x Events that are clearly related to the patient’s disease. 3.1.5 Phase I – Dose Escalation Subjects will receive their assigned dose level of NCI-4650 as IM injections on Days 0, 14, 28, and 42. The maximum dose of mRNA va ccine for each dose level will be: Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 25 Dose Level NCI-4650, mRNA Vaccine Dose Level 1 0.04 mg (0.0006 mg/kg based on a 70-kg subject) Level 2 0.13 mg (0.0019 mg/kg based on a 70-kg subject) Level 3 0.39 mg (0.0056 mg/kg based on a 70-kg subject) Moderna is the sponsor of a dose escalation tr ial utilizing a nearly identical vaccine (mRNA- 4157) as the mRNA vaccine used in this protocol (NCI-4650). The dose escalation in both protocols is the same (0.04 mg, 0.13 mg, and 0.3 9 mg). As indicated in the Moderna Safety Review Committee Dose Level Approval Form dated 21 December 2017, the Moderna Medical Monitor noted that for the three patients enrolled on Dose Level 1 (Part A) in the Moderna trial (0.04 mg mRNA-4157 monotherapy), no DLTs were experienced and the only AEs experienced were grade 1 injection site sorene ss, which is expected for the vaccine. As a result, Dose Level 1 was declared safe and tolerated. For this trial, because Dose Level 1 was shown to be safe and tolerated, the first two patients were enrolled on Dose Level 2. No DLTs were experienced and the only AEs experienced were grade 1 and 2 related events. As indicated in the Moderna Safety Review Committee Dose Level Approval Form dated 13 July 2018, the Moderna Medical Monitor noted th at for the six evaluable patients enrolled on Dose Level 2 (Part A) in the Moderna tria l (0.13 mg mRNA-4157 monotherapy), no DLTs were experienced and the only AEs experienced were mild, non-serious grade 1 events considered to be related to the experimental treatment. Additionally, as indicated in the Moderna Safety Review Committee Dose Level Approval Form dated 07 September 2018, four evaluable patients were enrolled on Dose Level 2 (Part B) in the M oderna trial (0.13 mg mRNA-4157 in combination with pembrolizumab). These four patients experienced only mild, non-serious grade 1 events considered to be related to the expe rimental treatment, and no DLTs were seen. The following table shows all grade 1 events experien ced by all patients enrolled on Dose Level 2 in the Moderna trial, for both parts A and B: Adverse Events Incidence # of Patients Injection site (edema, erythema, tenderness) 8 3 Diarrhea 2 1 Photosensitivity 1 1 Arthritis 4 3 Finger edema 1 1 Myalgias 4 3 Flu-like symptoms 1 1 For this trial, because Dose Level 2 has been show n to be safe and tolera ted, we propose to enroll our third patient (previously anticipated to be th e third patient in Dose Level 2) as the first patient in Dose Level 3. The protocol will enroll three patients at Dose Level 3 unless a patient experiences a DLT. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 26 In each dose level, if a patient experiences a DLT, a total of six patients will be treated at that dose to confirm that no greater than 1/6 patients have a DLT prior to proceeding to the next higher dose level. If a level with 2 or more DLTs in 3-6 patients has been identified, patients will be accrued at the next-lowest dose level, for a total of 6 patients. If two DLTs occur in the second dose level (0.13 mg), the dose will be de-e scalated to Dose Level 1 (0.04 mg). If two DLTs occur at Dose Level 1, the study will be temporarily halted, as described in Section 3.2. The MTD is the highest dose at which  1 of 6 patients experienced a DLT or the highest dose level studied if DLTs are not obser ved at any of the dose levels. Although the MTD may be reached during this cl inical study, determining the MTD is not the aim of the study as benefit from this therapeutic vaccine approach may be achieved at lower doses. 3.1.6 Rationale for Dose Level Regime n and Route of Administration As described in the repeat-dose GLP toxicolo gy studies in outbred Sprague Dawley rats described in Section 1.2.4 and IND 17390, the no-observed-adverse-effect-level (NOAEL) for mRNA-4157 was determined to be 0.035 mg base d on the clinical and microscopic findings observed in this study. Based on HED, this repres ents an approximate 24-fold safety margin over the proposed human starting dose of 0.04 mg. The highest dose in the rat toxicology study was 0.117 mg (0.32 mg/kg), which is an approximate HED of 3.51 mg (0.05 mg/kg based on a 70-kg subject) and is approximately 88-fold higher than the proposed starting clinical dose of 0.04 mg. Thus, it is considered that a starting dose of 0.04 mg has an adequate safety margin. The highest dose in the GLP rat toxicology study (0.117 mg; which is a HED of 3.51 mg based on a 70-kg subject) is approximately 9-fold high er than the highest planned clinical dose of 0.39 mg. The NCI-SB considers that this represents a sufficient safety margin to the dose levels in this clinical study. The 3+3-guided dose escal ation scheme of approxima tely half-log (3-fold) increments (i.e., 0.04, 0.13, and 0.39 mg) is consis tent with FDA guidance for early-phase clinical trials involving ce llular and gene therapy products 27. Subjects will receive their assigned dose level on Day 1 of each 14-day cycle (days 0, 14, 28, and 42). Four cycles of NCI-4650 are planned to boost the intended tumor-killing T-cell population over an extended period by promoting optimal development of T-effector memory cells. The 14-day cycle allows for maximal contraction of the antigen-specific CD8 + T-cells response before boosting, which found to produce best maintenance of CD8+ T-cells with high recall capacity. From a safety perspective, the Manufacturer as sessed the toxicity of mRNA-4157 in Sprague Dawley rats at the dose levels of 0.012, 0.035, and 0.117 mg via IM injection every week for 4 weeks (5 doses in total; refer to Section 1.2.4 and IND 17390). This preclinical weekly injection schedule is 2-fold more dose-dense over time than the clinical dosing schedule in this study, and thus provides an additional margin to potential cumulative toxicity. The IM route of administration was chosen for ease of administration. 3.1.7 Phase II Portion Patients in Phase II will receive the MTD as deter mined in Phase I. The Phase II portion of the protocol will consist of two c ohorts as defined in Section 2.3.3.1: Cohort 2a, patients with melanoma and Cohort 2b, patients with gastrointestinal or genitourinary cancer. Abbreviated Title: mRNA-Based, Cancer Vaccine Version Date: January 23, 2019 27The Phase II portion of the study will utilize an optimal design where initially 12 evaluable patients will be enrolled in each cohort. For each cohort, if 0 of the first 12 evaluable patients experience a clinical response, then no further patients will be enrolled; if 1 or more of the first 12 evaluable patients enrolled have a clinical response, then accrual will continue until a total of 21 evaluable patients have been enrolled in each cohort. Note: As stated in Section 8.2, up to 6 patients enrolled at the MTD will count towards the accrual in the Phase II portion of the trial if they are evaluable for response and if they would be fully eligible for enrollment in the Phase II portion of the trial. PROTOCOL STOPPING RULES New subject enrollment to the protocol will be temporarily halted if any of the following conditions are met, and consideration will be given to the need for protocol revisions regarding safety, if applicable. Co nsultation with the FDA may be arranged by the Sponsor before or after the protocol amendment is submitted to the NIH Intramural IRB, as needed. x During the Phase I portion of the study – If two DLTs, as described in Section 3.1.4, occur at Dose Level 1. x During the Phase II portion of the study – On ce five or more patient s have been enrolled, if 20% or more patients cumulatively enrolle d develop a DLT, as described in Section 3.1.4. x During the Phase I and II portions of the st udy – Any death that is at least possibly attributed to the investigational agent and which occurs within 30 days of receiving the investigational agent. DRUG ADMINISTRATION Patients will receive the mRNA vaccine on days 0, 14 (± 5 days), 28 (± 5 days), and 42 (± 5 days). The vaccine will be administered as an IM injection. The patient’s vaccine is delivered to the Patient Care Unit by the research nurse. Prior to the injection, the vaccine identity label is double-chec ked by two authorized staff (MD or RN), and an identification of the product and documentation of administrati on are entered in the patient’s chart. x The NCI-4650 mRNA vaccine will be administere d as an outpatient procedure in the 3SE Day Hospital in the NIH Clinical Center. x For all subjects, NCI-4650 will be administered by IM injection into the deltoid muscle (alternating deltoid muscle sites should be used for each subsequent injection, and injection sites may also be rotated to thigh muscles). x The dose volume for Dose Levels 1 and 2 will be held constant at approximately 0.3 mL per injection. The dose volume for Dose Level 3 (PN 85002) will be at least 0.39 mL. x The NCI Pharmacy Manual contains specific instructions for the preparation and administration of each dose level of NCI-4650.