1.4 Immunotherapeutic considerations in Ewing’s sarcoma Ewing’s sarcoma demonstrates elements of the immune escape mechanisms determined to be integral to the natural history of cancer. Patients with primary disseminated disease at presentation show an increased frequency of T-regulatory cells in the bone marrow compared to those with localized disease (Brinkrolf, Landmeier et al. 2009). Factors associated with survival are increased number of tumor infiltrating CD8+ T-lymphocytes (p=0.05) which inversely correlates with tumor volume (<200 ml) at diagnosis (Berghuis, Sa ntos et al. 2011) . Furthermore, the unique, defining EWS/FLI-1 fusion protein may be an effective immunogenic driver neoantigen less likely to be adaptively downregulated due to its driver status (Liu, Huang et al. 2012, Peng, Huang et al. 2014). Meyer -Wentrup et al. discovered the first immunogenic Ewing’s sarcoma specific peptide directly derived from the EWS/FLI1 fusion gene (Meyer - Wentrup, Richter et al. 2005). Ten (10) peptides derived from the 21-mer fusion region were used to prime T cell responses by pulsing single peptides onto mature dendritic cells. Gamma interferon (IFNγ ) production was measured in ELISPOT assays as a surrogate marker for T- and NK cell activation. QQSSSYGQQN -pulsed dendritic cells induced significant IFN γ immune response while the other 9 peptide-induced IFN γ production remained unspecific. Interestingly, the response to QQSSSYGQQN was highly peptide specific and could be inhibited by HLA -DR- blocking antibody, known to block HLA class II activation. Fusion-region specific neoantigens, such as described by Meyer -Wentrup and colleagues, could reveal new opportunities to induce and sustain cytotoxic immune responses. Re-activation of immunogenic antitumor response as a treatment strategy for EWS could be exploited either in a monotherapy or in combination with chemotherapy agents such as irinotecan and temozolomide, which produce lower rates of lymphopenia of shorter duration and allow for effective, concurrent immune responsiveness (Kushner, Kramer et al. 2006) . Preliminary results in 83% of Ewing’s sarcoma patients treated with Vigil immunotherapy demonstrated CD8+ T-cell activation, as defined by IFN γ ELISPOT assay using unmanipulated autologous tumor cells (verified by Mt. Sinai and plates are assessed for spots by Zellnet, Fort Lee, NJ). Notably, Vigil induced ELISPOT response has been shown to significantly correlate with survival and time to recurrence (TTR) in patients with a variety of solid tumors (Senzer, Barve et al. 2013, Oh, Barve et al. 2016) and specifically with tumor responses (1 CR/NED, 1 PR) in two Ewing’s sarcoma patients one of whom is described in a prior publication (Ghisoli, Rutledge et al. 2017) . The relationship of the sequential IFNγ ELISPOT level to clinical course in this patient is shown in Figure 1. Evidence of CD4+ immune activation has been identified by analysis of 24 different cancer testis (CT) antigens in Vigil treated Ewing’s sarcoma patients. Patient 062 ( Figure 1) showed seroconversion of CT47 during Vigil treatment with persistence of antibodies after treatment completion. CT47 expression has been observed to be elevated in sarcoma patients suggesting antigen targeted therapies (Chen, Iseli et al. 2006) . Another Ewing’s sarcoma patient (PID 095) and long-term survivor (1033 days since procurement) showed similar CT47 CD4 response during Vigil treatment in addition to CD8 T cell activation (ELISPOT response). Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 21 of 74 Confidential and proprietary information of Gradalis, Inc. Effective: 05-DEC-2018 It is the user’s responsibility to verify adequate training before executing a document. Do not make unauthorized copies. Figure 1 . Case Report 1 (F -062). Relapsed Ewing's sarcoma Response to Vigil. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 22 of 74 Confidential and proprietary information of Gradalis, Inc. Effective: 05-DEC-2018 It is the user’s responsibility to verify adequate training before executing a document. Do not make unauthorized copies. 2.0 VIGIL Vigil immunotherapy comprises 1) autologous tumor cells as a source of the full matrix of a patient’s tumor-related antigens and 2) a DNA plasmid with two genetic modifications in order to optimize a “triad” functionality i) patient tumor -specific antigen presentation, ii) dendritic cell activation (GMCSF), and iii) tolerance escape (blocking TGFβ 1, β2 activation) (Maples PB 2009, Nemunaitis 2011). To construct Vigil, autologous cancer cells are transfected with a multi -component expression vector encoding GMCSF and a downstream bi -functi onal small hairpin RNA for specific knockdown of furin, a proprotein convertase critical for maturational proteolytic processing of immune relevant TGF β isoforms. Since the start of a Phase I trial of Vigil in advanced cancer patients on 06/08/2009, strong evidence for safety and benefit was initially seen in a sub-analysis of 35 adult and pediatric patients given 176 vaccinations (Senzer, Barve et al. 2013). To determine optimal dosing regimen, analyses were carried out comparing survival, safety and ELISPOT response at 1 x 10 7 cells/injection vs. 2.5 x 107 cells/injection and at a range of Vigil dosing schedules between 4 and 12 monthly injections. This analysis showed similar results between dose levels and number of doses; thus 1 x 10 7 cells/injection with a minimum of 4 and a maximum of 12 monthly injections was selected as the treatment regimen for subsequent studies. Vector effectiveness was also confirmed via GMCSF transgene expression and knockdown of Furin, TGFβ1 and TGFβ2 expression (Senzer, Barve et al. 2012, Senzer, Barve et al. 2013) . Interestingly , a small sub-cohort (n=15) with a dose level of 1 x 10 6 cells/injection was enrolled and treated on the Phase 1 study with Vigil and showed ELISPOT conversion (12/12 evaluable patients) from baseline to treatment start in a recent analysis ( Figure 2 and Figure 3) (Manning 2017) . Six (6) of these patients enrolled with advanced, relapsed or refractory Ewing’s sarcoma and 5 of the 6 Ewing’s patients had prolonged survival of > 2 years (median OS 807.5 days, range 58-1090 days from time of procurement). Four (4) of the low -dose Ewing’s patients were also analyzed for ELISPOT response and all 4 showed positive conversion post treatment with Vigil ( Figure 4). The median survival from time of procurement of these 4 patients was 1288 days (range 840-1814 days). This data supports a 1x10 6 dose administration of Vigil in Ewing’s sarcoma. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 23 of 74 Confidential and proprietary information of Gradalis, Inc. Effective: 05-DEC-2018 It is the user’s responsibility to verify adequate training before executing a document.