5. Serum chemistries (creatinine, glucose, total protein, blood urea nitrogen (BUN), total carbon dioxide (CO2), albumin, total bilirubin, alkaline phosphatase, and aspartate transaminase (AST) and/or alanine transaminase (ALT)) and electrolytes (total calcium, chloride, potassium, sodium). 6. Blood collection for immune function analysis (ELISPOT) 3 months ±7 days from EOT and every 6 months ±7 days thereafter. 7. Assessment of Performance Status. Long Term Follow Up After progression, subjects and their physicians will be contacted quarterly for documentation of post study therapies and survival status. As the intent -to-treat population includes all randomized patients, should a subject be randomized and choose to not receive any treatment on this study, the subject should be asked if he or she is willing to allow for survival follow up through phone call and/or physician contact quarterly. This follow up would include any anti -cancer therapies received and survival status information. Based on findings during the study or during the follow up portion of the trial, Gradalis may request for additional blood and / or tissue samples from the research participant. Collection of whole blood (40ml) and / or tissue samples (via biopsy or clinically indicated surgical removal) will be optional and used to study the effects of the study agent (included, but not limited to testing of biomarkers, predictors or biological responses, toxicity, relationship between genotype and study agent responses). Should Gradalis request for additional blood or tissue and the subject consented to the collection, the clinical site will present the option of the procurement to the participant. 10.0 CROSS -OVER Subjects randomized to Group B may cross -over to Group A after discussion with, and approval from the Gradalis medical monitor or authorized designee. Within 6 weeks of second relapse or progression and if alternative therapy has not been given since EOT, subjects randomized to Group B, will be allowed to cross -over to receive single agent Vigil every 21 days following End of Treatment assessment. Subjects who cross -over may receive up to 12 doses of Vigil depending upon the quantity of Vigil manufactured. After the Group B EOT assessment, the subject will start “cross -over” from Assessments During Treatment onward. Vigil and all assessments will occur on Day 1 of each cycle. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 54 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. Cross -over must occur within 2 years of End of Treatment assessments of Group B enrollment. Subjects who progress ≥ 8 weeks post EOT assessment must re-screen and if they qualify will start “cross -over” from Assessments During Treatment onward. Eligibility criteria for cross -over subjects are listed below. 10.1 Inclusion Criteria for Cross-Over Subjects will be eligible for cross -over, if they meet all of the following criteria: 1. Evidence of radiologic disease progression by RECIST 1.1 after enrollment into Group B. 2. Successful manufacturing of at least 4 vials of Vigil. 3. Karnofsky performance status (KPS) / Lansky performance status (LS) ≥70%. 4. Normal organ and marrow function as defined below: Absolute granulocyte count ≥1,000/mm3 Absolute lymphocyte count ≥400/mm3 Platelets ≥75,000/mm3 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 5. Subject has recovered to CTCAE Grade 1 (except for parameters noted in I tem 4, 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. 6. 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 s tudy entry. 7. 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. 10.2 Exclusion Criteria for Cross-Over In addition to the Procurement and Study Enrollment exclusion criteria, Subjects will NOT be eligible for cross -over if meeting any of the following criteria: 1. With the exception of irinotecan and temozolomide while on study, 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. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 55 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. 11.0 CONDUCT OF THE STUDY 11.1 Ethics and Regulatory Considerations This study must have the approval of a properly constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC). Before the investigational drug is shipped to the investigator, the investigator will provide Gradalis, Inc. with a copy of the IRB or IEC approval letter stating that the study protocol and informed consent form have been reviewed and approved. 11.2 IRB This trial can be undertaken only after review and full approval of the protocol and a n 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 Informed Consent Form, and any applicable recruiting materials and subject compensation programs approved. The decision concerning the conduct of the study will be made in writing to the sponsor. Copies of this decision and of all IRB correspondence will be kept on file at the study site; copies will be provided to the Sponsor Office. During the trial, the PI is required to send various documents to the IRB for review: • All protocol amendments and Informed Consent Form revisions. • Reports of Serious Adverse Events. The PI provides Gradalis, Inc. with the necessary assurance that an IRB is responsible for the initial and continuing review and approval of the proposed clinical study in accordance with 21 CFR 312.60. At least once a year, the IRB 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 that the trial has been completed. 11.3 Written Informed Consent The informed consent document should meet the requirements of the latest version of the Declaration of Helsinki and any applicable regulations and guidelines. It must be approved by an IRB or IEC. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 56 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. Prior to entry into the trial and before any protocol -required procedures are performed, the Investigator must explain the nature of the trial, its intended purpose, and the implications of participation to potential subjects 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 subjects’ participation is voluntary, and that they may withdraw consent to participate at any time. They will also be informed that if subjects choose not to participate in the trial alternative treatments are available; such refusal will not prejudice further treatment of their disease. Potential subjects or their legal representatives must be given the opportunity to ask questions about the trial protocol and the procedures involved. Finally, each subject will be told that his or her records may be accessed by authorized personnel of Gradalis, Inc. and other authorized individuals without violating the subject’s confi dentiality, to the extent permitted by the applicable laws and/or regulations. By signing the written Informed Consent Form, the subject or his or her legal representative is authorizing such access. Following this explanation and prior to entry into the trial, the written, dated, and signed Informed Consent Form must be obtained from each subject or his or her legal representative; a copy will be given to the person signing the form. 11.4 Confidentiality of Records The Investigator is required to retain, in a confidential manner, sufficient information on each subject (i.e., full name, current address, and social security number) so that the subject may be contacted by the FDA, Gradalis, Inc., or by their affiliates should the need arise. 11.5 Modification of Pr otocol Any changes to this protocol that affect study objectives, study design, study procedures, patient population, or significant 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 amendment must be approved by the Gradalis, Inc., and approved by the applicable IRB or IEC. 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 agreed upon by the Gradalis, Inc., and will be documented in a memorandum. The applicable IRB or IEC will be notified of administrative changes according to applicable IRB guidelines. 11.6 Protocol Questions and Deviations When evaluating a potential patient or while a patient is on study, protocol questions can be directed to Gradalis via email or phone using the contact information provided in the Contact Information section of the Study Reference Manual. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 57 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. 11.7 Data Safety Monitoring Board An independent data monitoring committee (IDMC) or Data Safety Monitoring Board (DSMB) will be established for this trial. Membership will include physicians with appropriate areas of expertise in the therapeutic areas of most interest. A charter will be developed to document their operational methods and timing of safety reviews. 12.0 EVALUATION OF TUMORS 12.1 Tumor Measurements and Response (RECIST 1.1) Response and progression will also be evaluated in this study using the Response Evaluation Criteria in Solid Tumors (RECIST) Version 1.1. The guidelines are available online at: European Organization for Research and Treatment of Cancer (EORTC) RECIST Web page , https://www.eortc.be/Recist/documents/RECISTGuidelines.pdf . Radiological images should be submitted to WorldCare upon receipt from the imaging facility for third party review. 13.0 DRUG INFORMATION 13.1 Vigil Investigational Product Vigil is made up of irradiated autologous tumor cells which have been electroporated ex vivo with the Vigil plasmid designed to suppress expression of both the TGFβ1 and TGFβ2 proteins while simultaneously expressing rhGMCSF protein. Vigil Production The Vigil manufacturing process is identical to prior Vigil manufacturing (BB -IND 14205) (Maples 2010). Surgically excised tumor is collected in the surgical field and placed in 0.04mg/ml of gentamicin and sterile saline then packaged for transport to the manufacturing facility. The tumor is mechanically and enzymatically dissociated into a single cell suspension. The cells are counted and then transfected with the Vigil plasmid. The cells are incubated overnight to allow transcription of the Vigil plasmid. The following morning the cells are harvested, washed, and then irradiated at 10,000cGy in a standard Blood Bank irradiator. The irradiated cell suspension is then enumerated, aliquoted and frozen (1 x 10 6 cells). The freeze media consists of 10% DMSO (dimethyl sulfoxide; Cryoserv USP; Bionichepharma US), 1% Human Serum Albumin (ABO Pharmaceuticals) in Plasma-Lyte A at pH 7.4 (Baxter). After Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 58 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. freezing the cells are stored in the vapor phase of liquid nitrogen until all release testing is completed, all necessary approvals are obtained and the patient is ready for treatment. Safety Analysis Vigil plasmid employed in the generation of this product has been tested for identity, sterility, purity and strength. Irradiated Gene Modified Tumor Cells To ensure safety, all gene- modified tumor cells to be used in Vigil administrations must be irradiated 10,000 cGy prior to freezing. This is the same irradiation process as for the TAG vaccine, BB -IND 13650 and prior vaccines ( Belagenpumatucel -L and GVAX® published trial results and BB -IND 13401 and B B-IND 12118) (Kumar 2009, Maples PB 2009, Maples PB 2009). The selection of this radiation dose is based on the desire to utilize the lowest possible radiation dose for the transfected cells to optimize the level and duration of bifunctional shRNA furin transcription and GMCSF protein production and maximize the safety of vaccine cell injections at the same time. In addition, investigators have demonstrated that irradiating cultured tumor cells of different histologic origins at 10,000 cGy completely arrests tumor colony formation. Preparation Reference the Study Reference Manual for preparation and handling information. Vigil concentrate: 1.0 x 106 cells per injection in a volume of 1mL. Route of administration: Intradermal injection Storage and Shipping Frozen, unopened vials are stored in the vapor phase of Liquid Nitrogen below -150° C at Gradalis. Each Vigil concentrate will be shipped individually in a portable liquid nitrogen tank. This shipping container will be able to sustain temperature fluctuations for up to 7 days. This will enable sufficient time to reach the different clinical site pharmacies. Study medications are not expected to pose significant occupational safety risks to investigational staff under normal conditions of use and administration. However, precauti ons Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 59 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. should be taken to avoid direct contact with study medication. Biosafety Level 1 practices shall be employed with this study medication. Reference the Study Reference Manual. 14.0 ADVERSE EVENTS 14.1 Adverse Event and Serious Adverse Event Definitions 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 associated with the use of a medicinal (investigational) product, whether or not related 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 prolonged hospitalization (≥ 24 hours). This does not include hospitalizations which are part of routine medical practice. • 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. the 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 serious criteria, but may jeopardize the subject or may require surgical or medical 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. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 60 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. Grading Adverse Events Adverse events (AEs) will be recorded throughout the trial. Toxicities and AEs will be graded and reported using the Common Toxicity Criteria for Adverse Events (CTCAE) Version 5. All AEs, regardless of severity, will be followed by the Treating Physician until resolution is satisfactory. 14.2 Attribution of Causality The relationship of each event to treatment will be assessed by the Treating Physician and recorded on the CRF in the EDC. 14.3 Vigil Expected Side Effects Tumor cell vaccines have been previously administered 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 the site of injection: Erythema, tenderness, induration, urticaria/rash, pruritus. Other expected adverse events: Fever, myalgias/arthralgias, chills/rigors, nausea, fatigue, headache, thrombocytopenia and other cytopenias, hyperglycemia, vomiting, hypotension, infection at the immunization site. In addition, there may also be a risk 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. 14.4 Recording of an Adverse Event Adverse events will be recorded for the duration of a subject’s time on study: following procurement of the subject’s tissue for tissue manufacture and for up to 30 days following the last administration of Vigil. All AEs, regardless of causal relationship are to be recorded in the eCRF and source documentation. Additional information about each event, such as treatment Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 61 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. required, eventual outcome, and whether or not therapy had to be interrupted or dosages reduced, will also be recorded on the eCRF. 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. 14.5 Serious Adverse Event Reporting All SAEs will be reported to Gradalis, Inc. within 24 hours of notification by the site through email or facsimile. This includes any death from any cause following procurement of the subject’s tissue for tissue manufacture and for up to 30 days following the last dose of the protocol study agent (Vigil). The site will supply as much information as is available at the time of the initial notification (study number, patient initials, 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. 15.0 PATIENT COMPLETION A ND WITHDRAWAL 15.1 Indication for Taking Patients Off Study 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. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 62 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. 16.0 STATISTICAL CONSIDER ATIONS 16.1 Sample Size Justification This is an open label randomized controlled Phase III clinical trial. Information concerning the predicted survival of the control group is limited, based on rarity of disease and few published reports. However expert advisors in the EWS field estimate a conservative one-year survival rate of 25% in the chemotherapy control group. The one-year survival rate of 60% in the Vigil treated group is estimated from EWS patients treated on the Vigil phase 1 protocol. These estimates correspond to a hazard ratio (HR) of 0.383 favoring Vigil over control. Assuming 1:1 randomization and the use of a two-sided log rank test at the alpha=0.0476 level of significance, 67 events will provide 90% power to detect an PFS HR of 0.45. Assuming a Group B (control group) median PFS of 6 months, a one-year accrual period, and a six -month follow -up period after randomization of the last subject, it is estimated that the total sample size (number of subjects) required to achieve 67 events is 114 (57 patients in each arm). An unblinded interim analysis will be conducted by an independent data monitoring committee (IDMC) when 40 PFS events have occurred. Based on the results of this interim analysis, the planned sample size may be increased to 120 events. 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. Section 16.6 provides further details. 16.2 Definition of Progression Free Survival (PFS) • Pro gression free survival (PFS) is defined as the time from randomization to the event of disease recurrence/progression or death due to any cause. • Radiographic disease recurrence/progression will be assessed using the RECIST 1.1 criteria. If the disease recurrence/progression assessment involves more than one date, the earliest date will be used as the event date. • Patients who are alive and recurrence free at the time of analysis data cut-off, or who terminate the study for reasons other than disease progression or death, or who receive non-protocol treatment for Ewing’s Sarcoma, will be censored on the last assessment date at which a RECIST 1.1 evaluation of disease status was made. • Patients missing baseline disease assessment will be censored at date of randomization. • For equivocal findings of recurrence (e.g., very small and uncertain new lesions; cystic changes or necrosis in existing lesions), treatment may continue until the next scheduled assessment. If recurrence is confirmed at the next scheduled assessment, the date of recurrence should be the earlier date when recurrence was suspected. Ph III Ewing’s Sarcoma Vigil ± Irinotecan +Temozolomide IND 014205 CL-PTL-130 Version: 03 Page 63 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.