Do not make unauthorized copies. SYNOPSIS Summary : Ewing’s sarcoma is the second most frequently diagnosed primary malignant bone tumor in the US with an annual incidence, from birth to age 20, of 2.9 cases per million population. The survival rate for patients in first relapse is dismal (13% 5-year survival). Temozolomide and irinotecan is the consensus standard of care treatment regimen after relapse. Vigil immune response correlates with survival and time to relapse improvement in multiple solid tumors including Ewing’s sarcoma. Limited data has demonstrated no adverse effect of the combination therapy with irinotecan, temozolomide and Vigil (1x10 6 - 1x107 cells/injection (x4 or more)). Therefore, the combination regimen of Vigil, irinotecan, and temozolomide is hypothesized to increase anti -tumor responses and prolong time to progression in patients with relapsed or refractory Ewing’s sarcoma (first relapse). In an effort to overcome limitations of first-generation immunostimulatory cancer vaccines, we designed a novel autologous whole cell vaccine, Vigil, incorporating the rhGMCSF transgene and the bifunctional shRNA furin to: 1) address the inability to fully identify relevant strong-affinity cancer specific (and associated) antigens, 2) effect antigen recognition by the immune system (i.e. antigen→ immunogen), 3) enhance effector potency, and 4) subvert endogenous cancer -induced immune resistance. A Phase I assessment of Vigil in advanced solid tumor patients including 16 Ewing’s sarcoma patients receiving ≥1 cycle ( at a dose > 1.0 x 10 6 cells / injection / month for a minimum of 4 and maximum of 12 vaccinations) demonstrated safety of the Vigil immunotherapy. Furthermore, mechanism of action (i.e., vector effectiveness) was established in the manufactured products with increased mean GMCSF expression and knockdown of TGFβ 1 and TGFβ 2. The Vigil induced immunomodulatory effects against Ewing’s sarcoma was reflected in positive IFNγ ELISPOT response to autologous tumor cells in 10/12 patients, one RECIST criteria partial response (PR), a second patient achieving complete response (CR) and a total 1 -year overall survival (Kaplan Meier method) of 73% in comparison to expected 1-year survival of <25%. Objective(s) : Primary Objective: To compare the progression free survival (PFS) of subjects dosed with Vigil immunotherapy in combination with irinotecan and temozolomide vs. irinotecan and temozolomide. Secondary Objective(s) : • To determine and compare the overall survival (OS) of subjects with relapsed or refractory Ewing’s sarcoma dosed with Vigil immunotherapy in combination with irinotecan and temozolomide with patients that are treated with irinotecan and temozolomide. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 9 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. • To determine the objective response rate (RECIST 1.1) of patients with me tastatic Ewing’s sarcoma refractory or intolerant to 1 prior line of systemic chemotherapy treated with Vigil immunotherapy dosed with Vigil immunotherapy in combination with irinotecan and temozolomide. • To determine the Vigil manufacturing success rate by gathering data from tumor tissue collection, Vigil construction, and test procedures. Methodology : This is a multicenter, 1:1 randomized Phase III study of intradermal autologous Vigil immunotherapy (1.0 x 106 cells/injection; minimum of 4 to a maximum of 12 administrations) in combination with irinotecan and temozolomide in subjects with metastatic Ewing’s sarcoma Family of Tumors (ESFT) refractory /intolerant or recurrent to 1 prior line of chemotherapy. Participants undergoing a standard surgical procedure (e.g., tumor biopsy or palliative resection) may have tumor tissue harvested for manufacture of the investigational product, Vigil. Subjects meeting eligibility criteria including manufacture of a minimum of 4 immunotherapy doses of Vigil will be randomized to receive either: Group A (i) oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), (ii) oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), and (iii) Vigil 1.0 x 106 cells/injection cells/injection, intradermally on Day 15. or Group B (i) oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), and (ii) oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle). One cycle equals 21 days. Screening for the main portion of the study may occur as early as one week but no later than 8 weeks following tumor procurement. Vigil is typically released approximately 3 weeks after the completion of the two-day manufacturing process. At the time of analysis t he subjects will be stratified by initial response to frontline treatment (recurrent versus refractory (progressive)). Participants will be managed in an outpatient setting. Hematologic function, liver enzymes, renal function and electrolytes will be monitored. Blood for immune function analyses including IFNγ -ELISPOT analysis of cytotoxic T cell activation in response to autologous tumor antigens will be collected at tissue procurement, post-procurement screening and Day 1 (prior to chemotherapy administration) at Cycles 2, 4, and 6, end of treatment (EOT), 3 months after EOT, and every 6 months thereafter. Blood for ctDNA analysis will be collected Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 10 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. at tissue procurement, prior to chemotherapy administration at baseline and on Da y 1 prior to chemotherapy administration at Cycles 2, 3, 4, and 6, and EOT. Number of Patients : Approximately 140 subjects will have tissue procured so that 114 evaluable subjects will be registered for enrollment into the study. Tissue Procurement Inclusion Criteria: Subjects will be eligible for tissue procurement for the Vigil manufacturing process, if they meet all of the following criteria: 1. Histologically confirmed Ewing’s sarcoma Family of Tumors (ESFT). 2. Age ≥ 2 years. 3. Estimated survival ≥ 6 months. 4. Evidence of EWS translocation by FISH or RT-PCR or Next Generation Sequencing (NGS). If available, NGS sequencing report should be submitted to Gradalis. 5. Recurrence or refractory to 1 line of systemic chemotherapy including but not limited to doxorubicin, vincristine and ifosfamide. 6. Planned standard of care surgical procedure (e.g., tumor biopsy or palliative resection or thoracentesis) and expected availability of a cumulative soft-tissue mass of ~10-30 grams tissue (“grape” to “golf-ball” size / approximate 2 cm total diameter on imaging) and/or pleural fluid estimated volume ≥ 500mL (from a primary or secondary thoracentesis, yielding in a high volume of tumor cells) for immunotherapy manufacture. 7. Tumor intended for immunotherapy manufacture is not embedded in bone and does not contain luminal tissue (e.g. bowel, ureter, bile duct). 8. Ability to understand and the willingness to sign a written informed protocol specific consent for tissue harvest or a parental/guardian informed consent and pediatric assent when appropriate. Tissue Procurement Exclusion Criteria: Subjects meeting any of the following criteria are not eligible for tissue procurement for the Vigil manufacturing: 1. Medical condition requiring any form of chronic systemic immunosuppressive therapy (steroid or other) except physiologic replacement doses of hydrocortisone or equivalent (no more than 30 mg hydrocortisone or 10 mg prednisone equivalent daily) for < 30 days duration. 2. Known history of other malignancy unless having undergone curative intent therapy without evidence of that disease for ≥ 3 years except cutaneous squamous cell and Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 11 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. basal cell skin cancer, superficial bladder cancer, in situ cervical cancer or other in situ cancers are allowed if definitively resected. 3. Brain metastases unless treated with curative intent (gamma knife or surgical resection) and without evidence of progression for ≥ 2 months. 4. Any documented history of autoimmune disease with exception of Type 1 diabetes on stable insulin regimen, hypothyroidism on stable dose of replacement thyroid medication, vitiligo, or asthma not requiring systemic steroids. 5. Known HIV or chronic Hepatitis B or C infection. 6. Known hypersensitivity to any temozolomide component or to dacarbazine (DTIC). 7. Known hypersensitivity to irinotecan or its excipients. 8. Known history of allergies or sensitivities to gentamicin. 9. History of or current evidence of any condition (including medical, psychiatric or substance abuse disorder), therapy, or laboratory abnormality that might confound the results of the study, interfere with the patient’s participation for the full duration of the study, or is not in the best interest of the patient to participate, in the opinion of the treating Investigator. Study Enrollment Inclusion Criteria: Subjects will be eligible for registration if they meet all of the following inclusion criteria: 1. Completed manufacture of at least 4 vials of Vigil. 2. Karnofsky performance status (KPS) / Lansky performance status (LS) ≥ 80%. 3. Normal organ and marrow function as defined below: Absolute granulocyte count ≥1,000/mm3 Absolute lymphocyte count ≥400/mm3 Platelets ≥75,000/mm3 Hemoglobin ≥8.0 g/dL Total bilirubin ≤ institutional upper limit of normal * AST(SGOT)/ALT(SGPT) ≤2x institutional upper limit of normal Creatinine <1.5 mg/dL *documented Gilbert’s syndrome may be considered after medical monitor review 4. Subject has recovered to CTCAE Grade 1 (except for parameters noted in I tem 3, above) or better from all adverse events associated with prior therapy or surgery. Pre- existing motor, sensory neurologic pathology or symptoms, or dermatologic toxicities must be recovered to CTCAE Grade 2 or better. 5. If female of childbearing potential, has a negative urine or serum pregnancy test. If the urine test is positive or cannot be confirmed as negative, a negative serum test will be required for study entry. 6. Ability to understand and the willingness to sign a written informed protocol specific consent or a parental/guardian informed consent and pediatric assent when appropriate. Study Enrollment Exclusion Criteria: Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 12 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. In addition to the procurement exclusion criteria, subjects will NOT be eligible for study registration and randomization if meeting any of the following additional criteria: 1. Any anti -neoplastic therapy between tissue procurement for Vigil manufacture and start of study therapy. 2. Live vaccine used for the prevention of infectious disease administered < 30 days prior to the start of study therapy. 3. Post-surgery complication that in the opinion of the treating investigator would interfere with the subject’s study participation or make it not in the best interest of the patient to participate. Medication and Doses : Autologous Vigil will be supplied by Gradalis, Inc. Irinotecan will be supplied by Clinigen. Temozolomide will be ordered per standard institutional practice. Group A (i) oral temozolomide 100 mg/m2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), (ii) oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle), and (iii) Vigil 1.0 x 106 cells/injection or cells/injection, intradermally on Day 15. or Group B (i) oral temozolomide 100 mg/m 2 daily (Days 1 – 5, total dose 500 mg/m2/cycle), and (ii) oral irinotecan 50 mg/m2 daily (Days 1 – 5, total dose 250mg/m2/cycle). One cycle equals 21 days. Duration : Subjects may receive repeat cycles of treatment until disease progression, unacceptable toxicity, withdrawal of consent or other criterion is met for discontinuation from study. Subjects randomized to Group A, Vigil will receive up to 12 doses depending upon the quantity of Vigil manufactured from the surgical specimen. Within 6 weeks of second relapse or progression, subjects randomized to Group B, will be allowed to cross -over (see Section 10.0) to receive single agent Vigil every 21 days. Subjects who cross -over may receive up to 12 doses of Vigil depending upon the quantity of Vigil manufactured. Cross -over must occur within 2 years of End of Treatment assessments of Group B enrollment. For subjects crossing over, no alternative therapies will be given between End of Treatment and cross - over. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 13 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. Efficacy Assessments : • Progression Free Survival (PFS) • Objective Response Rate (ORR) (RECIST 1.1) • Overa ll survival (OS) Safety Assessments : • Laboratory assessments • Physical examination, performance status, height, weight, temperature, blood pressure, and pulse • Toxicity: CTCAE v 5.0 Exploratory Assessments: Circulating EWS FLI1 DNA detection and intratumoral immune function analysis including, but not limited to IFNγ -ELISPOT analysis. Evaluation of baseline tumor microenvironment including, but not limited to immunohistochemical evaluation of tumor PD -L1 expression and tumor infiltrating lym phocyte (TIL) populations. Statistical Considerations : The planned sample size of 114 randomized subjects is based on the following assumptions for the PFS endpoint: • 1:1 randomization; • Accrual period of 12 months; • Follow -up period of 6 months; • Assumed control group median PFS of 6 months from randomization; • Two-sided test at the alpha=0.0476 level of significance; • Power 90%; • Assumed hazard ratio (HR) of 0.45 The planned number of events is 67. An interim analysis for efficacy, futility, and potential sample size re-estimation will be conducted by an independent data monitoring committee (IDMC) when 40 PFS events have been achieved (60% information fraction). Based on the Lan-DeMets method for group sequential trials using O’Brien-Fleming boundaries (Reboussin, DeMets et al. 2000) , the levels of significance for the interim and final analyses are alpha=0.00762 and alpha=0.0476, respectively. Based on the results of the interim analysis, the IDMC will make one of the following four recommendations to the sponsor: • Terminate the study for efficacy (p<0.00762 at the interim analysis); Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 14 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. • Continue the study as planned; • Increase the sample size to 120 events; • Terminate the study for futility. If the sample size is increased to 120 events, then the study will have approximately 90% power to detect a true HR of 0.55. Note that if the sample size is increased to 120 events, then the accrual period will be extended to 24 months, with a 6-month follow-up period after randomization of the last patient. Under these assumptions, it is estimated that approximately 164 patients will need to be randomized in order to achieve 120 events. Provided that the IDMC does not recommend terminating the study early for efficacy, the conditional power of the planned final analysis at 67 events will be computed using the methodology of (Jennison and Turnbull 2000) under the assumption that the observed hazard ratio at the interim analysis represents the true effect. In terms of the HR observed at the interim analysis, these rules can be translated (approximately) as follows: • If the HR is less than 0.43, terminate for efficacy • If the HR is in the range from 0.43 to 0.51, continue as planned. • If the HR is in the range from 0.51 to 0.61 (i.e., conditional power in the range from 50% to 80%), increase the sample size to 120 events. • If the HR is greater than 0.61 (i.e., conditional power less than 50%), terminate for futility. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 15 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. STUDY SCHEMA Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 16 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. STUDY PROCESS FLOW Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 17 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. 1.0 INTRODUCTION 1.1 Summary Ewing’s sarcoma is the second most frequently diagnosed primary malignant bone tumor in the US with an annual age- adapted (birth – 20 years) incidence of 2.9 cases per million population (Esiashvili, Goodman et al. 2008). Approximately 20-30% of cases are diagnosed in the first decade and 10% after age 20. The median age at diagnosis is 14 – 15 years (Cotterill, Ahrens et al. 2000, Stahl, Ranft et al. 2011). In greater than 90% of patients there is an essentially specific translocation of the EWSR1 gene on chromosome 22q12 with FLI1 on chromosome 11q24 or, less commonly, other genes (ERG) of the ETS transcription factor family (Marino- Enriquez and Fletcher 2014). Up to 85% of Ewing’s tumors are characterized by the EWS/FLI1 Type 1 (EWS exon 7 to FLI-1 exon 6) or Type 2 (EWS exon 7 to FLI1 exon 5) translocation (Arvand and Denny 2001) . Patients with initially diagnosed localized disease have a disease-free survival rate (DFS) of 60- 70% with standard 5-drug chemotherapy regimen (VAC +IE or VIDE), while patients with metastatic disease have a DFS of <20%. The 5-year front line treatment survival rate for patients with localized disease approaches 70% with standard of care, but only ~30% for those with metastatic lesions isolated to the lung and <20% for those with bone or bone marrow involvement (Cotterill, Ahrens et al. 2000, Meyers, Krailo et al.